Thèse Pour

Obtenir Le Grade De

Docteur De L'université De Montpellier

Présentée Par

Amos Fumagalli

Philippe Marin

Séverine Chaumont-Dubel

Gwendal Lazennec

Marc Mesnil

Eric Reiter

Jean-Luc Galzi

Examinateur Rapporteur Examinateur

Potein Zo-1

Zonula Occludens-

Cxcl3 Groγ

CCR2 Ccr1

Mip-1α Ccr1

Aurélien Zarca

Maria Neves

Birgit Caspar

Stephen Hill

Federico Mayor

Martine Smit

Deciphering CXCR4 and ACKR3 interactomes reveals an influence of ACKR3 upon Gap Junctional Intercellular Communication

Keywords: chemokine, ACKR3, interactome, Connexin 43, Gap Junction, glial cell BRET, Bioluminescence Resonance Energy Transfer cAMP, Cyclic adenosine monophosphate CBX, CarBeneXolone Cdk, Cyclin-dependent kinase CDNA, complementary DeoxyriboNucleic, Co-ImmunoPrecipitation Cx, Connexin Cx43, Connexin 43 DMSO, Dimethyl sulfoxide DNA, DeoxyriboNucleic Acid EGF, Epidermal Growth Factor EGFP, Enhanced Green Fluorescent Protein EGFR, Epidermal Growth Factor Receptor ERK, Mitogen-activated protein kinase FRAP, Fluorescence Recovery After GJIC, Gap Junctional Intercellular Communication GLAST, Glutamate Aspartate Transporter GPCR, IP3, Inositol Trisphosphate LFQ, Label-Free Quantification LY, Lucifer Yellow MAPK, Mitogen-Activated Protein Kinases MEK, Mitogen-activated protein kinase mGlu2, Metabotropic ODDD, autosomal dominant occulodentodigital dysplasia PFA, paraformaldehyde PI3K, phosphoinositide 3 kinases PKC, Protein Kinase C PLA, Proximity Ligation Assay PTX, Pertussis ToXin RLuc, Renilla-luciferin 2-monooxygenase RNA, RiboNucleic TMZ, Temozolomide UV, UltraViolet vs., versus WHO, World Health Organization Y2H, Yeast two-Hybrid YFP, Yellow Fluorescent CXC class CXCL1 Groα CXCR2, CXCR1 CXCL9 MIG CXCR3, CXCR3B IP-10 CXCR3, CXCR3B BRAK, Bolekine GCP-2 CXCR1, CXCR2 CXCL16

Deciphering CXCR4 and ACKR3 interactomes reveals an influence of ACKR3 upon Gap junctional intercellular communication

RÉSUMÉ

récepteur ACKR3 s'associe également au récepteur de l'EGF pour induire la prolifération cellulaire par un mécanisme indépendant de sa stimulation par un agoniste. Ces données illustrent l'intérêt de caractériser de façon systématique l'interactome de ces récepteurs pour comprendre leurs rôles physiologiques et pathologiques. Cette thèse a poursuivi cet objectif grâce à la mise en oeuvre d'une approche protéomique combinant la purification des partenaires des deux récepteurs par affinité suivie de leur identification par spectrométrie de masse. J'ai ainsi identifié respectivement 19 et 151 partenaires protéiques potentiels des récepteurs CXCR4 et ACKR3 exprimés dans les cellules HEK-293T. Parmi les protéines recrutées par ACKR3, nous nous sommes focalisés sur la connexine 43 (Cx43, une des protéines constituant les jonctions Gap) du fait de la similitude des effets du récepteur et de la Cx43 dans la pénétration des leucocytes dans le parenchyme cérébral, la migration des interneurones et la progression des gliomes. J'ai confirmé par Western blot et par BRET l'association spécifique de la Cx43 à l'ACKR3 et non pas au CXCR4. De la même façon, j'ai montré une co-localisation de la Cx43 et de l'ACKR3 dans des cellules de gliome humain, ainsi que dans les astrocytes de la zone sous-ventriculaire et les pieds astrocytaires entourant les capillaires cérébraux chez la souris, suggérant que les deux protéines forment un complexe protéique dans un contexte biologique authentique. Des études fonctionnelles ont révélé que l'ACKR3 module les fonctions de la Cx43 par différents mécanismes. L'expression de l'ACKR3 dans les cellules HEK-293T (mimant la surexpression du récepteur dans les tumeurs), induit par elle-même une inhibition de l'activité jonctionnelle de la Cx43.

De même, la stimulation du récepteur par un agoniste réduit l'activité jonctionnelle de la Cx43 par un mécanisme impliquant l'activation d'une protéine Gi, la b-arrestine2 et l'internalisation de la Cx43. Cette thèse établit donc pour la première fois un lien fonctionnel entre le système constitué par les chimiokines CXCL11, CXCL12 et leur récepteur ACKR3 d'une part et les jonctions Gap d'autre part qui pourrait jouer un rôle critique dans la progression des gliomes.

Mots clés : chimiokine, ACKR3, interactome, connexine 43, jonction gap, cellule gliale.

ACKNOWLEDGEMENTS

It is a strange sensation to be here writing my PhD acknowledgments 10 years after almost dropping out school for trying to be a professional football player.

In these regards, I would like to thank Prof. Corrado Lodovico Galli and Prof. Johanna Fink-Gremmels for instilling in me the passion of science that motivated the choice to pursue a PhD here in Montpellier at IGF. Heartfelt thanks go to my thesis supervisor Dr. Philippe Marin. Your scientific guidance has been essential for all the results obtained in these three years. Furthermore, your dedication and passion for this work, your attention to the details, your scientific and logic reasoning will be forever an example for me.

My sincere thanks to my other thesis supervisor Dr. Severine Chaumont-Dubel. Despite your hectic agenda (thanks to your enviable organization) you were always there when I needed you.

In addition to my supervisors, I would like to thank the rest of my thesis committee: Prof. Marc Mesnil, Dr. Christian Giaume, Dr Eric Reiter, Dr. Jean-Luc Galzi and Dr. Gwendal Lazennec. I am looking forward to hearing your comments, suggestions and criticisms and I am eager, besides being a little anxious, to be engaged in a scientific discussion with such renowned experts in the field.

My deepest gratitude goes out to all the current and former members of Philippe Marin's team. Dr. Claeysen, Dr. Becamel, Dr. Thouvenot, Prof. Bockaert, Simon, Caroline, Vincent, Lucile, Coralie, Camille, Samy, Hugo, Emilie, Angelina, Bérénice, Geoffrey and Patrizia. Thank you all for your constant support and encouragement. Special thanks go to Dr. Vandermoere for finding the time to read and correct this manuscript.

I am grateful to all the ONCORNET members. Thanks to Prof. Martine Smit and Prof.

J.E. van Muijlwijk-Koezen for coordinating this amazing network. Thanks to all the students: Ilze, Birgit, Vladimir, Marta, Joyce 2 , Cristina, Maria, Davide, Aurelién, Ali, Carmen, Sebastian and Angeliki. It has been an immense pleasure to share such an amazing experience with you. Special mention goes to my "bench-mate" and roommate Vladimir. You cheered me up and helped me chilling out when I needed it the most.

A very special gratitude goes to Dr. Françoise Bachelerie and Dr Géraldine Schlecht-Louf for hosting me in their laboratory and for giving me the opportunity to perform very interesting experiments. Needless to say that I could not even find a pipette without the continuous help of Joyce and Carmen. I would also like to dedicate few lines to express my immense gratitude towards Dr.

Nathalie Guérineau and Enora. Both of you believed in my work and both of you helped me performing experiments that I would not have been able to carry out alone. Thank you again. Just a little disclaimer…I will still need your help.

A special thank goes to all the members of the Functional Proteomic Platform of Montpellier and in particular to Dr Martial Seveno. Despite the initial "Triton" problems we managed to finalize what was planned. That would not have been possible without your help.

Impossible not to include in my acknowledgments Dr. Bienvenue, Dr. Decorsière and Maëva who have been my office neighbours, companions of coffee breaks and lunch at the RU, scientific counsellors and audiences for evening singing shows (Yeah, sometimes I sing in my office and for this I need to apologize also to Edith and David).

My gratitude also goes to Julien that I encountered only few years ago but that I feel to have known since a lifetime.

Finishing up the IGF Sud tour I would like to thank Laia, Laura and Emma for accepting my incursions in their office and for their continuous support and help in my existential crisis.

I am also thankful to Dr. Thierry Durroux. Your scientific honesty, work ethics and altruism will always be an inspiration for me.

My sincere thanks to Joyce. Rarely in my life I found a person with your optimism and enthusiasm. Thank you for reading and correcting this manuscript for helping me in my experiments, in keeping a decent body shape and in all the bureaucratic issues I encountered. Keep this sprit and attitude and life will keep smiling at you!! With my entire heart I have to admit that my research would have been impossible without the aid and support of Elisabeth. You have been my technical-supervisor and my French teacher and now I am very pleased to call you friend.

A general thank you to all the IGF community. Forgive me if I have not mentioned you.

Yet, all little attentions that you have dedicated me have not been unnoticed.

With this I once again thank you all and wish you all the best for your future! P.S. I apologise but in the next part I will write in Italian for thanking my family.

Per terminare questi ringraziamenti vorrei prendermi qualche riga per ringraziare la mia famiglia.

Grazie a tutti, niente di tutto ciò che ho fatto sarebbe stato possibile senza di voi. Grazie a mamma e papà per avermi insegnato i valori chiave per uno scienziato quali perseveranza, coraggio e onestà. Grazie alla nonna e a zia Patrizia per dimostrarmi che bisogna sempre avere fede. Grazie ad Asia per ricordarmi che la vita è bella. Grazie a Siria che mi testimonia che non bisogna avere paura nel fare la scelta più difficile. Grazie a Giona che con la sua fantastica famiglia mi aiuta a capire che a seguire il cuore non si sbaglia mai.

Un grazie speciale al mio amico Giulio che con la sua incredibile tenacia mi stimola a non abbandonare mai i sogni in cui si crede.

Un grazie a Valentina mia compagna di vita e per la vita.

Un abbraccio a zio Marino, Enea, Loris e Bianca e un bacio a Zia Nives.

Activated GPCRs function as guanine nucleotide exchange factors catalysing the exchange of GDP with GTP on the Gα subunit that in turn triggers the dissociation of the Gα subunit from the Gβγ complex [START_REF] Goricanec | Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding[END_REF] . Subsequently, the intrinsic GTP hydrolysis of the Gα will lead to the re-association of the αβγ complex.

Gα subunits are divided in four principal families: Gαs, Gαi, Gαq and Gα12/13 [START_REF] Syrovatkina | Signaling, and Physiological Functions of G-Proteins[END_REF] . Gαs family is characterized for its ability to stimulate adenylyl cyclase (AC) (enzyme catalysing the transformation of ATP in cAMP). Stimulation of adenylyl cyclase results in elevated cAMP levels that in turn trigger the activation of several effectors such as protein kinase A, cyclic nucleotide-gated channels, and the exchange protein directly activated by cAMP (EPAC) [START_REF] Wettschureck | Mammalian G Proteins and Their Cell Type Specific Functions[END_REF] . On the other hand, Gαi proteins inhibit adenylyl cyclase. The Gαq family activates the β-isoform of phospholipase C (PLC) that catalyses the cleavage of phosphatidylinositol 4,5-bisphosphate into inositol triphosphate (IP3), which opens IP3-sensitive calcium channels present in the endoplasmic reticulum, and membrane-bound diacylglycerol, which activates the protein kinase C (PKC) family [START_REF] Wettschureck | Mammalian G Proteins and Their Cell Type Specific Functions[END_REF] (Figure 1). Gα12/13 family is responsible for the activation of a plethora of effectors such as Rho proteins, Btk family tyrosine kinases, Gap1, rasGAP and cadherins [START_REF] Syrovatkina | Signaling, and Physiological Functions of G-Proteins[END_REF] .

Figure 1 Schematic representation of G protein activation and G protein-activated pathways.

Agonist binding to GPCR triggers conformational changes in the receptor that catalyze the exchange of GDP with GTP on the Gα subunit that in turn triggers the dissociation of the Gα subunit from the Gβγ complex. These two subunits are then able to activate the available downstream signalling pathways.

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The βγ units were originally accounted only for the inactivation of the Gα unit. However, purified βγ units have been shown to activate a cardiac potassium channel in 1987 [START_REF] Logothetis | The βγ subunits of GTP-Binding proteins activate the muscarinic K+channel in heart[END_REF] . From this study, accumulating evidence has been showing that βγ complexes activate several other effectors such as PLC, AC, voltage-gated calcium channels, phosphoinositide 3 kinase (PI3K) and Mitogen-Activated Protein Kinases (MAPKs) [START_REF] Khan | The Expanding Roles of G Subunits in G Protein-Coupled Receptor Signaling and Drug Action[END_REF] .

GRKS AND β-ARRESTINS

Researchers rapidly realized that G proteins are not the only proteins interacting with GPCRs. In fact, almost simultaneously G protein coupled-receptor kinases (GRKs) [START_REF] Benovic | Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor[END_REF] and β-arrestins [START_REF] Kühn | Deactivation of photoactivated rhodopsin by rhodopsinkinase and arrestin[END_REF] (especially the ubiquitously expressed β-arrestin1 and 2 [START_REF] Attramadal | Beta-arrestin2, a novel member of the arrestin/betaarrestin gene family[END_REF] ) were identified as GPCR interacting proteins (GIPs) involved in the desensitization process. Ligand-induced activation of the GPCR promotes conformational changes that trigger the recruitment of GRKs, which phosphorylate the receptor leading to the recruitment of β-arrestins. β-arrestins impair receptor coupling to G proteins through steric hindrance and therefore receptor-operated signal transduction. Further studies revealed that β-arrestin roles are not limited to signal transduction termination. They in fact actively participate in the receptor endocytosis by clathrin coated pits, functioning as adaptor protein for the recruitment of clathrin and its adaptor protein AP2 [START_REF] Smith | The β-Arrestins: Multifunctional regulators of G protein-coupled receptors[END_REF] . Recent evidence showed that they are also essential scaffold proteins for the recruitment and activation of proteins of the MAP kinase ERK1/2 pathway [START_REF] Cassier | Phosphorylation of β-arrestin2 at Thr383 by MEK underlies β-arrestin-dependent activation of Erk1/2 by GPCRs[END_REF] . As for β-arrestins, GRKs have been involved in the activation of others effectors such as phosducin, AKT and Mitogen-activated protein kinase MEK [START_REF] Evron | 2: multiple roles beyond G proteincoupled receptor desensitization[END_REF] .

NON CANONICAL G PROTEIN-COUPLED RECEPTOR INTERACTING PROTEINS

A special class of GIPs is constituted by GPCRs themselves. In fact, accumulating evidence, principally collected in heterologous systems, has been showing that GPCRs can interact forming hetero and homomers [START_REF] Ferré | G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives[END_REF] . Heteromers are formed by at least two different GPCRs, whereas in homomers the monomers are the same receptor. Therefore, when engaged in heteromers, one GPCR can be considered as a GIP of the other. GPCRs may have a pharmacologically-distinct profile as a monomer, homomer and heteromer. Exemplificative is the case of the chemokine receptor ACKR3. ACKR3 is not coupled to G proteins in HEK-293 cells as monomer or homodimer but it is able to modify CXCR4 G protein activation and Ca 2+ mobilization when engaged in the ACKR3/CXCR4 heterodimer [START_REF] Levoye | CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling[END_REF] . Another example is ! the cross talk between the 5-HT2A and mGlu2 receptor. Activation of the 5HT2A receptor was shown to mediate the phosphorylation of mGlu2 at Ser 843 and to promote its receptor-operated Gαi/o signalling [START_REF] Murat | 5-HT2A receptor-dependent phosphorylation of mGlu2 receptor at Serine 843 promotes mGlu2 receptor-operated Gi/o signaling[END_REF] .

In addition to canonical GIPs (G proteins, β-arrestins and GRKs), and GPCRs themselves, GPCRs have been found to specifically interact with a large number of proteins that modulate their activity [START_REF] Ritter | Fine-tuning of GPCR activity by receptor-interacting proteins[END_REF] , trafficking [START_REF] Sauvageau | CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum[END_REF] , and signal transduction properties [START_REF] Deraredj Nadim | Physical interaction between neurofibromin and serotonin 5-HT6 receptor promotes receptor constitutive activity[END_REF] . GIPs can even open a new vista of signalization. For instance, the AT1 angiotensin receptor was thought to signal only via its coupling to Gαq proteins. Yet, upon stimulation it can recruit Jak2 that in turn will phosphorylate a member of the STAT family of transcription factors [START_REF] Godeny | The N-terminal SH2 domain of the tyrosine phosphatase, SHP-2, is essential for Jak2-dependent signaling via the angiotensin II type AT1 receptor[END_REF] . Another case is the agonist-dependent recruitment of the Na + /H + exchange regulatory factor 1 (NHERF-1) to the C-terminal domain of the β2-adrenergic receptor [START_REF] Hall | The beta2-adrenergic receptor interacts with the Na+/H+exchanger regulatory factor to control Na+/H+ exchange[END_REF] . This association allows the β2-adrenergic receptor to control the Na + /H + exchange in the kidney in a G protein-independent way. A more recent example is the interactions between the serotonin6 (5-HT6) receptor and the cyclin-dependent kinase (Cdk) 5. Cdk5 bound to the receptor constitutively phosphorylates its Ser 350 , a necessary step in the activation of the Cdc42 pathway to promote neuronal differentiation [START_REF] Duhr | Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth[END_REF] . In addition, the 5-HT6 receptor was found to physically interact with several proteins of the mammalian target of rapamycin (mTOR) pathway, including mTOR itself. Correspondingly, activation of the receptor increases mTOR signalling through a mechanism requiring physical interaction between the receptor and mTOR. Conversely, inhibition of mTOR by rapamycin prevented cognitive deficits induced by 5-HT6-receptor agonists [START_REF] Meffre | 5-HT 6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia[END_REF] .

However, interactions with GIPs can also tune the canonical signalling pathways such as G protein and β-arrestin activation. For instance, the association of GPCRs with proteins of the regulator of G protein signalling (RGS) family regulates the amplitude and time course of GPCR signalling by increasing the GTPase activity of the activated Gα subunit [START_REF] Kach | A finer tuning of G-protein signaling through regulated control of RGS proteins[END_REF] . Another example is the association of Calmodulin with the 5HT2C receptor that is critical for G protein-independent, but arrestin-dependent receptor signalling [START_REF] Labasque | Physical Interaction of Calmodulin with the 5-Hydroxytryptamine 2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling[END_REF] .

In addition GIPs can both modify GPCR targeting by clustering and anchoring the receptors [START_REF] Tu | Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins[END_REF] and modify GPCRs trafficking as in the case of dynein light chain component that facilitates the rhodopsin trafficking to the membrane or the case of GASP proteins which increase the trafficking of the D2 dopamine, CB1 cannabinoid and δ opioid receptors [START_REF] Ritter | Fine-tuning of GPCR activity by receptor-interacting proteins[END_REF] to the lysosomes. GPCRs can therefore signal through parallel pathways simultaneously. Agonists that have different efficacy for these different pathways are defined as "biased" (Figure 2). Whereas "balanced" agonists have equal efficacy to available downstream pathways, "biased" agonists will preferentially or selectively activate specific pathways [START_REF] Smith | Biased signalling: From simple switches to allosteric microprocessors[END_REF]

METHODS FOR THE IDENTIFICATION OF GPCR-INTERACTING

PROTEINS

As earlier reported considerable evidence has been accumulating supporting that GPCRs recruit a number of proteins called G receptor interacting proteins (GIPs) [START_REF] Magalhaes | Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins[END_REF] . This prompted numerous investigations aimed at identifying GIPs and at characterizing GPCR-GIP interactions, using either blind or targeted approaches. In blind methods no beforehand knowledge of the GIP is required and the GPCR of interest is used as bait for fishing GIPs, while targeted methods are devoted to the validation and characterization of a previously identified GPCR-GIP interaction.

Methods for identifying GIPs or characterizing GPCR-GIP interactions can be divided into three major classes: genetic, biophysical and proteomic ones.

GENETIC METHODS

The first method belonging to this class is the Yeast two-Hybrid (Y2H) assay [START_REF] Fields | A novel genetic system to detect protein-protein interactions[END_REF] . In Y2H, the protein of interest (bait protein) is expressed in yeast as a fusion to the DNA-binding domain of a transcription factor lacking the transcription activation domain. To identify partners of this bait, a plasmid library that expresses cDNAencoded proteins fused to a transcription activation domain is introduced into the yeast strain. Interaction of a cDNA-encoded protein with the bait protein results in the activation of the transcription factor and expression of a reporter gene, enabling growth on specific media or a colour reaction and the identification of the cDNA encoding the target proteins. A first disadvantage is the loss of spatio-temporal localization of the interaction; in fact, Y2H only captures a snapshot of potential interaction in an artificial biological system. A second disadvantage is the impossibility to investigate membrane-anchored proteins since the two proteins must cross the nuclear membrane for carrying the reconstituted transcriptional factor to the DNA. The Membrane Yeast two Hybrid assay (MYTH) [START_REF] Stagljar | A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo[END_REF] has been developed for overcoming this limitation. In this assay, the ubiquitin protein is split into two fragments, which are fused to the two proteins of interest. The ubiquitin C-terminal fragment is conjugated to a transcription factor that is released when the interaction occurs and the ubiquitin protein is reformed. However, as in Y2H, the spatio-temporal localization of the interaction is lost. A second limitation is that the ubiquitin Cterminus carrying the transcription factor cannot be fused to soluble proteins because they could diffuse into the nucleus. Recently, a mammalian version of the assay, Mammalian membrane two-hybrid (MaMYTH) [START_REF] Petschnigg | The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells[END_REF] , has been developed. The Kinase substrate sensor (KISS) [START_REF] Lievens | Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor[END_REF] assay, using STAT3 as transcriptional factor, can also be used for investigating protein-protein interactions implicating both cytosolic and membrane proteins in mammalian cells, but not for studying GPCR interaction with proteins involved in the STAT cascade.

BIOPHYSICAL METHODS

Energy transfer-based methods such as Bioluminescence and Fluorescent

Resonance Energy Transfer (BRET [START_REF] Xu | A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins[END_REF] and FRET [START_REF] Clegg | Fluorescence resonance energy transfer[END_REF] ) assays, are generally targeted methods used to investigate previously reported interactions. Both are based on the transfer of energy from a donor to a nearby acceptor (<100 Å) and their high sensitivity allows the study of weak and transient interactions. The high spatialtemporal resolution permits accurate kinetic studies for investigating interaction dynamics.

Proximity Ligation Assay (PLA) [START_REF] Fredriksson | Protein detection using proximity-dependent DNA ligation assays[END_REF] is an other powerful targeted fluorescent method. In the direct PLA technique a couple of DNA oligonucleotide conjugated antibodies against the proteins of interest are used. In the indirect PLA technique secondary DNA conjugated antibodies are used after targeting the proteins of interest with the ! appropriate primary antibody. If the two conjugated antibodies are close enough (30-40 nm) they can bind together with the addition of two connectors. The DNA connecting the two probes is then amplified and hybridized with fluorophores that allows the visualization of the interaction in the place where it occurs, at a single molecule resolution. Main disadvantages are the high costs and the necessity of specific antibodies that are not always available. In the bimolecular fluorescent complementation (BiFC) [START_REF] Hu | Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation[END_REF] assay, a fluorescent protein is divided into two nonfluorescent fragments that are fused to the proteins of interest. Interaction reconstitutes the entire fluorescent protein. This method allows the direct visualization of the interaction and can be used for soluble or membrane-bound proteins. In addition, several interactions can be investigated in parallel using different fluorescent proteins. Since there is a delay in fluorescence formation upon reconstitution of the fluorescent proteins and the fluorophore formation is irreversible, these methods are not well suited for kinetic studies.

PROTEOMIC METHODS

Proteomic methods consist in the identification of GIPs interacting with a receptor of interest by mass spectrometry (MS) after an Affinity Purification (AP) selection phase and are therefore often named AP-MS. AP-MS is usually employed as a blind method for screening virtually all the GIPs of a GPCR of interest. Targeted versions of the method also exist and rely on GIP identification by Western Blotting. However, the requirement of specific antibodies seriously limits their applications. Several strategies can be used for the affinity purification step. In Co-IP specific antibodies against the protein of interest are used for precipitating the bait from a protein lysate.

As specific GPCR antibodies providing high IP yields are rarely available, epitopetagged versions of the receptor of interest are often expressed in the cell type or the organism of interest and precipitated using antibodies against the tag. The main advantages of Co-IP are the purification of proteins interacting with the entire receptor (whenever possible the native receptor) in living cells or tissues and its ability to purify the entire associated protein complex. The main limitations are the necessity of specific antibodies for precipitating GPCRs, the loss of spatio-temporal information and the use of detergents for cell lysis that might disrupt weak interactions. Alternatively, pull-down assays using the receptor (or one of its domains) fused with an affinity tag (e.g. Glutathione S-transferase) and immobilized on beads as baits, can be performed to purify GPCR partners from a cell or tissue lysate. Such in vitro binding assays can also be used to prove the direct physically ! interaction between two protein partners. In that case, the bait is incubated with a purified protein instead of a cell or tissue lysate. Two-step versions of AP called Tandem Affinity Purification (TAP) [START_REF] Puig | The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification[END_REF] have also been reported [START_REF] Daulat | Purification and Identification of G Protein-coupled Receptor Protein Complexes under Native Conditions[END_REF] and apply to both co-IPs or pull-downs. Although TAP methods drastically reduce the number of falsepositive identifications, they require larger amounts of starting material.

In the proximity-dependent biotin identification (BioID) 43 method, the ''bait'' protein is fused to a prokaryotic biotin ligase molecule that biotinylates proteins in close proximity once expressed in cells. BioID can detect weak and transient interactions occurring in living cells and detergents do not affect the results. However, the fusion of biotin ligase to the bait might alter its targeting or functions.

CHEMOKINE AND CHEMOKINE RECEPTOR NETWORK

Chemokines and 7TM receptors form the chemokine signalling system.

Approximately, fifty chemokines and twenty chemokine receptors are encoded by the mammalian genome (Table 1). The complexity of this network is increased by the promiscuity of the chemokine-receptor binding: different chemokines can bind to the same receptor and different receptors can recognize the same chemokine. The intricacy is further enhanced by the ability of chemokines and chemokine receptors to form homo-and hetero-oligomers. This multi-level network tuning allows to accurately control the biological processes regulated by chemokines and chemokine receptors such as cell movement, cell-cell adhesion, embryonic development and immune cell development.

Table 1 List of known human chemokines with the associated receptors.!Adapted!from [START_REF] Zlotnik | The chemokine and chemokine receptor superfamilies and their molecular evolution[END_REF] .

CHEMOKINES

Chemokines are small proteins classified by the arrangement of conserved cysteine residues in their primary amino acid sequence. Chemokines are divided in four subfamilies: CC chemokines where the two cysteine residues are next to eachother, CXC whose cysteines are separated by one variable amino acid, CX3C chemokines have three amino acids separating the cysteine residues and XC chemokines lack the first and third cysteine of the motif. A systematic nomenclature named the chemokines based on their subfamily followed by the letter L (standing for "ligand") and a number indicating when the gene was first isolated [START_REF] Zlotnik | Chemokines: A New Classification Review System and Their Role in Immunity[END_REF] .

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Approximately fifty chemokines are encoded by the mammalian genome and several polymorphisms have been identified in chemokine genes [START_REF] Stone | Mechanisms of regulation of the chemokine-receptor network[END_REF] . For example, a single point polymorphism (G801A) has been reported for CXCL12 and homozygotes for this polymorphism have shown protection against the acquired immunodeficiency syndrome (AIDS) caused by the retrovirus known as human immunodeficiency virus (HIV) [START_REF] Winkler | Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant[END_REF] .

Once translated, chemokines must undergo deep rearrangements for exerting their biological functions. In fact, chemokines, due to their high affinity, bind to small linear polysaccharides consisting in repeated disaccharide subunits present on the cell surfaces of most cells called glycosaminoglycans (GAGs). GAGs promote the formation of the chemotactic gradient (BOX 2) anchoring chemokines and avoiding that they are washed out by the blood flow. In addition, GAGs are engaged in oligomerization that improves chemokine solubility [START_REF] Salanga | Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function[END_REF] . The vital role played by GAGs in chemokine activity is exemplified by CCL2, CCL4 and CCL5 that were found to be inactive in vivo when their binding site for GAGs is mutated [START_REF] Proudfoot | Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines[END_REF] . Furthermore, after their translation, chemokines can be deeply modified by posttranslational modifications (PTMs). Specifically, chemokines can be citrullinated [START_REF] Loos | Citrullination of CXCL10 and CXCL11 by peptidylarginine deiminase: a naturally occurring posttranslational modification of chemokines and new dimension of immunoregulation[END_REF] , nitrated [START_REF] Janssens | Natural nitration of CXCL12 reduces its signaling capacity and chemotactic activity in vitro and abrogates intra-articular lymphocyte recruitment in vivo[END_REF] , glycosylated [START_REF] Richter | Posttranslationally Processed Forms of the Human Chemokine HCC-1[END_REF] and cleaved [START_REF] Vanheule | How post-translational modifications influence the biological activity of chemokines[END_REF] . The paramount importance of PTMs for chemokine activity is clearly illustrated by CXCL7 and CCL14 that are active only after proteolytic processing [START_REF] Brandt | Neurphils can generate their activator neutrophil-activating peptide 2 by proteolytic cleavage of plateletderived connective tissue-activating peptide III[END_REF][START_REF] Detheux | Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties[END_REF] .

Modified and GAG-bound chemokines are ready for binding with their receptor. Early works on CXCL8 showed that the N-terminal domain of the chemokine is necessary for the activation and that binding and activation are un-coupled. Mutations on the 30s-loop of CXCL8 (N-terminal domain) were found to have a little effect on the affinity but a drastic effect on the activity [START_REF] Joseph | Dynamic conformational switching in the chemokine ligand is essential for G-protein-coupled receptor activation[END_REF] . On the receptor side, it was observed that the N-terminal domain is necessary for the binding of the chemokine into the receptor [START_REF] Pease | The N-terminal extracellular segments of the chemokine receptors CCR1 and CCR3 are determinants for MIP-1alpha and eotaxin binding, respectively, but a second domain is essential for efficient receptor activation[END_REF] . These observations have been later generalized into a two site model (Figure 3) where the site 1 (N-terminal domain of the receptor) is responsible for the recognition and the binding of the chemokines; after the binding, the flexible N-

BOX 2 CHEMOTAXIS

Chemotaxis is defined as "the directional locomotion of cells towards a source of a chemical gradient" 46 . Chemokines are shaped in a chemoattracting gradient by immobilization on GAGs, heparane sulfate and extracellular matrix [START_REF] Wang | Endothelial heparan sulfate deficiency impairs L-selectin-and chemokine-mediated neutrophil trafficking during inflammatory responses[END_REF][START_REF] Middleton | Leukocyte extravasation: Chemokine transport and presentation by the endothelium[END_REF] and by sequestration by chemokine receptor [START_REF] Boldajipour | Control of Chemokine-Guided Cell Migration by Ligand Sequestration[END_REF] . Cells, such as leukocytes, migrate towards higher chemokine concentrated areas.

The migration can be divided in three steps: gradient sensing, polarization and cell-mobility [START_REF] Iglesias | Navigating through models of chemotaxis[END_REF] .

After interaction of the chemokine with its receptor the cell undergoes cytoskeletal rearrangements forming a leading and a trailing edge enabling its movement along the gradient.

! 11!

terminal part of the chemokine (site 2) is free to "enter" the chemokine receptor and to trigger the structural rearrangements necessary for its activation [START_REF] Crump | Solution structure and basis for functional activity of stromal cellderived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1[END_REF] .

CXCL12

CXCL12 was initially thought to transduce signals exclusively through its binding to CXCR4 [START_REF] Oberlin | The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1[END_REF] . However, in 2005 it has been discovered that CXCL12 can also bind to another chemokine receptor named ACKR3 [START_REF] Balabanian | The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes[END_REF] . In order to be biologically active the pro-CXCL12 precursor needs to be cleaved, resulting in the elimination of the 21 amino acid signal peptide present at the N-terminal region. Six and three different CXCL12 isoforms have been identified in human and mice, respectively. CXCL12 isoforms are generated by alternative splicing [START_REF] Nagasawa | CXC chemokine ligand 12 (CXCL12) and its receptor CXCR4[END_REF] . CXCL12α (major isoform) is highly conserved among species [START_REF] Devries | Defining the origins and evolution of the chemokine/chemokine receptor system[END_REF] , suggesting an essential role in developmental processes. Accordingly, CXCL12 -/-mice are lethal (half the embryos die at E18.5 and neonates die within one hour after birth) [START_REF] Nagasawa | Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1[END_REF] . CXCL12 plays pivotal roles in hematopoietic and germ cell development, cardiogenesis and vascular formation, immune-response as well as neurogenesis [START_REF] Nagasawa | CXC chemokine ligand 12 (CXCL12) and its receptor CXCR4[END_REF] . It is highly expressed in homeostatic conditions in lymph nodes, lung, liver and bone marrow. Lower expression levels have been shown in small intestine, kidney, skin, brain and skeletal muscle [START_REF] Sánchez-Martín | CXCR7 impact on CXCL12 biology and disease[END_REF] .

CXCL11

As CXCL12, CXCL11 was originally thought to bind only to one chemokine receptor (CXCR3) [START_REF] Cole | Interferon-inducible T Cell Alpha Chemoattractant (I-TAC): A Novel Non-ELR CXC Chemokine with Potent Activity on Activated T Cells through Selective High Affinity Binding to CXCR3[END_REF] . However, as CXCL12, CXCL11 also binds ACKR3 [START_REF] Burns | A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development[END_REF] . CXCL11 is primarily found in leukocytes, pancreas, liver, thymus, spleen and lung. It has been found at lower level in the small intestine, placenta and prostate [START_REF] Sánchez-Martín | CXCR7 impact on CXCL12 biology and disease[END_REF] . CXCL11, as CXCL12, plays a pivotal role in the immune response, mediating the T-cell polarization and migration (via activation of STAT3 and STAT6) [START_REF] Zohar | CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis[END_REF] , as well as the migration of macrophages [START_REF] Torraca | The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection[END_REF] .

CHEMOKINE RECEPTORS

Chemokine receptors were originally named only according to the chemokine subfamily recognized, followed by the capital letter R (standing for "receptor") and a number according to their order of discovery [START_REF] Murphy | International union of pharmacology. XXII. Nomenclature for chemokine receptors[END_REF] . However, in 2014, a new functional nomenclature divided the chemokine receptor family into two subfamilies: conventional chemokine receptors G protein-coupled and atypical chemokine receptors unable to activate G proteins [START_REF] Bachelerie | LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors[END_REF] . The first group, that structurally shares the DRLYAIV motif at the end of transmembrane domain 3, follows the original nomenclature. The atypical receptors were named ACKR (standing for Atypical ChemoKine Receptor) followed by an identifier number. In spite of this different nomenclature all chemokine receptors are classified as Class A GPCRs.

Conventional chemokine receptors are able to activate G proteins upon chemokine binding. This subfamily includes ten CCR receptors (CCR1-10), six CXCR receptors (CXCR1-6), CX3C1 and XCR1 [START_REF] Bachelerie | LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors[END_REF] .

The atypical chemokine receptor family contains 7TM receptors highly homologous to typical chemokine receptors that binds chemokines but do not signal trough G proteins. Before 2014 they were called scavengers, decoys, interceptors or chemokine-binding proteins [START_REF] Bachelerie | LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors[END_REF] . ACKR1 (previously Duffy Antigen Receptor for Chemokines), ACKR2 (formerly D6 or CCBP2), ACKR3 (alias CXCR7), ACKR4 (formerly CCRL1 and CCX CKR), CCRL2 (ACKR5, reserved, pending confirmation; aliases CKRX, HCR, and CRAM), PITPNM3 (also known as the CCL18/PARC Receptor; new name: ACKR6, Reserved) and C5L2 belong to this family.

CXCR4

CXCR4 belongs to the conventional chemokine receptor family and it was identified for the first time in leukocytes. It is now appreciated that CXCR4 is expressed in several cellular types such as endothelial cells, lymphocytes, fibroblasts and hematopoietic stem cells [START_REF] Rossi | The Biology of Chemokines and their Receptors[END_REF] . As for CXCL12, the CXCR4 knock out is lethal [START_REF] Tachibana | The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract[END_REF] . Its crystal structure, in complex with its antagonist IT1t, has been resolved in 2010 [START_REF] Wu | Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists[END_REF] .

! 13!

Upon binding to its endogenous agonists CXCL12, CXCR4 activates Gαi proteins that subsequently inhibit adenylyl cyclase activity [START_REF] Pozzobon | CXCR4 signaling in health and disease[END_REF] . This leads to the activation of the Src tyrosine kinase family that stimulates the Ras/Raf/MEK/ERK pathway. In addition, CXCR4 can also activate PI3K by both the α and βγ subunits of Gαi proteins, leading to Ca 2+ mobilization and activation of PKC and MAPK. CXCR4 can also signals through GRKs and β-arrestins. CXCL12 stimulation triggers activation of GRK2, GRK6, GRK3, β-arrestin1 and 2. GRK2 and 6 as well as β-arrestin2 negatively regulate Calcium mobilization. GRK3 and 6 as well as β-arrestin1 increased ERK1/2 activation whereas GRK2 negatively regulates ERK1/2 activation 80 .

CXCR4 NON CANONICAL INTERACTING PROTEINS

Beside canonical interacting proteins, CXCR4 has been shown to interact with additional proteins that modulate CXCR4 trafficking, subcellular localization and signalling and proteins whose functions are still unknown. The CXCR4 interacting proteins, the methods used for the identification, the site of their interaction in the receptor sequence and their functional impact are indicated in Table 2.

PROTEINS CONTROLLING CXCR4 LOCALIZATION OR TRAFFICKING

Filamin A directly interacts with CXCR4 and stabilizes the receptor at the plasma membrane by blocking its endocytosis [START_REF] Gómez-Moutón | Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors[END_REF] . The association of the E3 Ubiquitin Ligase Atrophin Interacting Protein 4 (AIP4) has opposite consequences: ubiquitination of CXCR4 by AIP4 targets the receptor to multi-vesicular bodies, which is followed by receptor degradation. In addition, agonist treatment increases CXCR4/AIP4 interaction, as assessed by Co-IP and FRET experiments [START_REF] Bhandari | The E3 ubiquitin ligase atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 via a novel WW domain-mediated interaction[END_REF] , indicating that this interaction is dynamically regulated by the conformational state of the receptor.

Reticulon3 (RTN3) is another CXCR4 interacting protein that constitutively promotes its translocation to the cytoplasm 83 . 1.2.2.1.1.2 PROTEINS MODULATING CXCR4 SIGNALLING AND FUNCTIONS CD74, a single-pass type II membrane protein sharing with CXCR4 the ability to bind to the Macrophage migration inhibitory factor (MIF), was also shown to interact with CXCR4 [START_REF] Schwartz | A functional heteromeric MIF receptor formed by CD74 and CXCR4[END_REF] . The CXCR4/CD74 complex is involved in AKT phosphorylation. In fact, blocking either CXCR4 or CD74 inhibits MIF-stimulated AKT activation. Using FRET, an interaction between CXCR4 and the Toll Like Receptor 2 (TLR2) was observed in ! 14! human monocytes upon activation by Pg-fimbria (fimbriae produced by the major pathogen associated with periodontitis named Porphyromonas gingivalis). Analysis of a possible crosstalk between the two receptors showed that Pg-fimbria, directly binds to CXCR4 and inhibits TLR2-induced NF-kB activation by P. gingivali. [START_REF] Hajishengallis | Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function[END_REF][START_REF] Triantafilou | Chemokine receptor 4 (CXCR4) is part of the lipopolysaccharide "sensing apparatus&quot[END_REF] . In Jurkat cells, CD164 co-precipitates with CXCR4 in presence of CXCL12 presented on fibronectin [START_REF] Forde | Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells[END_REF] . CXCR4-CD164 interaction participates in CXCL12-induced activation of AKT and PKC isoform zeta (PKCζ). Downregulation of CD164 reduces the activation of both kinases measured upon exposure of Jurkat cells to CXCL12. CXCR4/CD164 interaction has been detected in additional cell lines, such as primary human ovarian surface epithelial (hOSE) cells stably expressing CD164 [START_REF] Huang | CD164 regulates the tumorigenesis of ovarian surface epithelial cells through the SDF-1α/CXCR4 axis[END_REF] .

The ability of CXCR4 to promote cell migration requires deep cytoskeletal rearrangements that can be modulated by CXCR4 interacting proteins. In J77 T cells, CXCR4 constitutively associates with Drebrin 89 , a protein known to bind to F-actin and stabilize actin filaments. Drebrin is also involved in CXCR4-and CD4-dependent Human immunodeficiency virus (HIV) cellular penetration [START_REF] Gordón-Alonso | Actin-binding protein drebrin regulates HIV-1triggered actin polymerization and viral infection[END_REF] . CXCR4 interacts with Diaphanous-related formin-2 (mDIA2). This interaction induces cytoskeletal rearrangements that lead to non-apoptotic blebbing. The mDIA2-CXCR4 interaction is only detected during non-apoptotic amoeboid blebbing and is confined to nonapoptotic blebs upon CXCL12 stimulation [START_REF] Wyse | mDia2 and CXCL12/CXCR4 chemokine signaling intersect to drive tumor cell amoeboid morphological transitions[END_REF] , suggesting a fine spatio-temporal regulation of the interaction. CXCR4 also constitutively associates with the motor protein non-muscle myosin H chain (NMMHC) via its C-terminal domain [START_REF] Rey | Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes[END_REF] . The authors showed hat NMMHC and CXCR4 are co-localized in the leading edge of migrating lymphocytes, suggesting that this association might have a role in lymphocyte migration. The PI3-kinase isoform p110g co-precipitates with CXCR4 in CXCL12-stimulated human myeloid cells. This interaction contributes to receptoroperated integrin activation and chemotaxis of myeloid cells [START_REF] Schmid | Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression[END_REF] . Finally, CXCR4 was found to be part of a junctional mechano-sensitive complex through its interaction with PECAM-1 [START_REF] Dela Paz | Heparan sulfates mediate the interaction between platelet endothelial cell adhesion molecule-1 (PECAM-1) and the Gαq/11 subunits of heterotrimeric G proteins[END_REF] . 1.2.2.1.1.3 PROTEINS WITH UNKNOWN FUNCTIONS Other potential CXCR4-interacting proteins have been identified using blind methods.

These include the lysosomal protein ATP13A2 [START_REF] Usenovic | Identification of novel ATP13A2 interactors and their role in α-synuclein misfolding and toxicity[END_REF] and the nuclear protein Myb-related protein B that is involved in cell cycle progression [START_REF] Wang | Toward an understanding of the protein interaction network of the human liver[END_REF] . In a study aimed at characterizing the human interactome by Co-IP of 1,125 GFP-tagged proteins and ! 15! MS analysis, CXCR4 was found to co-precipitate with the potassium channel subfamily K member 1, the CSC1-like protein 2 and the Vesicle transport protein GOT1B 97 . Blood cells are generated in the bone marrow from hematopoietic stem cells. CXCR4 plays a pivotal role in the colonization of the bone marrow by hematopoietic stem cells (HSCs), as well as in HSCs homeostasis. In fact, the hematopoietic stem cell niche is maintained by the high CXCL12 concentration in the bone marrow [START_REF] Sugiyama | Maintenance of the Hematopoietic Stem Cell Pool by CXCL12-CXCR4 Chemokine Signaling in Bone Marrow Stromal Cell Niches[END_REF] . In addition, CXCR4 negatively regulates the proliferation of hematopoietic stem cells [START_REF] Stein | Chemokine control of lymphocyte trafficking: a general overview[END_REF] .

Similarly to its effect upon hematopoietic stem cells, CXCR4 also controls lymphocyte and myeloid cell homeostasis 100,101 . Besides regulating bone marrow homeostasis, CXCR4 is essential for coordinating both adaptive and innate immune responses. It controls leucocyte dissemination and trafficking, contributes to the organization of lymph nodes [START_REF] Stein | Chemokine control of lymphocyte trafficking: a general overview[END_REF] CXCR4 involvement in cancer has first been suggested by its overexpression in more than 23 types of different cancers including kidney, lung, brain, prostate, breast, pancreas, ovarian, and melanomas and its involvement in tumour growth, angiogenesis, metastasis, and therapeutic resistance 105 is now well established.

ACKR3

ACKR3 belongs to the atypical chemokine receptor family and it was identified for the first time in 1990 as the orphan receptor RDC1 107 . It was deorphanized in 2005 [START_REF] Balabanian | The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes[END_REF] when CXCL12, originally identified as the exclusive ligand of CXCR4, was found to bind to ACKR3. Only one year later, CXCL11, originally exclusive ligand of CXCR3, was identified as the second ligand of ACKR3 108 . Interestingly, CXCL12 has a 10-fold higher affinity for ACKR3 than CXCL11 (Kd = 0.4 nM vs. 4 nM, respectively) [START_REF] Balabanian | The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes[END_REF]108 .

A recent study has investigated the structural basis of the interaction between ACKR3 and its endogenous ligand CXCL12 109 . Consistent with the previously described two-step model (see page 11), the N-terminal domain of the receptor binds to the N-loop and 40s loop of the chemokine, then the N-terminus of the chemokine interacts with the second extracellular loop and trans-membrane domain pocket of the receptor. Additionally, the authors showed that the conformational changes of ACKR3 upon activation are strikingly similar to those observed for other GPCRs.

Partial proofs of a possible binding of adrenomedullin to ACKR3 have also been obtained 110 . Although a possible cross-talk between both proteins has been shown 111 , further and more precise binding studies are needed before adding adrenomedullin to the ACKR3 ligand list. Likewise, though a functional link between ACKR3 and Macrophage migration-Inhibitory Factor (MIF) 112 , the ligand of CD74, has been suggested, further studies are needed for confirming the direct binding of MIF to ACKR3. Some ACKR3 ligands were also synthetized such as CCX773, CCX771, CCX451 and CCX754. Originally they were all classified as antagonists [START_REF] Burns | A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development[END_REF]113,114 . However, when these molecules were tested, ACKR3 was thought to be a silent receptor. Not surprisingly, at least one of them, CCX771, was later identified as a potent agonist for β-arrestin recruitment 115 . Despite this agonistic effect the CCX771 compound can also be considered as a functional antagonist 116 due to its ability to inhibit CXCL12 induced CXCR4 mediated chemotaxis. Further experiments should be conducted with the other molecules for assessing their actual pharmacological profile. However, in this manuscript all "antagonists" will be considered as antagonists when no proof of agonism is provided.

! 20!

ACKR3 EXPRESSION

The first pioneering study aimed at mapping ACKR3 expression showed that ACKR3 is expressed in astrocytes, neutrophils, kidney, spleen and heart of mice 117 . ACKR3 mRNA expression in kidney was confirmed using in situ hybridization 118 . The receptor is expressed in the ureter, the region of the renal capsule, immature/mature glomeruli of kidneys from E12.5 and E14.5 mice. ACKR3 expression in the heart was confirmed using ACKR3 -/-mice. Although the gene deletion was lethal for more than the 95% of the new-born mice, the survivors exhibited severe abnormal heart formation 119 . In wild type mice, the expression of ACKR3 in the endothelial layer of the forming heart (E=9.5) and in the microvasculature of the myocardium and valves (from E=14.5) was confirmed using in situ hybridization 119 .

Consensus on the expression of ACKR3 in spleen has not yet been reached. In fact, immunohistochemistry on mouse, rat and human spleen revealed that ACKR3 is expressed only in B cells of the marginal zone of the rat and not in human or mouse.

These results were confirmed by flow cytometry and by β-galactosidase enzymatic staining of spleen sections from ACKR3 +/lacZ mice 120 . In contrast with these results,

Wang et al 114 and others 121,122 observed ACKR3 expression in mouse splenic marginal zone B cells. These different results might be due to the different protocols used in cell ex vivo isolation that could have affected ACKR3 expression.

Contrasting results were obtained regarding ACKR3 expression by peripheral blood cells. In fact, ACKR3 was found in primary T cells [START_REF] Balabanian | The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes[END_REF]123 and B cells 124 . On the other hand, other studies failed to detect ACKR3 in T cells 125 or human lymphocytes 126 .

However, in the latter study the authors failed to detect CXCR4 expression on leukocytes, which express high CXCR4 levels, raising doubts on the quality of the data. In addition to B and T cells, platelets isolated from healthy volunteers and patients suffering from both acute coronary syndrome and stable coronary artery disease express ACKR3, as shown by Western Blot, immunofluorescence and flow cytometry 127,128 .

ACKR3 was also detected in human placenta Although ACKR3 expression was found to be essential for in vivo migration of zebrafish primordial germ cell [START_REF] Boldajipour | Control of Chemokine-Guided Cell Migration by Ligand Sequestration[END_REF] the receptor was originally described as a silent receptor unable to activate any downstream signalling upon CXCL12 or CXCL11 binding (Figure 4). This idea rose from two early studies where the authors failed to observe any ACKR3-operated calcium mobilization 108 or PIK3 activation [START_REF] Boldajipour | Control of Chemokine-Guided Cell Migration by Ligand Sequestration[END_REF] .

ACKR3 function was limited to the shaping of the chemokine gradient by binding with CXCL12 or CXCL11 and concomitant internalisation 124

. Repression or inhibition of ACKR3 would lead to an absence of gradient, which would result in the disruption of CXCR4-dependent migration. GRK2 but not GRK3, 5 or 6 was found to play a pivotal role in CXCL12-mediated activation of ACKR3 in astrocytes 147 .

Expanding the signalling pathways activated by the receptor, ACKR3 increases the expression of Cyclin D1, D3 and E1 and decreases the expression of p27 in CD34 + cells, thus altering cell cycle and increasing cell proliferation 148 (Figure 5). In addition to triggering ERK1/2 phosphorylation, ACKR3 increases the expression of Vascular endothelial growth factor A in endothelial cells 149 . Although several groups of investigators have shown that ACKR3 is unable to activate G proteins, few studies showed that the receptor might directly or indirectly interact with and activate G proteins. In fact, ACKR3 was found to constitutively recruit, but not to activate, Gαi proteins 150 . In addition, ACKR3 influences CXCR4operated G protein activation when engaged in the CXCR4/ACKR3 heterodimers 119,150,151 . Furthermore, ACKR3 induces cAMP production after adrenomedullin activation, 110 suggesting a possible Gαs coupling of the receptor. showed that conditional knock-out of ACKR3 in interneurons leads to neuronal laminar positioning defects similar to the ones observed in CXCR4 -/-mice (migrating interneurons express also CXCR4). In both ACKR3 and CXCR4 conditional mutants more interneurons were found in the cortical plate and less are present in the marginal zone and sub-ventricular one in vivo. In the cortical plate, movements of interneurons from the two mutants exhibited opposite phenotypes compared to the wild type. ACKR3 -/-interneurons were much less motile with a shorter leading process, whereas CXCR4-deficient neurons were highly motile with longer and complicated processes. In vitro, CXCL12 mediated migration was inhibited by the ACKR3 functional antagonist CCX771 and by the CXCR4 antagonist AMD3100

suggesting that both CXCR4 and ACKR3 are required for the correct migration.

Importantly, AMD3100 did not exacerbate the ACKR3 In contrast to its induction by pro-inflammatory cytokines, ACKR3 was not induced by hypoxia in colon cancer 200 .

Although the heterogeneity of cancer models used in these studies produced heterogeneous results, there is consensus on few ACKR3-regulated signalling pathways. In fact, ACKR3 principally regulates two pathways: the MAPK ERK1/2 pathway 155,173,174,183,193,197 and the mTOR 173,178,193,194 pathway, leading to the phosphorylation of P38, AKT, JNK and PI3K. In addition, ACKR3 induces and activates metalloproteinase-9 (MMP-9) 174,178 , to modulate the expression of proteins involved in the regulation of cell cycle (cyclin A and B1, Cdk2 and 4, p21 and p57) 155,183,201 as well as to induce N-cadherin and repress E-cadherin expression 181,197 Through the activation of these pathways ACKR3 has been linked with increased invasion, adhesion and tumour growth of several cancer types.

Both expression and activation of ACKR3 have been correlated with cancer progression. In fact, its genetic suppression inhibited tumour growth invasion and adhesion in several studies 171,178,181,183,184,202,203 . In addition, its pharmacological inhibition by CCX771 (biased agonist but functional antagonist 115 ) reduced CXCL12and CXCL11-mediated ERK1/2 phosphorylation 178,204 (also in CXCR4 -/-cells 205 ) and AKT phosphorylation 148 resulting in lower proliferation 187,206,207 , invasion 160,184,185,187,206,208 , adhesion 175 of cancer cells. "GJ" (for Gap Junction), whereas the most common nomenclature uses "Cx" (for connexin) followed by a number indicating the predicted molecular mass in kDa of the protein.

Although differing in molecular weight, all connexins share the same topology consisting in two highly

Human Mouse

Gene symbol Protein name Gene symbol Protein name Opening and closure will determine the passage of ions and small molecules (<1KDa) from one cell to the other. Connexins are differently permeable to monovalent ions including Rb + , Cs + , K + , Na + and Li +230 as well as second messengers and other cytoplasmic molecules such as ATP, ADP, AMP, Adenosine, Aspartate, cAMP, cGMP, Ca 2+ , Glucose, Glutamate, Glutathione, IP3, NAD + , Prostaglandin E2 and miRNA 231 . Interestingly, there are dramatic connexin-specific differences in the channel permeability for different molecules. For example, there is a 3.4-fold difference between ADP/ATP and glutamate in Cx43 junctional channels and a 33fold difference between Cx43 and Cx36 junctional channels in the permeability to cAMP.

GJB1 CX32 Gjb1 Cx32 GJB2 CX26 Gjb2 Cx26 GJB3 CX31 Gjb2 Cx26 GJB4 CX30.3 Gjb4 Cx30.3 GJB5 CX31.1 Gjb5 Cx31.1 GJB6 CX30 Gjb6 Cx30 GJB7 CX25 - - GJA1 CX43 Gja1 Cx43 GJA3 CX46 Gja3 Cx46 GJA4 CX37 Gja4 Cx37 GJA5 CX40 Gja5 Cx40 - - Gja6 Cx33 GJA8 CX50 Gja8 Cx50 GJA9 CX59 - - GJA10 CX62 Gja10 Cx62 GJC1 CX45 Gjc1 Cx45 GJC2 CX47 Gjc2 Cx47 GJC3 CX30.2/31.3 Gjc3 Cx29 GJD2 CX36 Gjd2 Cx36 GJD3 CX31.9 Gjd3 Cx30.2 GJD4 CX40.1 Gjd4 Cx39 GJE1 CX23 Gje1 Cx23
As earlier reported, connexins are transported to the plasma membrane as hemichannels. Originally, hemichannels were thought to remain closed. This notion started to be challenged by the discovery that Cx hemichannels could be opened either following lowering the extracellular calcium concentration 232 or a large

METHODS FOR STUDYING GAP JUNCTIONS AND HEMICHANNELS

Since gap junctions are permeable to ions and small molecules, their activity can be investigated measuring the passage of current or tracer between coupled cells.

ELECTROPHYSIOLOGICAL MEASUREMENT OF THE JUNCTIONAL CURRENT

The first recording of the current passing between cells coupled through gap junctions was made using the dual whole-cell patch clamp method between rat lacrimal gland isolated cells. In that case, each cell was implanted with two microelectrodes 246 . Nowadays, the most widely used electrophysiological method to record junctional current is the double whole-cell voltage-clamp with one patch pipette positioned on each coupled cell. In this case, the two cells are voltage- Electrophysiological recordings can also be used for investigating hemichannel activity using voltage-clamp in the whole-cell configuration. In this case the goal is to record total and single channel current of isolated cells 249 . In addition hemichannels were found to have approximately twice the conductance of the corresponding gap junction channels 235 .

TRACER DIFFUSION

Six techniques have been developing for assessing gap junctional communication using the diffusion of a tracer. Three of them differ in the way that the tracer is introduced into the cells. These are the scrape loading, microinjection and electroporation (Figure 11).

A. The most widely used method for assessing the gap junctional activity is the scrape loading technique. In this approach a confluent monolayer of cells is scraped in the presence of a membrane-impermeable, but gap junctional permeable, tracer. The tracer will enter the damaged cells and then diffuse to neighbouring cells trough gap junctions. The magnitude of the diffusion reflects the gap junctional activity. One of the most used tracers is the fluorescent Lucifer Yellow (LY) dye 250 . Gap junctional impermeable dyes, such as rhodamine-dextran, can be used for assessing the unspecific diffusion of Gap junctional permeable dyes. For the quantification of the diffusion, several methods have been used, including measurement of the area of diffusion 251 , the diffusional distance 252 and the distance at which the Intuitively, it is not appropriate for studying cells at low confluence and for studying connexins impermeable to dyes such as Cx45 that is impermeable to LY 254 .

B. The microinjection is a more suitable technique for investigating the coupling of cells at low density. In this case, the tracer is injected in one single cell using a micropipette 255 . The dye then diffuses from one cell to the other and the number of neighbouring cells receiving the dye is quantified. Compared to the scrape loading technique, its lower invasiveness makes its suitable for studies of intact tissues 247 . Its main limitation is the possible bias introduced by visually counting dye-positive cells. In fact, cells could have a very different morphology and in addition the dye becomes more diluted the more it is diffusing.

C. The tracer can also be introduced into the cells via electroporation as proposed by Raptis et al 256 . A glass slide, half covered with an electric conductive material, is used for this experiment. Cells are grown on the glass slide in order that half of them are in contact with the conductive part. Then, an electric pulse is applied in the presence of the tracer. This transient pulse generates "pores" on the cellular membrane of the cells growing on the conductive part. The tracer penetrates the cells through these "pores" and then diffuses to the half of the cells growing on the non-conductive part of the glass slide. This method has the advantage to study gap junctional activity in a cell population as the scrape loading. Compared to the latter, it is less invasive. However, it is not suitable for cells, which poorly adhere on glass. then analysed. The advantage of this technique is its non-invasiveness and the ability of repeated measures on the same cells. In fact, after the diffusion reaches equilibrium, more tracer can be un-caged from the same cell and the measurement can be repeated.

Cx hemichannel activity can also be studied using tracers. In this case, cells are exposed to a solution of ethidium bromide. Its uptake by hemichannels is then registered using a fluorescence microscope and quantified 259 .

PHARMACOLOGICAL TOLS

Generating specific pharmacological tools to modulate specific connexin subtypes and distinguish between gap junctional and hemichannel activity has been stimulating scientists for a long time. Since gap junctions are formed by two hemichannels the major difficulty was first to specifically target only hemichannel and not gap junction. The second major obstacle was then to specifically target a subtype of connexin that is structurally similar to the others. In the 80s, connexins were blocked using long-chain alcohols, volatile anaesthetics or glycyrrhetinic acid derivatives. However, all of them turned out to be unspecific. Nowadays, the most frequently used connexin inhibitor is carbenoxolone (CBX) that was shown to be a are not blocked 263 . However, since all these products block both gap junctional as well as hemichannel activities of connexins, they cannot be used for discriminating between them 234 . A cation, La 3+ , was found to block hemichannels without affecting gap junctional activity in astrocytes 264 . However, it also blocks maxi-anion channel and Ca 2+ channels 234 .

In order to gain specificity, mimetic peptides against specific connexin sequences have also been generated (Figure 13). Since the two extracellular loops of connexin hemichannels dock together for forming a gap junction they were chosen as first target sequences for the generation of Gap26 and 27 peptides. These two peptides were therefore designed for blocking gap junctional activity without blocking the hemichannel one. However, both peptides were found to inhibit both hemichannels and gap junctions 267 . In addition, although they were designed based on Cx43 sequences, they also block Cx37 268 . Later on, less conserved intracellular connexin sequences were chosen as targets. That led to the design of the L2 peptide against the cytoplasmic loop of Cx43. As already mentioned, during connexin closure, the C-terminal tail of Cx43 interacts with the L2 sequence in the particle-receptor model; therefore, the L2 peptide was designed for

BOX 5 Src

Src is a 60-KDa tyrosine kinase. Both Src keeping Cx43 open. Unexpectedly, L2 was found to block Cx43 hemichannels 269 but not gap junctional activity of Cx43. Likewise, the Gap19 peptide designed against a sequence within the L2 domain was found to block hemichannels but not gap junctions 270 . The PEP-2 peptide designed against the interaction site between Src and Cx43 (BOX 5) was found to inhibit hemichannel activity 271,272 . Two peptides against the 10 and 9 C-terminal aminoacids of Cx43 able to block hemichannels activity were also generated 273 . The latter peptides named αCT1, also referred to as aCT1 or ACT1 in publications, have passed phase II clinical trials for wound healing 274 . The biotinylation technique also allows to discriminate between Cx gap junction and hemichannel activities [275][276][277] . Using a cell-impermeable amine-reactive biotinylation reagent, lysine residues present in the extracellular space are biotinlyated. In the case of Cx, only hemichannel present at the cell surface will be biotinylated 278 , whereas engaged in gap junctions will remain non-biotinylated, since the three lysines are involved in the head-to-head docking of the two hemichannels.

In addition, Cx in gap junctions and hemichannels can be separated based on their solubility in Triton X-100 279 . Gap junctions are insoluble whereas hemichannels are soluble.

CONNEXIN 43 (CX43)

As it will be detailed in the "Results" chapter Cx43 was identified as a potential ACKR3 interacting protein. In the brain Cx43 and ACKR3 have overlapping biological functions in physio-pathological conditions and they are also co-expressed in cellular sub-populations. Therefore, the next chapters will focus on the expression and biological functions of Cx43 in the brain.

CX43 EXPRESSION

Cx43 is the most ubiquitously expressed member of the connexin family. It is highly expressed in the skin Ranvier and astrocytes end feet surrounding blood vessels 282 . Astrocyte Cx43 can form heteromeric or homomeric gap junctions between astrocytes themselves, astrocytes and other glial cells, astrocytes and neurons as well as astrocytes and cancer cells 283 .

CX43 TRAFFICKING AND DEGRADATION

Cx43 has a very short half life (1.5 to 2 hours) 284 . Therefore, its turnover must be tightly controlled (see Figure 14

POST-TRANSLATIONAL MODIFICATIONS

Several post-translational modifications have been involved in each step of Cx43 trafficking. The most studied one is the phosphorylation. Some phosphorylation processes increase Cx43 gap junctional activity (Table 5). In fact, formation of the gap junction requires its phosphorylation mediated by Casein kinase 1 (CK1) 295 . In addition, elevated cAMP levels and activation of protein kinase A leads to phosphorylation of Ser 364 and increases gap junctional activity of Cx43 296 . AKTdependent phosphorylayion at Ser 373 hinders Cx43 interaction with ZO-1 and thereby increases gap junctional activity 297 after wounding or in ischemic conditions. On the other hand, phosphorylation of Cx43 by other kinases leads to a decrease in gap junctional communication (Table 5).

PKC activation was found to phosphorylate Ser 368 and induce closure and internalisation of gap junctions 298 . However, dephosphorylation of the same residue was also found to decrease gap junctional activity 299 . The protein tyrosine kinase v-Src phosphorylates and inhibits Cx43 300 via the phosphorylation of Tyr 247 "truncated" isoforms originated from AUG starting codons located into the Cx43 gene. Smyth and Shaw showed that at least four of these internally translated Cx43 isoforms, characterized by an N-truncation, arise from translation initiated from internal AUG start codons in a cap-independent fashion 305 . They also showed that one of these isoforms, the 20 kDa fragment corresponding to the C-terminal domain of Cx43 is necessary for the correct translocation of Cx43 into the plasma membrane

CX43 INTERACTOME

As earlier reported, Cx43 trafficking is regulated by its association with interacting proteins. Several studies have shown that trafficking of Cx43 and its channeldependent and independent functions are controlled by interacting proteins (Table 6). In fact, Cx43 regulates important processes such as cell cycle progression, cell motility, cell fusion, autophagy, membrane permeability and mitochondrial redox state through its interaction with other proteins. In turn, interacting proteins might regulate Cx43 activity and turnover.

Interacting protein Functional role of the interaction

FUNCTIONAL INTERPLAY BETWEEN GPCRS AND CX43

As reported in Table 6, β-arrestin, PKA and PKC were found to interact with Cx43.

Notably, these proteins are also involved in the signalling cascade evoked by GPCRs. In addition protein kinases that control Cx43 trafficking, such as MAPK and AKT, can also be activated by GPCRs. Consequently, several GPCRs were found to inhibit gap junctional communication such as the α1 and β-adrenergic receptors, the purinergic P2X receptor, cannabinoid, endothelin and lysophospholipid receptors.

• Activation of the α1-adrenergic receptor by noradrenaline inhibits Lucifer Yellow diffusion via activation of PLC in astrocytes 347 . On the other hand, activation of the β-adrenergic receptor with isoproterenol alone did not modify the gap junctional communication but it became effective thanks to the cotreatment with the phosphodiesterase inhibitor IBMX 347 .

• ATP inhibits electrical coupling and Lucifer Yellow diffusion through the binding to the P2X receptor in primary cultured astrocytes. This inhibition was increased by pre-treatment with IL-1β 348 .

• The cannabinoid agonist anandamide reversibly inhibits gap junctional communication and calcium wave propagation in primary cultured astrocytes.

PTX reverses the inhibitory effect of anandamide, suggesting a possible role of Gαi/o proteins 349 .

• Activation of endothelin receptors by endothelin-1 and 3 also inhibits diffusion of Lucifer Yellow in cultured astrocytes 350 . Endothelin- Cx43 also participates in neuroprotection. Uncoupling of gap junction increases neuronal vulnerability in neurons co-cultured with astrocytes and exposed to oxidative stress or glutamate 282 . In addition Cx43 plays a pivotal role in migration and proliferation processes as well as in the infiltration of leukocytes into the brain 358 .

The brain was traditionally considered as an immune-privileged organ isolated via the blood brain barrier (BBB) from normal inflammatory processes. However, it is now recognized that immune cells penetrate the brain in pathological conditions such as neuroinflammation 359 . In addition, the BBB originally thought to be a pure endothelial barrier is now seen as a modulatory interface regulated by integrative signalling between endothelial cells, pericytes, neurons and astrocytes. Cx43 expressed either on endothelial or astrocyte cells have been found to regulate BBB permeability (Figure 15). As already reported, Cx43 is associated to tight junctions in endothelial cells. Inhibition of Cx43 via endothelial growth factor (VEGF) or

BOX 6 CALCIUM WAVES

Almost 30 years ago, it was shown that astrocytes are not only able to increase intracellular Ca 2+ in response to extracellular stimuli, but that they also transmit these Ca 2+ signals to adjacent and non-stimulated astrocytes, as intracellular Ca 2+ waves 354 . There are two possible routes of Ca 2+ transmission from cell to cell: one involves the direct transfer of second messengers mobilizing Ca 2+ through gap junctional-coupled cells, the second relies on the "de novo" generation of second messengers in neighbouring cells via membrane receptors 355 . Independent from their way of propagation, Ca 2+ waves will activate Ca 2+dependent signalling pathways. The most discussed functional consequence is the release of transmitters from astrocytes that regulate neuronal and vascular function (gliotransmission) 356 . Interestingly, Cx43 hemichannels also participate in the diffusion of Ca 2+ waves through the release of ATP 357 and activation of purinergic receptors.

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endothelins induces a rupture of the tight junctions and increases BBB permeability 359 . Astrocyte end feet processes completely wrap around capillaries of the BBB and regulate blood flow and water homeostatis 359 . Cx43 expressed in astrocytes is also necessary for BBB 360 . Invalidation of Cx30 and Cx43 expression specifically in astrocytes leads to a weakening and subsequently higher permeability of the BBB, as measured by sucrose and HRP permeability. This effect was explained by the loss of the dystrophin-associated protein complex (DAPC) necessary for the anchoring of astrocytic end feet around the vessel and the decreased expression of the Aquaporin 4 channel. Single invalidation of Cx30 did not reproduce this phenotype, suggesting that Cx43 is the principal connexin involved in the maintenance of BBB permeability 361 .

Cx43 was also shown to play a direct role in leucocyte invasion of the brain. In mice where Cx43 was conditionally KO in GFAP-positive astrocytes, leukocytes were detected in the brain parenchyma. In contrast with previous results, the authors failed to observe a higher BBB permeability or inflammatory processes but they observed a higher production of chemoattractant chemokines, such as CXCL12, that was responsible for the leukocyte infiltration 358 . In addition, Cx43 hemichannels could also be involved in the maintenance of the BBB through the secretion of paracrine signals such as ATP, glutamate, prostaglandins and cytokines 359 . several, multi-directional protrusions. Interestingly, this effect was mediated by a defect of Cx-mediated adhesion in a channel-independent fashion (Figure 16).

Cx43 was also linked to the migration of astrocytes since its invalidation induces an increase in migration in trans-well assays 364 . In addition, Cx43 invalidation using siRNA altered the expression of cytoskeletal proteins involved in cell migration. The release of ATP trough Cx43 hemichannels and calcium wave propagation have also been involved in the migratory process 365 . Contrasting pieces of evidence are linking Cx43 with astrocytes proliferation. On one hand, acute Cx43 inhibition by endothelin-1 or arachidonic acid increases proliferation rate in astrocytes 366 . On the other hand, primary cultures isolated from Cx43 KO mice grow less rapidly than WT ones 367 . Since the role of Cx43 in cell proliferation has been extensively characterized in carcinogenic models, it will be discussed in details below. Regarding cell growth, the first evidence of a possible role of Cx43 in brain cancer progression was collected by transfecting rat C6 glioma cells with cDNA encoding Cx43. These cells proliferate less both in vitro and in vivo 375 upon Cx43 overexpression. The role of Cx43 as a tumour suppressor in glioma was confirmed when Cx43 was found to be down-regulated in glioma in different studies [376][377][378] . In a recent study involving the analysis of 474 tumour samples from patients with glioma, Cx43 expression was found to decrease concomitantly with an increase in glioma grade 379 . Based on accumulating evidence, Cx43 is consensually considered as an inhibitor of glioma cell growth that slows down the transition from G1 to S phase 380 .

This is achieved via two mechanisms. In the first one, Cx43 mediates the exchange of growth suppressor factors in a channel-dependent fashion. In the second one, Cx43 regulates the expression of proteins involved in the cell cycle, such as Cyclins and Cdks, or by scavenging important kinases involved in cell growth such as Src 381 , in a channel-independent fashion. In some studies the Cx43 C-terminal tail alone was found to translocate into the nucleus for slowing down cell proliferation 379 .

!

The role of Cx43 in glioma invasion is more heterogeneous. In fact, suppression or inhibition of Cx43 functional activity in U87MG glioma cells decreases their invasiveness indicating that gap junctional communication between glioma cells has a tumour suppressor role. However, gap junctional communication between glioma and astrocytes as well as between astrocytes themselves increase glioma invasiveness 382 . Dissecting the molecular mechanism underlying these effects revealed that inhibition miRNA (miR-5096) transfer between glioma cells and astrocytes decreased invasiveness of glioma cells 382 . Accordingly, suppression of Cx43 expression in mice implanted with GL261 glioma cells decreased the number of cancer cells in the brain parenchyma adjacent to the tumour core and the percentage of infiltrative tumour edge 379 . In contrast with previous observations, these effects were independent of the formation of functional gap junctions 379 .

As

. Although these effects are mediated by channel-dependent functions of Cx43, several studies link Cx43 channel independent functions to migration. In fact, Cx43 was found to mediate the adhesion and migration of glioma cells as it happens for neuronal progenitor cells (NPCs) 388 . In addition, it appears that the C-terminal tail of Cx43 may be sufficient to promote migration 238,389 , an effect mediated by the activation of P38 and ERK1/2 signalling 238 .

Recent studies have also connected Cx43 with resistance of glioma cells, to the oral alkylating agent used to treat glioblastoma multiforme Temozolomide (TMZ).

Intriguingly, suppression or inhibition of Cx43 increase TMZ sensitivity 390 . Gielen et al. 391 found that full-length, wild type Cx43 was necessary for the acquired resistance. In addition, inhibition of Cx43 hemichannels using the αCT1 peptide restored TMZ sensitivity 392 . All these results converge to the idea that Cx43 mediates TMZ resistance by decreasing intracellular concentration of TMZ either by diffusing it to neighbouring cells through gap junctions or by extruding it via hemichannels.

The role of Cx43 in apoptosis is quite controversial. Some reports indicate Cx43 as anti-apoptotic whereas others demonstrate a pro-apoptotic action. Both effects are partially mediated by the diffusion of molecules that in the first case are "survival messengers", such as ATP and reduced glutathione 393 , whereas in the latter by the diffusion of "death messengers", such as calcium 390 .

In summary:

• Cx43 down-regulation and inhibition have been linked with higher proliferative states of glioma.

• Cx43 inhibition and suppression of communication between glioma cells has been linked with higher migration and invasiveness. However, increased

Cx43-mediated Gap junctional communication between astrocytes and glioma-astrocytes induces higher invasiveness and motility.

This opposing role of Cx43 in glioma could be partially explained by the heterogeneity of Cx43 expression encountered in a recent article 394 . In fact, despite

Cx43 expression relatively and inversely correlates with the tumour grade it was heterogeneously expressed within the same grade. Therefore, within the same tumour some cells express Cx43 and they would be expected to migrate and not proliferate whereas others do not express Cx43 and would expect to proliferate but not migrate. Consistent with the fact that heteromeric Cx43 gap junctions between glioma and surrounding astrocytes would promote invasion, Cx43 has been identified in the non-tumoural area surrounding glioma 394,395 and in the tumour cells at the invading front 387 . In addition, cancer cells were found to shunt cGAMP in astrocytes.

cGAMP binds to the adaptor protein the Stimulator of interferon genes (STING) triggering a conformational change in STING that translocates from the ER to the Golgi apparatus. This leads to the a release of pro-inflammatory cytokines, such as

IFNα and TNF, that provide a growth advantage for brain metastatic cells by protecting against physiological and chemotherapeutic stress 283 .

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2.AIM & OBJECTIVES

As described in the introduction, CXCR4 activates canonical GPCR signalling pathways involving Gαi protein and β-arrestins. In contrast, the cellular pathways underlying ACKR3-dependent effects remain poorly characterized. Both receptors were shown to interact with several proteins able to modify and mediate either their signalling, trafficking or localization.

Therefore, the aim of the work described in this thesis was to identify novel interacting proteins involved in the signalisation cascade of the two receptors.

The study objectives included:

• Identification of intracellular partners (GIPs) of CXCR4 and ACKR3 using an AP-MS proteomics strategy.

• Validation of the interaction between the receptor and selected GIPs via other methods and in authentic tissues.

• Determination of the functional consequences of the association between the receptor and identified GIPs.

• Elucidation of the cellular mechanisms involved in the functional consequences observed. ACKR3 interacting proteins were identified in human embryonic kidney HEK-293T cells transiently expressing hemagglutinin (HA)-tagged ACKR3 using an affinitypurification coupled to mass spectrometry (AP-MS) proteomic strategy. ACKR3interacting proteins were immunoprecipitated using an anti-HA monoclonal antibody immobilized onto agarose beads and identified by nano-flow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS), as described in the "Materials and Methods" chapter (see page114). Control immuneprecipitations were performed using cells transfected with an empty plasmid (mock). Systematic analysis by tandem MS of the immunoprecipitates identified a total of 4,009 proteins in the three independent experiments performed on different sets of cultured cells. This number was reduced to 1516 after filtering out the proteins either identified only by site, or labelled as contaminant, or not identified in all three biological replicates in at least one group (ACKR3 or mock). Label-free quantification (LFQ) of the relative protein abundances in immunoprecipitates obtained from cells expressing ACKR3 and mock cells showed that 151 proteins were significantly more abundant in immunoprecipitates from ACKR3-expressing cells, using a t-test conducted on both sides and setting a stringent False Discovery Rate (FDR) of 1%, in comparison with mock condition (Table 7). These proteins were considered as potential ACKR3 interacting protein partners.

As expected, ACKR3 (bait protein) was the most enriched one (Figure 17).

Consistent with its constitutive internalisation 143 , we identified Clathrin as an ACKR3

interacting protein as well as accessory proteins of the Rab5 and Rab3 complexes that are involved in ACKR3 internalisation 396 . In addition, we identified several enzymes involved in the ubiquitination process, which might be responsible for the basal ubiquitination of the receptor 142 . ACKR3 was also found able to phosphorylate ERK1/2 via activation of MAP2K2 397 that was also retrieved in our interactomic screen. Consistent with a previous study showing a constitutive interaction between ACKR3 and G proteins 150 , we identified Gαi3 as a putative ACKR3 partner. signalling. Consistent with these findings, all these proteins were also identified in ACKR3 interactome (Figure 17). ACKR3 and Cx43 share several important biological functions, including the control of cell migration 362,402 . In addition both proteins are involved in glioma progression during which there is an up-regulation of ACKR3 210 and a concomitant suppression of Cx43 activity and expression 376 .

In line with these findings, we decided to focus on ACKR3/CX43 interaction. We first confirmed the interaction between these two proteins by immunoprecipitation followed by Western blotting. We also compared the ability of ACKR3 and CXCR4, another chemokine receptor known to heterodimerize with ACKR3, to recruit Cx43.

As shown in

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We then investigated the interaction between each receptor and Cx43 in living HEK-293T cells using BRET 2 . We transiently co-transfected a constant amount of cDNA encoding ACKR3 or CXCR4 C-terminally tagged with Nano Luciferase with an increasing amount of Cx43 C-terminally tagged with YFP. In line with our coimmunoprecipitation experiments, the BRET 2 ratio between ACKR3 and Cx43 increased hyperbolically, indicating a specific and constitutive interaction, whereas the BRET 2 ratio between CXCR4 and Cx43 increased only linearly (Figure 19A).

ACKR3 activation by CXCL12 (10 -8 M) did not affect the interaction between ACKR3 and Cx43 (Figure 19B) (p = 0.79, n = 4). ! 69!

ACKR3 ACTIVATION INHIBITS CX43-MEDIATED GAP JUNCTIONAL INTERCELLULAR COMMUNICATION (GJIC) IN PRIMARY CULTURE OF MOUSE

ASTROCYTES.

Since ACKR3 was shown to be expressed in primary culture of astrocytes 153 , which are exclusively coupled by Cx43 248,406 , we investigated the effect of ACKR3 activation on GJIC in primary cultures of cortical astrocytes from E15.5 mouse embryos. Cells were maintained in culture for five weeks. Under these conditions, cultures showed high enrichment in astrocytes, as assessed by GFAP immunostaining (not shown).

Astrocytes were then starved overnight before proceeding to the experiment. GJIC increased cell resistance indicating that the membrane resistance value faithfully reflects a change in gap junction-mediated coupling between two cells. In the same way when cells were challenged with CXCL12, the membrane resistance significantly increased, suggesting that CXCL12 might decrease electrical coupling between astrocytes (Figure 24).

To further confirm the possible role of ACKR3 in the regulation of astrocyte electrical coupling, we calculated the coupling ratio between coupled astrocytes in the presence of either vehicle, CXCL12 (10 -8 M), CXCL11 (10 -7 M) or CBX (100μM)

(Figure 25A). The coupling ratio remained stable during the 15 min recording of vehicle-treated cells. Consistent with a blocking effect of the ACKR3 agonist CXCL12 on gap junction-mediated electrical coupling between astrocytes, the coupling ratio decreased as soon as 5 min after the onset of bath-applied CXCL12 (0.495 ± 0.06 and 0.48 ± 0.07 for saline before vs. saline after treatment, p = 0.045, n = 6 vs.

saline) and remained reduced as far as 11 minutes after application. However, the kinetics of CXCL12 action in this experiment must be interpreted cautiously, because CXCL12 (as the other treatments) was added by bath application and reached the recording chamber after 4 min of perfusion. In the same way, preliminary results

indicate that CXCL11 treatment also decreased the coupling ratio between astrocytes (data not shown), but this effect must be confirmed on a larger cell number.

ACKR3-MEDIATED INHIBITION OF GJIC IN PRIMARY ASTROCYTES IS

DEPENDENT ON BOTH DYNAMIN AND β-ARRESTIN2.

ACKR3 interacts with Cx43 and inhibits its GJIC activity upon activation. Cx43 activity is often regulated by alteration of its trafficking 224 . We therefore decided to investigate if the ACKR3-mediated inhibition of GJIC is mediated by Cx43 internalisation. We first examined whether treating cells with the chemical inhibitor of dynamin Dynasore prevents ACKR3 mediated inhibition of GJIC in primary cultures of astrocytes, using again the scrape loading technique (Figure 22). Pre-treatment with astrocytes for 30 min with 80 μM Dynasore abolished the ACKR3-mediated inhibition of GJIC (p = 0.99, n = 3 for control vs. dyn+CXCL12 and control vs dyn+CXCL11), but not the effect of CBX that was still able to inhibit GJIC (Figure 22C). Thus, inhibition of dynamin reverses inhibition of GJIC induced by agonist stimulation of ACKR3.

ACKR3 is known to signal through β-arrestins, which are also involved in its internalisation 143 . In astrocyte cultures prepared from β-arrestin2 KO mice, neither CXCL12 nor CXCL11 inhibited GJIC (p = 0.99, n =3 for control vs. βarr2 -/-+CXCL12 and p = 0.90, n = 3 for control v.s βarr2 -/-+CXCL11) (Figure 22C). In addition, the magnitude of LY diffusion through vehicle-treated astrocytes was similar to that measured in astrocytes from WT mice (p = 0.99, n = 3). Collectively, these observations indicate that ACKR3 stimulation by its two natural agonists CXCL12 and CXCL11 inhibits GJIC in primary cultures of astrocytes. Both inhibition of dynamin and suppression of β-arrestin2 expression completely reverse this inhibition suggesting that Cx43 internalisation underlies the ACKR3-mediated GJIC inhibition.

ACKR3 ACTIVATION TRIGGERS CX43 INTERNALISATION IN PRIMARY CULTURES OF MOUSE ASTROCYTES.

To further confirm this hypothesis and demonstrate that ACKR3 activation triggers Cx43 internalisation, we performed Cx43 immunostaining on primary astrocyte cultures. As expected, Cx43 was mainly localized at the interface of cell-cell contacts in cultures treated with vehicle, resulting in a typical "pavement-like" immunostaining (Figure 26), though a little proportion of Cx43 was detected inside the cells. Treating cultures with CXCL12 (10 -8 M) or CXCL11 (10 -7 M) for 30 min profoundly modified this staining pattern: Cx43 was only marginally detected at the cell interface but mainly in intracellular vesicles (Figure 26).

ACKR3 ACTIVATION INCREASES SURFACE LOCALIZATION OF CX43

HEMICHANNELS.

As reported in the introduction, Cx43 also forms hemichannels that mediate the release of ATP, glutathione, glutamate and aspartate in the extracellular space 234 .

Hemichannels and gap junctions have previously been shown to be differently regulated by pro-inflammatory cytokines 249 . Specifically, cytokines diminish Cx43 hemichannels activity and concomitantly increase GJIC. Using a biotinylation technique, we investigated the amount of Cx43 hemichannel present at the cellular surface upon ACKR3 activation by CXCL12 or CXCL11. In fact, using the biotinylation technique it is possible to discriminate between Cx43 forming gap junctions and hemichannels [275][276][277] : only Cx43 in hemichannels, but not Cx43 engaged in gap junctions can be biotinylated by a cell-impermeable amine-reactive biotinylation reagent 278 . Astrocyte cultures were exposed to CXCL12 (10 -8 M) or CXCL11 (10 -7 M) for 30 min prior to biotinylation of cell-surface proteins. Afterwards, biotin was quenched, cell lysed and biotinylated proteins purified on streptavidin beads. We have collected preliminary data showing that activation of ACKR3 by CXCL12 increases the amount of biotinylated Cx43, i.e. Cx43 hemichannels at the cell surface of astrocytes (Figure 27). Yet, these results must be confirmed on a larger replicate number.

Since more Cx43 hemichannels are present at the cellular surface, we next examined whether this was due to reduced internalisation. Proteins expressed at the cell surface were biotinylated at 4°C. After quenching and removing the biotin in excess, astrocytes were either kept at 4°C or 37°C or exposed to CXCL12 (10 -8 M) or CXCL11 (10 -7 M) for 30 min at 37°C. Thereafter, cells were exposed to a cellimpermeable reducing agent (MESNA) in order to cleave the biotin from cell-surface proteins, so that the remaining biotinylated fraction represents intracellular (internalised) proteins. For each condition, some cells were not exposed to MESNA to measure total biotinylated proteins (internalised + non-internalised). Biotinylated proteins were then purified on streptavidin beads. Neither CXCL12 (p = 0.56, n = 3 vs. total 37°C) nor CXCL11 (p = 0.95, n = 3 vs. total 37°C) influenced the total expression of Cx43. Consistent with its constitutive internalisation, Cx43 was found to be more internalised at 37°C compared to 4°C. Both CXCL12 and CXCL11

inhibited the internalisation of Cx43 hemichannels when compared to the constitutive internalisation at 37°C (Figure 28).

ACKR3 IS CO-LOCALIZED WITH CX43 IN GLIOBLASTOMA.

As reported in the introduction, both Cx43 and ACKR3 play pivotal role in glioma progression. Co-localization analysis after double labelling of ACKR3 or CXCR4 and Cx43 indicates that ACKR3, but not CXCR4, is co-localized with Cx43 in two different glioma-initiating cell lines (R633 and TG1) isolated from human glioblastoma endogenously expressing ACKR3, CXCR4 and Cx43. Specifically, using the overlap approach 408 and setting an automatic threshold 409 we defined the overlapping volume where Cx43 co-localizes with ACKR3 or CXCR4 (Figure 30). To quantify colocalization we firstly calculated the percentage of the signal intensity of Cx43 in the overlapping volume when compared to the total Cx43 signal. We found that 68 ± 8.2

(n = 5) and 55 ± 3.8 (n = 4) % of Cx43 co-localizes with ACKR3 in R633 and TG1 cells, respectively. On the other hand, a much smaller fraction of Cx43 co-localizes with CXCR4 in the same cell lines (21 ± 4.5, n = 5 and 13 ± 3.2 %, n = 7 colocalization in R633 and TG1 cells, respectively) (Figure 30B). In order to confirm these results, we also quantified the percentage of either receptor present in the overlapping volume. 54±12.3 and 42 ± 7.5% of ACKR3 signal was co-localized with Cx43 in the R633 and TG1 cells, respectively; whereas only 25 ± 7.0 and 7 ± 2.7 % of CXCR4 signal was co-localized with Cx43 (Figure 30C).

ACKR3 LIGAND INDEPENDENTLY INHIBITS CX-MEDIATED GJIC IN HEK293T

CELLS.

ACKR3 is overexpressed in various cancer types, including glioblastoma [START_REF] Sánchez-Martín | CXCR7 impact on CXCL12 biology and disease[END_REF] . After

showing that ACKR3 activation inhibits GJIC in astrocytes primary cultures triggering Cx43 internalisation, we examined whether ACKR3 overexpression alone would also be able to modulate Cx43 activity. Therefore, we transiently transfected HEK-293T cells with cDNAs encoding either ACKR3 or CXCR4. Using a glass patch pipette we microinjected single transiently transfected HEK-293T cell with a 1mg/ml LY solution.

Cells were perfused with LY for 5 min. LY diffused for an additional 5-min period.

GJIC was then quantified counting the number of neighbouring cells stained with LY after the 10 min period (Figure 31A). LY injected into cells transfected with empty vector diffused into more than 14 ± 1.05 neighbouring cells (n = 17) (Figure 31B).

This value slightly but not significantly decreased to 11 ± 1. ACKR3 is known for its high degree of constitutive and ligand-induced internalization 142 . We confirmed the constitutive internalization using a DERET internalization assay 410 . In brief, HEK-293T cells were transfected with cDNA encoding N-terminally SNAP-tagged ACKR3 or CXCR4. Cell surface expressed receptors were labelled with SNAP-Lumi4 ® -Terbium (energy donor) at 16°C, which upon excitation with a laser at 347 nm emits at 620 nm. Cells were then exposed to a solution of cell impermeable fluorescein. Fluorescein quenches the signal coming from membrane-expressed receptors, by absorbing the energy transferred from terbium and then emit at 520 nm because of an energy transfer between the Terbium and fluorescein. Internalized receptors will be free to emit at 620 nm when excited.

Therefore, recording the change over time in the ratio of the signal at 620 nm (internalized receptor) divided by the signal at 520 nm (receptor at the membrane)

will allow quantifying the receptor internalization. In this assay ACKR3 constitutively internalized at higher rate, compared to CXCR4 (Figure 32A). Corroborating the higher constitutive internalisation rate of ACKR3 internalisation, compared with CXCR4 demonstrated by DERET, immunocytochemistry experiments showed a diffuse cytoplasmic staining of ACKR3 (Figure 32B), whereas CXCR4 staining was mainly confined into the plasma membrane. DERET experiments also confirmed that ACKR3 internalisation is induced by CXCL12 (Figure 32C).

ACKR3 C-TERMINAL DOMAIN IS NOT ESSENTIAL FOR INTERACTION WITH CX43 BUT IS REQUIRED FOR ACKR3-MEDIATED GJIC INHIBITION.

To further explore the molecular mechanisms underlying Cx43-mediated GJIC inhibition induced by ACKR3 expression, we tried to characterize the site of interaction between Cx43 and ACKR3. Given that the C-terminal domain of GPCRs vs. ACKR3Δ16 and ACKR3ΔCt, respectively) (Figure 34C), suggesting that the Cterminal domain of ACKR3 is not involved in the interaction.

This domain also plays a pivotal role in signal transduction, especially β-arrestindependent signalling, as well as receptor internalisation. Therefore we next explored whether expression of the truncated ACKR3 mutants inhibits GJIC (Figure 35A).

Expression of the two mutants did not inhibit LY diffusion in HEK-293 cells (p = 0.99, n = 10 for ACKR3Δ16 and p = 0.83, n = 9 for ACKR3Δ16 both vs. mock) (Figure 35B), indicating that the C-terminal domain of ACKR3 is essential for inhibiting but not for interacting with Cx43.

ACKR3 ACTIVATES GΑI/O PROTEINS IN ASTROCYTES

Intrigued by the possible involvement of Gi/o proteins in the ACKR3-mediated inhibition of GJIC, we investigated the ability of the receptor to engage Gαidependent signalling proteins by measuring the inhibition of cAMP production in astrocytes challenged with the adenylyl cyclase activator forskolin. Treating cells with CXCL12 (10 -8 M) or CXCL11 (10 -7 M) for 5 min inhibited cAMP production (-36.51 ± 6.44%, n = 4 and -19.25 ± 5.83%, n = 3) (Figure 37). As expected and reminiscent of the scrape loading studies, PTX (100 ng/ml, 18 h) reversed this inhibition. Pretreatment of cells with the CXCR4 antagonist AMD3100 (10 -6 M) for 30 min was unable to prevent the CXCL12-induced inhibition of cAMP production (-39.4 ± 3.62%, n = 5), suggesting that ACKR3 (and not CXCR4) is involved in the CXCL12 effect.

Taken together, these results show that ACKR3 stimulation by CXCL12 and CXCL11 triggers activation of Gαi/o proteins in primary cultures of murine astrocytes.

ACKR3 CAN CONSTITUTIVELY RECRUIT G PROTEINS BUT IT IS UNABLE TO ACTIVATE THEM IN HEK-293T CELLS.

We found that ACKR3 activates G proteins in astrocytes and that G proteins are necessary for the inhibition of GJIC. We then tested if this is also valid in another context such as HEK-293T cells in line with the results of our interactomic screen that demonstrated agonist-independent interaction of ACKR3 with Gαi3. We first confirmed this interaction by saturation BRET (Figure 38A). HEK-293T cells were co-transfected with increasing amounts of cDNA encoding ACKR3 C-terminallytagged with YFP and a constant amount of RLuc-tagged Gαi3. The data show a constitutive interaction between ACKR3 and Gαi3 proteins (Figure 38A). Neither PTX nor CXCL12 (10 -8 M) treatments modified the interaction (p = 0.79, n=3 for vehicle vs.

CXCL12 and p = 0.86, n = 3 for vehicle vs. PTX) (Figure 38B).

MAPPING CXCR4 INTERACTOME

DECIPHERING THE CXCR4 INTERACTOME IN HEK-293T CELLS BY AP-MS.

As for ACKR3, CXCR4 interacting proteins were identified in human embryonic kidney HEK-293T cells transiently expressing hemagglutinin (HA)-tagged CXCR4 using an affinity-purification coupled to mass spectrometry (AP-MS) proteomic strategy. CXCR4-interacting proteins were immunoprecipitated using an anti-HA monoclonal antibody immobilized onto agarose beads and identified by nano-flow liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS).

Control immunoprecipitations were performed using cells transfected with empty plasmid (mock). Systematic analysis by tandem MS of the immunoprecipitates identified a total of 3,802 proteins in the three independent experiments performed on different sets of cultured cells. This number reduced down to 1,203 after filtering out the proteins either identified only by site, or labelled as contaminant, or not identified in all three biological replicates in at least one group (CXCR4 or mock).

Label-free quantification (LFQ) of the relative protein abundances in immunoprecipitates obtained from cells expressing CXCR4 and mock cells showed that 19 proteins were significantly more abundant in immunoprecipitates from CXCR4-expressing cells, using a t-test conducted on both sides and setting a False Discovery Rate (FDR) of 1%, in comparison with mock condition (Table 8).

Analysis of the relative abundance of proteins in immunoprecipitates showed, as expected, that CXCR4 is the most abundant protein (Figure 41). Identified CXCR4 partners included Glutaredoxin-3 (GLRX3), an enzyme belonging to the protein disulphide isomerase (PDI) family. Another PDI, GLRX1, is known to reduce intramolecular disulphide bonds of HIV envelope glycoprotein gp120 during virus entry 411 . Since gp120 interacts with CXCR4, GLRX enzymes could in turn indirectly interact with CXCR4 412,413 . CXCR4 also recruits COUP-TF 2 (NRF2F2), an orphan nuclear receptor that plays a critical role in organogenesis. Overexpression of COUP-TF1, characterized by 84.13% identity with COUP-TF 2 when aligned 414 , inhibits expression of both CXCR4 and its endogenous ligand CXCL12 in breast cancer cells through EGFR activation 415 . The Minor histocompatibility antigen HM13, a peptidase required for the genesis of monomeric peptides, was also detected as a CXCR4 interacting protein. HM 13 that is a member of the HIV-1 envelope interactome as CXCR4 416 .

EPHRIN B1 INTERACTS WITH CXCR4.

Ephrin B1 (EFNB1), a cell surface anchored ligand for Ephrin B receptors, likewise co-immunoprecipitated with CXCR4 in our interactomic screen. Binding of Ephrin B1

to its receptor by direct cell-cell contact triggers both a forward and a backward signalling cascade in two adjacent cells. EFNB1 inhibits, in its backward signalling, G protein activation elicited by CXCR4 upon activation by CXCL12 417 and influences chemotaxis of HUVEC cells 418 . We next collect preliminary results, which must be repeated on a larger replicate number, confirming by western blotting the interaction between CXCR4 and Ephrin B1 using ACKR3 as negative control (Figure 42).

DISCUSSION

PROTEOMIC SCREENING OF CXCR4 AND ACKR3

Although ACKR3 has been found playing a pivotal role in fundamental biological processes such as migration [START_REF] Boldajipour | Control of Chemokine-Guided Cell Migration by Ligand Sequestration[END_REF] , proliferation 148 and differentiation 167,168 of different cellular populations, the cellular pathways transducing these effects remain poorly characterized. The receptor was initially described as a silent receptor able only to shape the CXCL12 and CXCL11 chemo-attractant gradients and to lower the extracellular chemokine concentrations by binding and internalising them 124 . Several studies [139][140][141][142] showing that ACKR3 activates β-arrestin dependent signalling have now overcome this simplistic conception of the role of ACKR3. In addition, the notion that ACKR3 physically and functionally interacts with non-canonical G Protein

Interacting Proteins (GIPs) such as the other chemokine receptor CXCR4 119 and the Epidermal Growth Factor Receptor (EGFR) 155 has expanded the possible signalling pathways activated by the receptor. CXCR4 shares with ACKR3 the capacity to bind to CXCL12. Upon activation CXCR4 triggers canonically associated GPCR signalling pathways [START_REF] Pozzobon | CXCR4 signaling in health and disease[END_REF] , including Gαi and β-arrestin pathways. As ACKR3, CXCR4 has also been found to interact with several proteins able to modify its signalling, trafficking and localization.

Collectively, these findings provided the impetus for ACKR3 and CXCR4 interactome characterization using an AP-MS proteomics strategy. Thus, we immunoprecipitated (HA)-tagged ACKR3 or CXCR4 expressed in human embryonic kidney (HEK)-293T cells and we systematically identified co-immunoprecipitating proteins by mass spectrometry.

Analysis of the CXCR4 interacting network based on Label Free Quantification (LFQ)

and setting a stringent False Discovery Rate (FDR) of 1% revealed that 19 proteins specifically co-immunoprecipitated with the receptor (they were not detected in control immunoprecipitations performed from cells transfected with empty plasmid).

In accordance with its constitutive internalisation 142 and presence in intracellular compartments, the same analysis performed on the ACKR3 interacting network resulted in 151 proteins that were significantly more abundant in immunoprecipitates from ACKR3-expressing cells compared to mock cells. Therefore, by AP-MS, we identified 19 and 151 potential interacting proteins for CXCR4 and ACKR3, respectively. Indeed, it is important to keep in mind that interacting proteins identified

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following AP-MS protocols, especially when performed in cells overexpressing the protein of interest, must be considered as potential partners whose interaction must be further validated via other assays including functional ones. In fact, overexpressed proteins might interact with more proteins (false positive) only because of their mislocalization in different subcellular compartments compared to the endogenously expressed ones. Despite this limitation, our group has already applied the same strategy with other GPCRs whose potential interacting proteins firstly identified with a similar AP-MS screen have been further validated [START_REF] Duhr | Cdk5 induces constitutive activation of 5-HT6 receptors to promote neurite growth[END_REF][START_REF] Meffre | 5-HT 6 receptor recruitment of mTOR as a mechanism for perturbed cognition in schizophrenia[END_REF] . In addition, the Functional Proteomic Platform (FPP) of Montpellier has generated a database including the proteins considered as "contaminants", due to their frequent appearance as coimmunoprecipitating proteins, in overexpressed system, that helps in the discrimination between "false" and "real" interacting proteins.

As expected, and validating the relevance of the co-immunoprecipitation strategy used for affinity-purification of partners, the two bait receptors (CXCR4 and ACKR3)

were the most abundant proteins in each Co-IP. In addition, consistent with the constitutive internalisation and ubiquitination 142 of ACKR3, clathrin, Rab3 and Rab 5 complexes were retrieved in the ACKR3 complexes together with ubquitinin enzymes.

Amongst the 19 potential CXCR4-interacting proteins identified, we decided to validate by Western Blotting (WB) its interaction with Ephrin B1 in line with its high relative abundance in the co-immunoprecipitating complex and previous results 417 showing that Ephrin B1 inhibits G protein signalling triggered by CXCR4 upon activation by CXCL12 417 . Despite these promising preliminary results, further studies validating the interaction in endogenous settings are needed before considering EphrinB1 as a CXCR4-interacting protein Only two studies based on large screening showed a correlation between ACKR3 and gap junctions. In fact, Cx43 mRNA levels were up-regulated in ACKR3

expressing papillary thyroid carcinoma cells 424 and the receptor was previously found to interact with the gap junction beta-2 protein GJB2 95 in a large interactomic screen.

Therefore, these studies clearly show that Cx43 mediates the release of CXCL12 and suggest that CXCR4 might influence the Cx43 phosphorylation state. However, a functional link between ACKR3 and Cx43 has never been demonstrated. In line with these considerations and the aforementioned overlapping biological functions of these two proteins in interneuron migration, leukocyte entry into the brain and glioma progression, we decided to further explore and validate the interaction between ACKR3 and Cx43. ! 100!

CX43-ACKR3 INTERACTION VALIDATION

As a first step, we confirmed the interaction by WB and BRET. Overexpressed CXCR4 was chosen as negative control, as Cx43 was not identified in the CXCR4 interactome. Both experiments showed that Cx43 preferentially interacts with ACKR3 compared to CXCR4. Yet, a weak though statistically non-significant interaction was observed between CXCR4 and ACKR3 in WB. Cx43 was probably not identified by MS-MS in the CXCR4 immune complex due to the lower sensitivity of MS detection compared to immune detection. Since ACKR3 and CXCR4 are known to heterodimerize 119 it is possible that Cx43 is indirectly recruited to CXCR4 via its interaction with ACKR3. This point must be addressed using two sequential immunoprecipitations where ACKR3 and CXCR4, tagged with two different tags, are co-expressed in the same cell population. Starting from the same lysate, the two receptors can then be immunoprecipitated one after the other in two steps. The resulting co-immunoprecipitating proteins would be the one exclusively associated with the CXCR4/ACKR3 heterodimer.

Since the experiments conducted so far were all performed in HEK-293T cells transiently expressing the receptor, we decided to validate the interaction in an authentic biological context endogenously expressing both Cx43 and ACKR3. In line with previous studies showing that ACKR3 (see Table ! 3) and Cx43 234 are both expressed in the brain we performed immunohistochemistry (IHC) on mouse brain slices. Since there is no commercially available antibody against the mouse ACKR3 isoform we performed the IHC on brains obtained from BAC mice expressing EGFP under the ACKR3 promoter 402 . Immunostaining revealed that ACKR3 and Cx43 are co-expressed in GFAP-positive astrocytes of the Sub Ventricular Zone (SVZ). Both proteins 363,402 have been shown to play a pivotal role in the migration of neuronal progenitor cells and interneuron migration along the radial glia cells of the SVZ.

Interestingly the phenotypes obtained from the suppression of either Cx43 or ACKR3

show opposite effects upon interneuron migration: in ACKR3 -/-mice, interneurons leave the migratory streams and enter the cortical plate prematurely, which disrupts their regional distribution within the neocortex 160,402 , whereas suppression of Cx43 363 leads to the accumulation of the interneurons in the ventricular zone with very few interneurons reaching the cortical plate. Furthermore, Cx43 and ACKR3 were found in our study to co-localize in end-feet surrounding blood vessels from GFAP-positive astrocytes. Previous studies showed that Cx43 suppression increases the Blood Brain Barrier (BBB) permeability and leukocyte entry into the brain via disrupting the

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DAPC complex, deregulating Aquaporin channel 4 360 and increasing CXCL12 production 358 . In contrast, CCX771-mediated ACKR3 activation was found to reduce leukocyte entry 169 . Given our results on the site of ACKR3/Cx43 interaction, it is tempting to speculate that an ACKR3-Cx43 cross talk might control interneuron migration and the BBB permeability. Regarding its potential role in interneuron migration, it is of utmost importance to repeat the IHC in the embryonic brain.

Considering their role in the BBB permeability more studies are needed to better dissect the BBB cellular populations co-expressing the two proteins. Specifically, CD31 and CD13 should be used for assessing a potential interaction of both proteins in endothelial cells and pericytes, respectively.

ACKR3 ACTIVATION AND EXPRESSION INHIBIT GJIC

To investigate a possible functional relationship between Cx43 and ACKR3, we firstly found that ACKR3 activation by its two endogenous ligands CXCL12 and CXCL11 inhibits Cx43 gap junctional-mediated dye diffusion in primary cultured astrocytes.

Since CXCL12 affinity for ACKR3 is 10-fold higher than that of CXCL11 [START_REF] Balabanian | The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes[END_REF]108 , CXCL12 was used at 10 -8 M, whereas CXCL11 was used at 10 -7 M. CXCL12 binds to both CXCR4 and ACKR3, whereas CXCL11 also binds to CXCR3. Therefore, the CXCR4 antagonist AMD3100 and the CXCR3 antagonist NB-74330 425 were used for blocking CXCR4 and CXCR3, respectively. Since AMD3100 was found to have allosteric agonistic properties on ACKR3 at 10 -5 M 426 , we used it at 10 -6 M. Both antagonists did not have any effect on GJIC when applied alone and did not reverse the CXCL12-and CXCL11-induced GJIC inhibition, suggesting that only ACKR3 and not CXCR4 or CXCR3 is involved in GJIC inhibition. Since one of our collaborators completed the selection and characterization of ACKR3 nanobodies with antagonistic properties against the mouse ACKR3 receptor, we are planning to investigate if these nanobodies are able to reverse the CXCL12 and CXCL11-mediated GJIC inhibition. Several studies showed that Cx43 is rapidly and transiently inhibited by GPCR agonists. For example GJIC inhibition by SP-1 and ET1 was evident as soon as 3 min after their addition and was completely reversed 30 min after agonist washout 299 . Both agonists were found to promote dephosphorylation of Cx43 Ser 368 .

On the other hand, in our experimental conditions, CXCL12 and CXCL11 inhibited GJIC 30 min after the onset of chemokine application and GJIC was still inhibited 20 min after their withdrawing (data not shown). As it will be discussed in the next sections, we have collected data supporting the hypothesis that ACKR3 inhibits GJIC by promoting Cx43 internalization. Therefore, this persistent effect might be due to

! 102!
the fact that Cx43 is targeted to lysosomes for degradation after internalization. This hypothesis must be tested by investigating if firstly Cx43 co-localizes with lysosome markers and secondly if even longer chemokine exposures would lead to Cx43 degradation. It would be also interesting to investigate if the different kinetics between chemokines and SP-1 or ET1 is due to a different mechanism of action and if CXCL11 and CXCL12 are able to induce dephosphorylation of Cx43 Ser 368 using a specific antibody.

After establishing that ACKR3 activation inhibits GJIC in primary astrocytes, as assessed indirectly by the diffusion of Lucifer Yellow, we then investigated the possibility that ACKR3 inhibits gap junctional mediated electrical coupling that is a more direct measure of GJIC 259 . Confirming what was already observed by measuring LY diffusion, activation of ACKR3 by CXCL12 significantly inhibited electrical coupling of astrocytes. Interestingly, CXCL12 significantly inhibited electrical coupling in four out of the six coupled cell pairs that were patched, whereas CBX was effective in all six pairs. This might either reflect a heterogeneous distribution of the receptor in primary cultured astrocytes or a lower efficacy of CXCL12. The first hypothesis can be investigated generating primary cultures from BAC EGFP mice. In addition, as already mentioned in the "Results" section, these findings must be confirmed by the demonstration that CXCL11 also inhibits electrical coupling of astrocytes. Furthermore, the chemokines were delivered by bath application, making the interpretation of the kinetics of their effect challenging.

Therefore, further experiments must be performed using faster drug delivery systems for better interpreting the kinetics of the ACKR3-mediated inhibition of electrical coupling.

ACKR3-MEDIATED GJIC INHIBTION: MECHANISM OF ACTION

Cx43 activity is often regulated by alteration of its trafficking 224 . Thus, we investigated if ACKR3 could inhibit Cx43 by increasing its internalisation. Previous studies showed that inflammatory cytokines, including interleukin-1β and tumour necrosis factor-α, reduce gap junctional communication and concomitantly increase Cx43 hemichannel-mediated membrane permeability 249 . In line with these results, we found that ACKR3 activation oppositely regulates Cx43 gap junctions and hemichannel trafficking. Specifically, we observed that ACKR3 activation triggered internalisation of Cx43 involved in gap junctions, while it inhibited Cx43 hemichannel internalisation, increasing their expression at the cell surface. Further studies investigating the activity of Cx43 hemichannels, by ethidium bromide uptake, are

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planned. Inhibition of Cx43 internalisation by the dynamin inhibitor Dynasore completely reversed the ability of ACKR3 stimulation to inhibit GJIC and to promote Cx43 internalisation, suggesting that internalisation is needed for blocking GJIC.

Consistent with the ability of the receptor to interact with and to activate βarrestins 139 , the GJIC inhibition and concomitant Cx43 internalisation elicited by ACKR3 stimulation was dependent on β-arrestin2. In fact, activation of ACKR3 in primary cultured astrocytes obtained from β-arrestin2 -/-mice neither inhibited GJIC nor induced Cx43 internalisation.

Therefore, ACKR3 activation by its two endogenous agonists CXCL12 and CXCL11 inhibits GJIC through a mechanism involving Cx43 internalisation. In addition, these data show that both β-arrestin2 and dynamin are necessary for the ACKR3dependent inhibition of GJIC. However, an important question remains unanswered:

is ACKR3 co-internalising with Cx43 or is ACKR3 indirectly inducing Cx43 internalisation?

Regarding an indirect inhibition, upon stimulation ACKR3 might activate β-arrestin2

that in turn will engage an intracellular signalling cascade resulting in an ACKR3indirect Cx43 internalisation and GJIC inhibition. However, this hypothesis is in contrast with previous results showing that inhibition of dynamin increases β-arrestindependent ERK1/2 phosphorylation 141 .

In the second hypothesis, consistent with the aforementioned interaction between ACKR3 and Cx43, Cx43 might directly co-internalise with activated ACKR3 as an ACKR3/Cx43 complex. Indeed, as shown by the DERET assay and by previous results, ACKR3 internalises upon activation. In addition, both inhibition of dynamin 141 and β-arrestin suppression 142 were shown to reduce ACKR3 internalisation.

Consolidating the importance of ACKR3 internalisation in Cx43 inhibition, transient expression in HEK-293T cells of C-terminally truncated ACKR3 mutant that are not capable of internalising 143 , does not inhibit GJIC even though they retain ability to interact with Cx43.

Collectively, these results suggest that the direct internalisation of the ACKR3/Cx43 might be more plausible than the indirect one. However, very recent and unpublished results from two independent laboratories (Prof Martine Smit, University of Amsterdam and Prof Ralf Stumm, University of Jena, personal communications)

suggest that β-arrestin is not essential for ACKR3 internalisation. Therefore we are currently initiating experiments in collaboration with Prof Ralf Stumm to investigate whether ACKR3 and Cx43 are co-localized in intracellular vesicles using HA-tagged

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ACKR3 knock-in mice. In addition, since β-arrestin2 can directly interact with Cx43 345 it would be interesting to investigate if ACKR3 and Cx43 form a multi-protein complex with β-arrestin2.

ACKR3-DEPENDENT GJIC INHIBTION IN TUMOUR PROGRESSION

Both Cx43 380 and ACKR3 211 play an important role in the progression of several cancer types, including glioma. ACKR3 is overexpressed in glioma 211 and its expression correlates with higher proliferative state of cancer cells 210,[212][213][214] . On the other hand, Cx43 expression and activity is inversely correlated with the proliferative state of cancer cells 379 . Our interactomic screen, Co-IP followed by WB and BRET studies revealed that Cx43 preferentially interacts with ACKR3, compared to CXCR4.

Further supporting these biochemical studies, Cx43 is strongly co-localized with ACKR3 but not CXCR4 in glioma-initiating cell lines isolated from human glioblastoma. In next steps, we are planning to check if Cx43 and ACKR3 are also co-localized in biopsies obtained from human glioma.

ACKR3 is characterized by a high level of constitutive activity and internalisation in various cancer cell types 143 . Thus, we investigated if expression of ACKR3 would by itself inhibit GJIC. Single cell microinjection of Lucifer Yellow in HEK-293T cells transiently expressing ACKR3 or CXCR4 showed that only ACKR3 expression inhibits dye diffusion. As already reported, transient expression of two C-terminally truncated ACKR3 mutants did not inhibit GJIC even though they retain ability to interact with Cx43, suggesting that ACKR3 trafficking and internalisation are key components for the GJIC inhibition. However, in HEK-293T cells, Dynasore did not reverse the ACKR3 effect though it does inhibit its internalisation 143 , suggesting that newly synthesized ACKR3 might interact with Cx43 in the synthetic pathway. This interaction might affect Cx43 forward trafficking to the plasma membrane and thereby retain it in intracellular compartments, explaining the inhibition of GJIC observed in cells expressing ACKR3. Immunofluorescence microscopy and BRET experiments will be performed to characterize the intracellular compartments where ACKR3 interacts with Cx43. In addition, before completely ruling out the involvement of ACKR3 internalisation in GJIC inhibition, other conditions known to inhibit ACKR3 internalisation (sucrose, dynamin K44A mutant) will be tested.

Several lines of evidence suggest that engagement of a β-arrestin2-dependent signalling cascade might be responsible for the agonist-independent ACKR3mediated inhibition of GJIC in HEK293T cells: i) ACKR3 is known to constitutively ! 105! activate β-arrestin-dependent signalling, ii) the two C-terminally truncated ACKR3

mutants are characterized by lower β-arrestin recruitment 143 and iii) Dynasore does not reverse the ACKR3 mediated inhibition of GJIC, consistent with the aforementioned higher ACKR3-dependent β-arrestin activation upon dynamin inhibition 143 . Therefore, the impact of β-arrestin2 knockdown using RNA interference on the ACKR3-mediated ligand independent GJIC inhibition will be investigated to further explore this hypothesis.

The fact that the two ACKR3 C-terminally truncated mutants interact with Cx43 but do not inhibit its activity raise questions about the role of the physical interaction between the two proteins. Clearly, the interaction is not sufficient for mediating the ACKR3-dependent GJIC inhibition but is it necessary? The identification of peptide sequences mediating the interaction is essential to address that issue. In this regard, considering that the C-terminal domain of ACKR3 is not involved in the interaction and that CXCR4 only marginally interacts with Cx43, the best strategy would be to swap ACKR3 domains with CXCR4 ones until the peptide sequence(s) mediating the interaction would be found. Once this (these) sequences are identified, interfering peptides could be designed to disrupt the interaction and to investigate its functional impact. In line with these results showing that Gαi/o protein activation is needed for GJIC inhibition, we investigated if ACKR3 is indeed able to activate them. We thus examined the ability of ACKR3 to activate Gαi proteins and consequently to inhibit cAMP production in astrocytes. Consistent with a previous study 153 showing that CXCL12, induces Gαi protein activation in primary cultured astrocytes, we found that ACKR3 activation by CXCL12 inhibited cAMP production induced by the adenylyl cyclase activator Forskolin. However, in contrast with the same study, we found that CXCL11 also inhibited cAMP production in astrocytes. This discrepancy might result from the 10 time higher concentration of CXCL11 used in our experiment. In order to check this point, the same experiments will be repeated using increasing concentrations of both chemokines. Future experiments employing other readouts for measuring G protein activation, such as monitoring Ca 2+ mobilization, are also planned.

Although these data suggest that ACKR3 might directly activate Gαi proteins a possible indirect activation of another Gαi coupled GPCR cannot be completely excluded. In fact, chemokine challenge might induce the release of GPCR agonists, such as glutamate or adenosine, which would in turn activate their endogenously expressed receptors, thereby inhibiting cAMP production. Although this process seems unlikely due to the short duration of the chemokine challenge (5 min), this certainly warrants further exploration. Specifically, astrocytes will be treated for 5 min

! 107!
with the chemokine before collecting the cell supernatant. A novel astrocyte culture will then be challenged with the conditioned supernatant in presence of CXCR4 and ACKR3 antagonists (AMD3100 and the aforementioned nanobodies). If one rules out an indirect effect, cAMP levels should remain unchanged.

Though this control experiment remains to be carried out, our results suggest that ACKR3 activates Gαi protein in primary cultured astrocytes obtained from embryonic mouse brains. In our interactomic screen, we identified Gαi3 as a potential ACKR3 interacting protein. In line with previous results showing that ACKR3 interacts with Gαi proteins [START_REF] Levoye | CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling[END_REF] , we validated by BRET that ACKR3 constitutively recruits Gαi3 in HEK-293T cells. Neither CXCL12 nor PTX could modulate the interaction, already suggesting that ACKR3 might be unable to activate them in HEK-293T cells.

Accordingly, only CXCR4 but not ACKR3 (transiently expressed in HEK-293T cells)

was able to inhibit cAMP production upon CXCL12 stimulation. In line with these results, PTX was unable to reverse the ligand-independent, ACKR3-mediated inhibition of GJIC in HEK-293T cells, confirming that Gαi/o proteins are not involved.

Intrigued by this discrepancy between mouse astrocytes and human HEK-293T cells we investigated if only the mouse isoform of ACKR3 and not the human one was able to activate Gαi/o proteins. Therefore, we transiently expressed mouse ACKR3 and CXCR4 in HEK-293T cells. However, only the mouse isoform of CXCR4 and not the ACKR3 one was able to trigger G protein, as previously demonstrated for human receptors.

Therefore, these results suggest that ACKR3 might be coupled to Gαi/o proteins only in astrocytes. The peculiarity of this cellular environment could be granted by the astrocyte expression of scaffolding proteins necessary for the activation of G proteins. It would be interesting therefore to study the interactome of ACKR3 expressed in astrocytes using, once again, the HA-ACKR3 knock-in mouse model just generated in the Ralf Stumm's laboratory.

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5.CONCLUSIONS

Starting from a large-scale interatomic study, we identified Ephrin B1 and Connexin 43 (Cx43) as novel potential interacting proteins of CXCR4 and ACKR3, respectively.

Validation of the ACKR3-Cx43 interaction showed that the two proteins can constitutively interact in living cells and they are co-expressed in specific astrocyte populations of authentic in physiological conditions. In addition, Cx43 and ACKR3

were found to co-localize in glioma-initiating cell lines isolated from human glioblastoma.

Functional validation of the ACKR3-Cx43 interaction revealed that ACKR3 influences and maintained in humidified atmosphere containing 5% CO2 at 37°C. Cells were used between passage 10 and 20 and passed twice a week.

Primary cultures of cortical astrocytes were prepared from Swiss mice (purchased from Janvier labs) embryos at embryonic day 15.5 and grown in a 1:1 mixture of DMEM and F-12 nutrient supplemented with glucose (30 mM), glutamine (2 mM), NaHCO3 (13 mM), HEPES buffer (5 mM, pH 7.4) and penicillin-streptomycin (100 unit/ml-0.1 mg/ml) and 10% heat inactivated Nu-Serum (Corning 355500). Cells were maintained in atmosphere containing 5% CO2 at 37°C. Medium was not changed for one week after seeding and then changed twice a week. Cultures were used after 5 weeks from seeding. The enrichment in astrocytes was routinely checked via GFAP staining. Only cultures where GFAP positive cells represented at least the 75% of the cellular population were used for experiments. Cultures were starved in DMEM medium supplemented with penicillin-streptomycin (100 unit/ml-0.1 mg/ml) overnight before experiment.

Secondary cultures of cortical astrocytes were prepared starting from five-week old primary cultures. After PBS washing primary cultures were incubated for 15 min with trypsin-EDTA 0.05% (GIBCO 25300-054) in humidified atmosphere containing 5% CO2 at 37°C. After resuspension in DMEM/F-12 medium cells were plated on coated glass coverslips.

β-arrestin2 -/-astrocytes cortical primary cultures were obtained from C57BL/6J β-arrestin2 -/-mice (provided by Dr Gyslaine Bertrand (Institut de Génomique Fonctionnelle) embryos at embryonic day 15.5 and grown as the WT cultures.

R633 and TG1 glioblastoma cancer stem cells were provided already fixed in PFA by Dr M.P Junier (Institut de Biologie Paris Seine, Paris, France). Their isolation and characterization are described elsewhere [428][429][430] .

Mice. EGFP-ACKR3 mice are BAC mice from GENSAT already characterized in 118,161 . They express EGFP under the promoter of ACKR3, which has been inserted

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into a random location of the genome ensuring germline transmission. They are maintained heterozygous on a Hsd:ICR (CD-1®) background.

PRINCIPAL REAGENTS USED

Chemokines. Recombinant mouse CXCL11 and Human/Feline/Rhesus Macaque CXCL12 were purchased from R&D System (572-MC and 350-NS). Both were dissolved in PBS with 0.1% BSA to a concentration of 10 -5 M, aliquoted and stored at -80°C.

Antagonist. AMD3100 (purchased from Tocris, ref 3299) was dissolved in PBS to a final concentration of 20 mM, aliquoted and stored at -20°C. NBI-74330 was purchased from R&D (4528/10) dissolved to a final concentration of 0.01 M in DMSO, aliquoted and stored at -20°C.

Principal chemicals used. Lucifer Yellow CH dilithium salt (LY) was purchased from Sigma (L0259) as Dynasore hydrate (D7693) and Carbenoxolone disodium salt (CBX) (C4790). Pertussis Toxin from Bordetella pertussis was purchased from Calbiochem (516560).

METHODS

Coating was performed incubating plates and coverslips with a solution of Poly-Lornithine hydrobromide 10mg/ml (Sigma P3655) for 2hrs in humidified 5% CO2 incubator at 37 °C. Coverslips and plates were washed twice with PBS before usage.

All plates and coverslips were coated in all experiments.

Transfection of cDNA was performed using Polyethylenimine (PEI) from Polyscience (24765). Cells were seeded one-day prior transfection and used two days after for all experiments but BRET. For BRET cells were transfected in suspension 24hrs before experiment. In both cases, a solution of optiMEM purchased from Gibco (11058-021) containing the cDNA was mixed with a solution of PEI. The ratio maintained for all experiment was 4ng PEI for each ng of cDNA. In all conditions of every experiment the total amount of cDNA was equilibrated using empty plasmid. For mock cells only empty plasmid was transfected. The solution was gently mixed. After 20min the solution was added to the cells. database for Homo sapiens (release 2017_10) using the following parameters:

Co

enzyme specificity was set as Trypsin/P, and a maximum of two missed cleavages and a mass tolerance of 0.5 Da for fragment ion were applied. A second database of known contaminants provided with the MaxQuant suite was also employed. The "match between runs", "iBAQ" and "LFQ" options were checked. Oxidation (M) and Phosphorylation (STY) were specified as variable modification and carbamidomethyl (C) as fixed modification. Database searches were performed with a mass tolerance of 20 ppm for precursor ion for mass calibration, and with a 4.5 ppm tolerance after calibration. The maximum false peptide and protein discovery rate was specified as 0.01. Seven amino acids were required as minimum peptide length. The MaxQuant software generates several output files that contain information about identified peptides and proteins. The "proteinGroups.txt" file is dedicated to identified proteins:

each single row collapses into protein groups all proteins that cannot be distinguished based on identified proteins. An in-house bioinformatics tool have been developed to automatically select a representative protein ID in each protein group.

First, proteins with the most identified peptides are isolated in a so called "match group" (proteins from the "Protein IDs" column with the maximum number of "peptides counts (all)"). For 1% of the remaining match groups where more than one protein ID existed, the "leading" protein has been chosen as the best annotated protein according to the number of Gene Ontology annotations (retrieved performed from UniProtKB 03/10/2017). The Perseus software (v 1.5.6.0 435 ) enabled protein quantification (label free quantification) via the intensity values and performed subsequent statistical analysis of these data. Quantification were performed using LFQ values for further analysis, after elimination of reverse and contaminant entries.

Experiments were repeated three times to assess biological reproducibility. For comparison this intensity was divided by the total intensity of each group before plotting.

Analysis

IHC. Mice were anesthetized with pentobarbital (100 mg/kg i.p., Ceva SA) and perfused transcardiacally with fixative solution containing 4% w/v paraformaldehyde, 0.1 M sodium phosphate buffer (pH 7.5), sodium fluoride (10 mM), and sodium orthovanadate (2 mM). Brains were post fixed for 48 h in the same solution. Brains were then dehydrated by immersion in ice-cold solutions containing first 10 then 20 and 30% sucrose. Solutions were changed when brains were sinking. Brains were then embedded in OCT and rapidly frozen with SnapFrost ® (Excilone). Brains were then stored at -80°C. 50μM brain slices were cut using a Cryostat Leica CM3050

and IHC was performed on floating slices. Specifically, slices were permeabilized and blocked with a PBS solution of 10% heat inactivated goat serum and 0.3 % Triton X-100 for 20min. Primary antibodies were then incubated overnight at 4°C in a 3 and 0.1% PBS solution of goat serum and Triton X-100, respectively. After ! extensive PBS washings cells were incubated for 2hrs, at room temperature and protected from light, with a PBS solution with 3% goat serum and 1% Triton X-100 PBS solution containing the appropriate secondary antibody and Hoechst 33342.

Scrape loading. Five week old astrocytes were starved overnight in presence, if necessary, of PTX (100 ng/mL) or CBX (50μM). Culture when then pre-treated with the appropriate antagonist or Dynasore (80μM) for 30min at 37°C. Removed from the incubator cells were exposed to the appropriate concentration of chemokine dissolved in a solution containing 130mM CaCl2, 2.8mM KCl, 1mM CaCl2, 2mM

MgCl2 and 10mM HEPES (pH=7.2), for 30min at room temperature. For the dynasore and CBX treated conditions the two chemicals were added also in this step. Cells were then exposed to the same solution without CaCl2 for 1min. After that, monolayer is scraped using a razor blade in the presence of Lucifer Yellow (1mg/ml) dissolved in the same calcium-free solution. LY is let diffusing for 1min. Cells are then washed 5 times with the calcium-containing solution. After ten minutes picture were taken using the inverted microscope Zeiss Axiovert 40CFL equipped with Axiocam ICCL1 (Zeiss) with the green filter using the Zeiss Axiovision 4.8 software.

For the quantification picture were analyzed with the Fiji software. were bath-applied through the perfusion system at a rate of 2 ml/min. Control cell pairs were challenged with saline, using the same protocol.

Cx43 internalisation via IF. Primary astrocyte cultures isolates from WT or β-arrestin2 -/-mice were seeded on glass coverslips for 5 weeks. After overnight starvation, when necessary, cells were pre-treated for 30min with 80μM Dynasore.

Cells were then exposed to either CXCL12 or CXCL11 for other 30min. Cells were then fixed and IF performed. Antibody 2 and Alexa Fluor 594-conjugated anti-rabbit were used. Pictures were taken with a Leica SP8-UV confocal microscope.

Quantification of cAMP production. Astrocytes or HEK293T cells were cultured in 24 well plates. HEK293T were transfected 48hrs prior experiment. Both cell types were starved overnight in presence or absence of PTX (100 ng/mL). When necessary cells were pre-exposed for 30min to the CXCR4 antagonist. Cells were then stimulated for 5min with CXCL11 or CXCL12 dissolved in a DMEM solution with 1% BSA before stimulation of cAMP production using Forskolin (10 -6 M) in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1methylxanthine (IBMX). After 5 minutes cells were then lysed in 1% Triton X-100 and cAMP production was quantified using the cAMP dynamic kit (Cisbio Bioassays) according to the manufacturer's instructions. All conditions were performed in triplicate within each biological replicate.

Biotinylation. Biotinylation experiments were performed using the Pierce™ Cell Surface Protein Isolation Kit (Thermo Fisher) according to the according to the In addition, we provide an overview of the current knowledge of CXCR4 and ACKR3 posttranslational modifications and their consequences upon receptor functional status and an exhaustive list of previously identified CXCR4 or ACKR3 interacting proteins. Finally, we present an original dataset comprising a CXCR4 interactome deciphered using an affinity purification coupled to mass spectrometry strategy, which revealed novel CXCR4 interacting proteins potentially involved in oncogenic signalling. A deeper knowledge of the CXCR4/ACKR3 interactomes along with their phosphorylation and ubiquitination status would shed new lights on their regulation and pathophysiological functions.

Introduction

The C-X-C chemokine receptor type 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3), earlier referred to as CXCR7, are class A G protein-coupled receptors (GPCRs).

Stromal cell-derived factor-1/ C-X-C motif chemokine 12 (CXCL12) binds to both CXCR4 and ACKR3 receptors, whereas CXCL11 binds only to the latter and the C-X-C chemokine receptor type 3. CXCR4 and ACKR3 are co-expressed in various cell types (e.g. endothelial cells (Volin., 1998; Berahovich, 2014), neurons (Banisadr, 2002; Sánchez-Alcañiz, 2011) and glial cells (Banisadr, 2002, 2016; Odemis, 2010)) where they play a pivotal role in migration, proliferation and differentiation. They are also over-expressed in various tumours and control invasion and metastasis (Sun, 2010; Zhao, 2015; Nazari, 2017). There is now considerable evidence indicating that GPCRs do not operate as isolated proteins within the plasma membrane. Instead, they physically interact with numerous proteins that influence their activity, trafficking, and signal transduction properties (Bockaert, 2004; Ritter and Hall, 2009; Magalhaes, 2012). These include proteins canonically associated with most GPCRs such as G proteins, G protein-coupled receptor kinases (GRKs) and b-arrestins, specific partner proteins and even GPCRs themselves. In fact, in comparison to monomers, GPCRs can form homo and heteromers with specific pharmacological and signal transduction properties (Ferré, 2014). Phosphorylation is another key mechanism contributing to the regulation of GPCR functional activity (Tobin, 2008). Again, GPCR phosphorylation can be elicited by canonical GRKs but also by other specific protein kinases (Luo, 2017). This review will describe recent data highlighting the influence of the CXCR4 and ACKR3 interactome on their functional activity and signal transduction properties. A special focus will be paid to the influence of the interactome on CXCR4/ACKR3 phosphorylation and ubiquitination and their impact on the functional status of each receptor.

Methods for the identification of GPCR-interacting proteins

Considerable evidence suggests that GPCRs recruit GPCR-interacting proteins (GIPs) (Maurice, 2011). This prompted investigations aimed at identifying GIPs and at characterising GPCR-GIP interactions, using either unbiased or targeted approaches. In unbiased methods, no knowledge of the GIPs is required beforehand and the GPCR of interest is used as bait to purify unknown GIPs. Meanwhile, targeted methods are devoted to the validation and characterisation of previously identified GPCR-GIP interactions. Methods for identifying GIPs or characterising GPCR-GIP interactions include genetic, biophysical or proteomic approaches and are summarised in Table 1.

! a) Genetic methods

The first method belonging to this class is the yeast two-hybrid assay (Fields and Song, 1989). In this method, the protein of interest (the bait protein) is expressed in yeast as a fusion to the DNA-binding domain of a transcription factor lacking the transcription activation domain. To identify partners of this bait, a plasmid library that expresses cDNA-encoded proteins fused to a transcription activation domain is introduced into the yeast strain.

Interaction of a cDNA-encoded protein with the bait protein results in the activation of the transcription factor and expression of a reporter gene, enabling growth on specific media or a colour reaction and the identification of the cDNA encoding the target proteins. A first disadvantage is the loss of spatial-temporal localisation of the interaction; in fact, the yeast two-hybrid assay only captures a snapshot of potential interactions in an artificial biological system. A second disadvantage is that it is not possible to investigate membrane-anchored proteins since the two proteins must cross the nuclear membrane to carry the reconstituted transcription factor to the DNA. To overcome this issue, the membrane yeast two-hybrid assay (Stagljar, 1998) was developed. In this assay, the ubiquitin protein is split into two fragments, which are fused to the two proteins of interest. The ubiquitin C-terminal fragment is then conjugated to a transcription factor that is released when the interaction occurs, and ubiquitin protein is reformed. However, as in the yeast two-hybrid assay, the spatial-temporal localisation of the interaction is lost. A second limitation is that the ubiquitin C-terminus carrying the transcription factor cannot be fused to soluble proteins because they could diffuse into the nucleus. Thereafter, a mammalian version of the assay called mammalian membrane two-hybrid (Petschnigg, 2014) has been developed. The kinase substrate sensor assay (Lievens, 2014), using the signal transducer and activator of transcription (STAT)3 as transcription factor, can also be used for investigating protein-protein interactions including those involving cytosolic and membrane proteins in mammalian cells. However, the kinase substrate sensor assay cannot be used for studying GPCR interaction with proteins involved in the STAT3 cascade.

b) Biophysical methods

Energy transfer-based methods, such as bioluminescence and fluorescent resonance energy transfer (BRET (Xu, 1999) and FRET (Clegg, 1995)) assays, are targeted methods that are generally used to investigate previously reported interactions. Both are based on the transfer of energy from a donor to a nearby acceptor (<100 Å). Their high sensitivity allows the study of weak and transient interactions. The high spatial-temporal resolution permits accurate kinetic studies for investigating interaction dynamics.

!

The proximity ligation assay (Fredriksson, 2002) is another powerful targeted fluorescencebased method. In the direct version of the technique, two DNA oligonucleotide-conjugated antibodies are used against the proteins of interest. In the indirect version, secondary DNAconjugated antibodies are used after targeting the proteins of interest with an appropriate primary antibody. If the two conjugated antibodies are close enough (30-40nm), they can bind together. The DNA connecting the two probes is then amplified and hybridised with fluorophores. This allows the visualisation of the interaction in the place where it occurs, at a single molecule resolution. The main disadvantages of the approach are the high costs and the necessity for specific antibodies that are not always available.

In the bimolecular fluorescent complementation assay (Hu, 2002), a fluorescent protein is divided into two non-fluorescent fragments that are fused to the proteins of interest.

Interaction reconstitutes the entire fluorescent protein. This method allows the direct visualisation of the interaction and can be used for soluble or membrane-bound proteins. In addition, several interactions can be investigated in parallel using different fluorescent proteins. Since there is a delay in fluorescence formation upon reconstitution of the fluorescent proteins, and the fluorophore formation is irreversible, these methods are not well suited for kinetic studies.

c) Proteomics methods

Proteomic methods aim at identifying GIPs of a receptor of interest via the use of affinity purification combined with mass spectrometry (AP-MS). This approach is usually employed as a unbiased method for screening virtually all the GIPs of a GPCR of interest. Targeted versions of the method also exist and rely on GIP identification by Western blotting.

However, the requirement for specific antibodies seriously limits its application. Several strategies can be used for the affinity purification step. In co-immunoprecipitation (Co-IP), specific antibodies against the protein of interest are used for precipitating the bait from a protein lysate. As specific GPCR antibodies providing high immunoprecipitation (IP) yields are rarely available, epitope-tagged versions of the receptor of interest are often expressed in the cell type or the organism of interest and precipitated using antibodies against the tag.

The main advantages of Co-IP are the purification of proteins interacting with the entire receptor (whenever possible the native receptor) in living cells or tissues and its ability to purify the entire associated protein complex. The main limitations are the necessity for specific antibodies to precipitate GPCRs, the loss of spatial-temporal information and the use of detergents for cell lysis that might disrupt weak interactions. Alternatively, pull-down assays can be performed to purify GPCR partners from a cell or tissue lysate. This approach uses the receptor (or one of its domains) fused with an affinity tag (e.g. glutathione S-! transferase) and immobilized on beads as bait. Such in vitro binding assays can also be used to prove direct physical interaction between two protein partners. In this case, the bait is incubated with a purified protein instead of a cell or tissue lysate. In all methods, affinity purified proteins are systematically identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A two-step version, named tandem affinity purification (Puig, 2001), has also been reported (Daulat, 2007) and applies to both Co-IPs or pulldowns. Although tandem affinity purification methods drastically reduce the number of falsepositive identifications, they require larger amounts of starting material.

In the proximity-dependent biotin identification method (Roux, 2013), the bait protein is fused to a prokaryotic biotin ligase molecule that biotinylates proteins in close proximity once expressed in cells. The method can detect weak and transient interactions occurring in living cells and detergents do not affect the results. However, the fusion of biotin ligase to the bait might alter its targeting or functions.

Methods for the identification of GPCR phosphorylated sites

GPCR phosphorylation is a key regulatory mechanism of receptor function (Lefkowitz, 2004).

Over the past years, numerous techniques have appeared with increasing resolution to pinpoint phosphorylated residues (summarized in Table 1), which consist of serines, threonines or tyrosines.

a) Radioactive labelling method

The first method that was introduced for deciphering the phosphorylation status of GPCRs is a radioactivity-based technique, consisting of culturing cells in a medium in which phosphate is replaced with its radioactive counterpart 32 P, resulting in radioactive phosphorylated residues (Meisenhelder, 2001). After culturing, cells are lysed and receptors are immunoprecipitated using specific antibodies and then resolved by SDS-polyacrylamide gel electrophoresis. Receptors are then digested using an enzyme, such as trypsin, and the resulting peptides are separated by 2D migration using electrophoresis and chromatography.

The radioactivity of the digested peptides in the gel is finally measured using a phosphorimager yielding a phosphorylation map for a given receptor in a given cell line (Chen, 2013). This method is very sensitive but cannot give information on the number of phosphorylated sites nor their position.

b) Liquid chromatography coupled to tandem mass spectrometry method

!

More recently, radioactive labelling-based methods have been progressively supplanted by the identification of phosphorylated residues by LC-MS/MS. In this method, the GPCR of interested is digested enzymatically, using one or several proteases, to generate peptides that cover a large part of the receptor sequence. The resulting peptides are then analysed by LC-MS/MS (Dephoure, 2013). Although this approach can pinpoint any phosphorylated residue with high confidence, few limitations complicate phosphorylated residue identification. Firstly, phosphorylation can be lost during fragmentation. Secondly, since phosphorylation sites have a limited level of phosphorylation, only a small percentage of peptide is actually phosphorylated (Wu, 2011). Thirdly, the identification of the phosphorylated residues in peptides with multiple adjacent phosphorylated residues can be challenging. For each modified site, a phosphorylation index can be estimated by dividing the ion signal intensity corresponding to the phosphorylated peptide by the sum of the ion signals of the phosphorylated peptide and its non-phosphorylated counterpart. Absolute quantification, and thus the stoichiometry of phosphorylation, can also be determined for each modified residue by spiking the sample with a known concentration of high purity heavy isotope-labelled peptides (AQUA peptides) corresponding to the phosphorylated peptide and not phosphorylated one and comparing the respective ion signals of un-labelled and labelled peptides (Gerber, 2003).

c) Mutagenesis method

Another approach that can be used as a stand-alone technique or in complement with the previously described methods is to mutate potential or predicted phosphorylated residues to assess functional differences compared to the wild-type receptor. Mutating residues to alanine prevents phosphorylation (Canals, 2012) while mutating residues to aspartic acid mimics phosphorylation (Okamoto and Shikano, 2017). Nevertheless, introducing those mutations can potentially alter expression of the receptor, its conformation or its cellular localisation.

d) Phospho-specific antibody method

To be able to detect and assess phosphorylation of residues in cells or tissues, antibodies that specifically target previously identified phosphorylated residues of GPCRs can be generated by immunising animals with purified synthetic phosphorylated peptides encompassing the phosphorylated residues (Chen, 2013). After selection and functional validation, those antibodies can be used in Western blot or immunohistochemistry experiments. Phosphorylation can also be indirectly detected using antibodies specific to the unphosphorylated GPCR, showing decreased binding to the target when residues are ! phosphorylated (Busillo, 2010), and recovery of the binding by using a protein phosphatase to dephosphorylate the receptor (Hoffmann, 2012).

Association of CXCR4 and ACKR3 with canonical GPCR interacting proteins

G proteins, GRKs and b arrestins are the protein families considered as canonical GPCR interacting proteins controlling receptor activity or being involved in signal transduction.

GPCR activity is a result of a tightly regulated balance between activation, desensitisation and re-sensitisation events. After receptor activation and interaction with G proteins, several mechanisms integrate to trigger GPCR desensitisation and/or modulate additional signalling cascades including phosphorylation by GRKs and recruitment of b-arrestins (Penela, 2010; Nogués, 2018).

a) G proteins

CXCR4 is known to couple to the pertussis toxin sensitive Gai protein family that mediate most of its signalling pathways (Busillo and Benovic, 2007). The coupling of CXCR4 to other G proteins such as Ga13 (Kumar, 2011) and Gaq (Soede, 2001) has been described but studied to a lesser extent.

As an atypical chemokine receptor, ACKR3 is missing the DRYLAIV (Asp-Arg-Tyr-Leu-Ala-Ile-Val) motif necessary for interaction with G proteins. Nevertheless, some reports have shown the interaction of the receptor with G proteins (Levoye, 2009; Ulvmar, 2011). ACKR3

was shown to form a heterodimer with CXCR4 in transfected cell lines (Levoye, 2009).

Furthermore, the organisation of CXCR4 and ACKR3 in heterodimers appears to induce a down regulating effect on the CXCR4 interaction with G proteins, shifting the signalling to barrestin mediated pathways.

b) GRKs

Agonist-occupied GPCRs are specifically phosphorylated by different GRKs, a family of 7 serine/threonine kinases (Ribas., 2007; Petronila Penela, 2010). GRK 2, 3, 5 and 6 phosphorylate CXCR4 in the C-terminus, which contains 15 serine and 3 threonine residues that are potential phosphorylation sites (Figure 1). At least six of these residues were shown to be phosphorylated following receptor activation by CXCL12 (Busillo, 2010; Barker and Benovic, 2011; Mueller, 2013). In human embryonic kidney 293 cells (HEK293) cells, Ser 321 , Ser 324 , Ser 325 , Ser 330 , Ser 339 , and two sites between Ser 346 and Ser 352 were shown to be phosphorylated in response to CXCL12 in the CXCR4 C-terminus using LC-MS/MS and ! phosphosite-specific antibodies (Busillo, 2010). GRK6 is able to phosphorylate Ser 324/5 , Ser 339 and Ser 330 , the latter with slower kinetics, whereas GRK2 and GRK3 phosphorylate residues between Ser 346 and Ser 352 (Figure 2) (Busillo, 2010), and specifically Ser 346/347 (Mueller, 2013). Interestingly, the latter study suggested a hierarchy in such phosphorylation events, since Ser 346/347 phosphorylation is achieved faster and is needed for the subsequent phosphorylation of Ser 338/339 and Ser 324/325 . Notably, ligand washout resulted in rapid Ser 324/325 and Ser 338/339 de-phosphorylation, whereas Ser 346/347 residues did not exhibit major dephosphorylation during the 60-minute period studied (Mueller, 2013) (Mueller, 2013). In this regard, a subpopulation of patients affected by WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome, a rare primary immunodeficiency disease, display C-terminally truncated CXCR4, leading to refractoriness to desensitisation and enhanced signalling (Balabanian, 2005). On the contrary, increased CXCR4 phosphorylation at Ser 339 is associated with poor survival in adults with B-acute lymphoblastic leukaemia and correlates with poor prognosis in acute myeloid leukaemia patients (Konoplev, 2011; Brault, 2014). Altered GRK expression/activity can also impair CXCR4 phosphorylation patterns. GRK3 suppression may contribute to abnormally sustained CXCR4 signalling in classical types of glioblastomas (Woerner, 2012), some WHIM patients (Balabanian, 2008) and in triple negative breast cancer, thus potentiating CXCR4-dependent migration, invasion and metastasis (Billard, 2016; Nogués, 2018). It is interesting to note that, although GRK2 and 3 share a high homology and are able to phosphorylate the same residues in CXCR4 in model cells, their function is not redundant. Whereas both CXCR4 and GRK2 levels are increased in breast cancer patients, GRK3 is decreased, suggesting a differential role for both GRKs in a cancer context (Billard, 2016; Nogués, 2018). In fact, deregulation of GRK2 potentiates several malignant features of ! breast cancer cells, and its level positively correlates with tumour growth and increased metastasis occurrence (Nogués, 2016), but whether these roles involve changes in CXCR4 modulation is still under investigation. On the other hand, impaired chemotaxis of T and B cells towards CXCL12 is noted in the absence of GRK6, whilst GRK6 deficiency potentiates neutrophil chemotactic response to CXCL12 (Fong, 2002; Vroon, 2004), suggesting that the occurrence of highly cell-type specific mechanisms in the control of the CXCL12-CXCR4-GRK6 axis. Overall, these data indicate the complexity of CXCR4 modulation by GRKs and support the need for a better characterization of cell type or disease-specific CXCR4-GRKs interactions.

ACKR3 has lately been the focus of many studies, in particular because of its role in cancer progression and metastasis. However, the mechanisms underlying its regulation are still not well understood, although this receptor has been shown to interact with GRKs and arrestins and to associate with other partners. The C-terminus of ACKR3 contains five serine and four threonine residues that can potentially be phosphorylated (Figure 2). Contrary to CXCR4, little is known about their actual phosphorylation status during the activation of the receptor, as no mass spectrometry data is available to date and only few mutational studies have been conducted (Canals, 2012; Hoffmann, 2012). In fact, only one study conducted in astrocytes showed that ACKR3 is phosphorylated by GRK2, but not other GRKs, and that this phosphorylation is essential for subsequent ACKR3-operated activation of ERK1/2 and AKT pathways (Lipfert, 2013). This study suggests that ACKR3 is indeed phosphorylated by

GRKs, but the isoform(s) involved and subsequent responses are likely cell type-dependent and remain to be investigated in detail.

c) Arrestins

GRK-modified GPCRs in turn recruit b-arrestins, an event that attenuates G protein-mediated signalling and promotes receptor internalisation (Smith and Rajagopal, 2016). Moreover, barrestins are scaffold proteins for several signalling mediators, thus eliciting additional GPCR signalling pathways (Shenoy and Lefkowitz, 2011; Peterson and Luttrell, 2017).

Since in HEK293 cells b-arrestin recruitment and association with CXCR4 seems to be driven by phosphorylation of the C-terminal residues, this long-term phosphorylation may be a key event in promoting the formation and maintenance of stable CXCR4/b-arrestin complexes (Oakley, 2000; Busillo, 2010; Mueller, 2013).

Upon activation by its cognate ligands CXCL11 and CXCL12, ACKR3 recruits b-arrestin-2

both in vitro (Rajagopal, 2010; Benredjem, 2016) and in vivo (Luker, 2009), a process leading to receptor internalisation (Canals et al., 2012) and degradation of both ACKR3 and ! receptor-bound chemokine (Hoffmann, 2012). Systematic mutation of C-terminal serine/threonine residues to alanine abolishes ligand-induced b-arrestin2 recruitment to ACKR3, as monitored by BRET (Canals, 2012), and decreases ACKR3 internalisation and subsequent degradation of radiolabelled CXCL12 in HEK293 cells (Hoffmann, 2012).

Selective mutations of the two C-terminal serine/threonine clusters to alanine revealed differences in their functional properties. Mutating Ser 335 , Thr 338 and Thr 341 (first cluster) or Ser 350 , Thr 352 and Ser 355 (second cluster) to alanine decreased CXCL12 internalization only after a 5-min challenge but not following longer agonist receptor stimulation, but only mutation of the second cluster prevented CXCL12 degradation. In addition, ACKR3 appears to undergo ligand-independent internalisation to a much greater extent than CXCR4 (Ray, 2012), and residues 339-362 (the two serine/threonine clusters) are essential for this peculiar cell fate in HEK293 cells.

Association of CXCR4 with non-canonical GPCR interacting proteins

Functional interaction of CXCR4 with second messenger-dependent kinases and receptor tyrosine kinases

Accumulating evidence indicates that phosphorylation of CXCR4 by second messengerdependent kinases such as protein kinase A and protein kinase C (PKC) (Lefkowitz, 1993; Ferguson, Barak,, 1996; Ferguson, Downey,, 1996; Krupnick and Benovic, 1998) as well as modulation by members of the receptor tyrosine kinase family (Woerner, 2005) participate in the regulation of CXCL12 signalling via CXCR4. CXCR4 is phosphorylated by PKC at Ser 324/5 upon CXCL12 stimulation (Busillo, 2010), and this kinase has also been involved in Ser 346/7 phosphorylation (Luo, 2017), although these results are not entirely consistent with a previous study using different PKC inhibitors (Mueller et al, 2013). In some glioblastoma cell types, CXCR4 is phosphorylated at Ser 339 in response to the PKC activator Phorbol myristate acetate. This suggests that Ser 339 is also a PKC phosphorylation site (Woerner, 2005; Busillo and Benovic, 2007), but the functional impact of such modifications remains to be fully established. Epidermal growth factor (EGF) through activation of its receptor can also promote CXCR4 phosphorylation at Ser 339 in glioblastoma cells (Woerner, 2005), and both EGF and heregulin trigger Ser 324/325 and Ser 330 phosphorylation in the breast cancer T47D cell line. Interestingly, in MCF7 breast cancer cells, heregulin also promotes CXCR4 phosphorylation on tyrosine residues via Epidermal Growth Factor Receptor (EGFR), leading to b arrestin2 association with CXCR4 and downstream activation of the PRex1/Rac1 axis.

However, it is still unclear whether the EGFR-CXCR4 functional interaction is direct or depends on other kinases (Sosa, 2010). In another breast cancer line, BT-474, CXCR4 is ! phosphorylated on tyrosine residues in response to CXCL12, but specific residues or kinases responsible were not described (Sosa, 2010). The crosstalk between CXCR4 and EGFR remains an interesting avenue for future research, given the involvement of both receptors in cancer.

Physical interaction with non-canonical GPCR interacting proteins

Beside canonical GIPs, CXCR4 has been shown to interact with additional proteins that modulate CXCR4 trafficking, subcellular localisation and signalling and proteins whose functions are still unknown. CXCR4 interacting proteins, the methods used for the identification of these proteins, the site of their interaction in the receptor sequence and their functional impact are summarised in Table 2.

a) Proteins controlling CXCR4 localization or trafficking

Filamin A directly interacts with CXCR4 and stabilises the receptor at the plasma membrane by blocking its endocytosis (Gómez-Moutón, 2015). CXCR4 association with the E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) has opposite consequences: ubiquitination of CXCR4 by AIP4 targets the receptor to multi-vesicular bodies, which is followed by receptor degradation. In addition, agonist treatment increases CXCR4/AIP4 interaction, as assessed by Co-IP and FRET experiments (Bhandari, 2009), indicating that this interaction is dynamically regulated by a receptor conformational state. Reticulon-3 is another CXCR4 interacting protein that promotes its translocation to the cytoplasm (Li, 2016).

b) Proteins modulating CXCR4 signalling and functions

CD74, a single-pass type II membrane protein that shares with CXCR4 the ability to bind to the macrophage migration inhibitory factor (MIF), was also shown to interact with CXCR4.

The CXCR4/CD74 complex is involved in AKT activation (Schwartz, 2009). In fact, blocking either CXCR4 or CD74 inhibits MIF-induced AKT activation. Using FRET, an interaction between CXCR4 and the toll like receptor 2 was observed in human monocytes upon activation by Pg-fimbria (fimbriae produced by the major pathogen associated with periodontitis named Porphyromonas gingivalis). Analysis of a possible crosstalk between the two receptors showed that Pg-fimbria, directly binds to CXCR4 and inhibits toll like receptor 2-induced NF-kB activation by P. gingivali (Hajishengallis, 2008; Triantafilou, 2008). In Jurkat cells, CD164 co-precipitates with CXCR4 in the presence of CXCL12 presented on fibronectin (Forde, 2007). CXCR4-CD164 interaction participates in CXCL12-induced activation of AKT and protein kinase C zeta (PKCζ). In fact, the down-regulation of CD164 reduces the activation of both kinases measured upon exposure of Jurkat cells to CXCL12. CXCR4/CD164 interaction has been detected in additional cell ! lines, such as primary human ovarian surface epithelial cells stably expressing CD164 (Huang, 2013).

The ability of CXCR4 to promote cell migration requires deep cytoskeletal rearrangements that can be modulated by CXCR4 interacting proteins. In Jurkat J77 cells, CXCR4 constitutively associates with drebrin (Pérez-Martínez, 2010), a protein known to bind to Factin and stabilise actin filaments. Drebrin is also involved in CXCR4-and CD4-dependent HIV cellular penetration (Gordón-Alonso, 2013). CXCR4 interacts with diaphanous-related formin-2 (mDIA2). This interaction induces cytoskeletal rearrangements that lead to nonapoptotic blebbing. mDIA2-CXCR4 interaction is only detected during non-apoptotic amoeboid blebbing and is confined to non-apoptotic blebs upon CXCL12 stimulation (Wyse, 2017), suggesting a fine spatio-temporal regulation of the interaction. CXCR4 also constitutively associates with the motor protein non-muscle myosin H chain (NMMHC) via its C-terminus (Rey, 2002). The authors showed that NMMHC and CXCR4 are co-localised in the leading edge of migrating lymphocytes, suggesting that this association might have a role in lymphocyte migration. The PI3-kinase isoform p110g co-precipitates with CXCR4 in CXCL12-stimulated human myeloid cells. This interaction contributes to receptor-operated integrin activation and chemotaxis of myeloid cells (Schmid, 2011). Finally, CXCR4 was found to be part of a junctional mechano-sensitive complex through its interaction with the platelet endothelial cell adhesion molecule (PECAM-1) (Dela Paz, 2014).

c) Proteins with unknown functions

Other potential CXCR4-interacting proteins have been identified using unbiased methods.

These include the lysosomal protein ATP13A2 (Usenovic, 2012) and the nuclear protein Myb-related protein B that is involved in cell cycle progression (Wang, 2014). In a study aimed at characterising the human interactome by Co-IP of 1,125 GFP-tagged proteins and LC-MS/MS analysis, CXCR4 was found to co-precipitate with the potassium channel subfamily K member 1, the CSC1-like protein 2 and the vesicle transport protein GOT1B (Hein, 2015).

CXCR4-interacting proteins identified in HEK293 cells by AP-MS

To get a global overview of CXCR4 interacting proteins, we transiently expressed hemagglutinin (HA)-tagged CXCR4 in HEK293 cells, immunoprecipitated the receptorassociated complex with an agarose bead-conjugated anti-HA antibody and systematically identified co-immunoprecipitated proteins by LC-MS/MS. These studies identified 79 proteins that co-immunoprecipitate with the receptor and are not detected in control ! shown to be down-regulated by CXCR4 1013 (Meuris, 2016), a CXCR4 WHIM variant. The CXCR4-associated proteins identified also include COPS5, a protein of the COP9 signalosome complex that interact with casein kinase 2 and protein kinase D both involved in p53 phosphorylation (Uhle, 2003). COPS5 is also part of the MIF-CXCR4 axis, that is responsible for the regulation of gene transcription involved in adhesion in multiple myeloma (Zheng, 2016). In addition to protein kinases, the CXCR4 interactome included protein phosphatase 6 (PP6) that is necessary for DNA repair (Zhong, 2011) and is known to negatively regulate NF-kB (Ziembik, 2017). Active NF-kB directly binds to CXCR4 promoter and regulates CXCR4 expression affecting CXCL12-mediated migration of cells (Helbig, 2003).

Association of ACKR3 with non-canonical GPCR interacting proteins

Contrary to CXCR4, only few proteins are described as ACKR3 interacting proteins. Given the described role of ACKR3 in cancer, several studies have addressed ACKR3 crosstalk with well-known pro-oncogenic growth factor receptors. ACKR3 co-localises with and phosphorylates EGFR in breast and prostate cancer cells (Singh and Lokeshwar, 2011; Salazar, 2014; Kallifatidis, 2016), via cell-type specific mechanisms. However, a potential role of EGFR in ACKR3 cross-activation was not assessed in these studies. Some reports also suggest a possible functional interaction between ACKR3 and Transforming growth factor beta (TGF-b) (Rath, 2015) or Vascular endothelial growth factor (VEGF) (Singh and Lokeshwar, 2011) receptors, but whether they involve physical interaction with ACKR3 and/or ACKR3 phosphorylation and activation was not assessed. ACKR3 weakly interacts with the MIF receptor CD74 (Alampour-Rajabi, 2015). Moreover, ACKR3 co-localizes with PECAM-1, the cell adhesion molecule required for leukocyte transendothelial migration in human coronary artery endothelial cells (Dela Paz, 2014). Using a Membrane Yeast two Hybrid assay screen, ATP13A2 was identified as a putative ACKR3 interacting protein (Usenovic, 2012). In the study aimed at characterizing the human interactome of 1,125 GFPtagged proteins, ACKR3 was found to interact with the gap junction beta-2 protein (GJB2), the 54S ribosomal protein L4, mitochondrial MRPL4, different ATP synthases (ATP5H, ATP5B, ATP5A1, ATP50), ACKR3 itself, the caspase Separin ESPL1, the probable E3 ubiquitin-protein ligase HECTD2 and the Putative E3 ubiquitin-protein ligase UBR7 (Hein, 2015). Ubiquitination is an essential mechanism of receptor regulation (Marchese and Benovic, 2001; Shenoy, 2007). ACKR3 can undergo ubiquitination in an agonist-dependent and independent manner, regulating receptor trafficking. Ubiquitination is promoted by three enzymes, E1 E2 and E3 that ubiquitinate proteins on lysine residues (Dores and Trejo, 2012;

! Alonso and Friedman, 2013). Unexpectedly, ACKR3 is ubiquitinated by E3-ubiquitin ligase (E3) in the absence of an agonist and undergoes deubiquitination upon CXCL12 activation (Canals, 2012). Mutation of the five lysines in the receptor C-terminus to alanine, to prevent ubiquitination, impaired ACKR3 cell trafficking and decreased ACKR3-mediated CXCL12 degradation (Hoffmann, 2012).

Conclusions

The identification of GPCR-interacting proteins and residues subjected to post-translational modification is of utmost importance. Loss of spatial-temporal information.

Membrane anchored proteins cannot be investigated. Performed in yeast.

MYTH

Highly suitable

Easy to perform. Membrane anchored proteins can be investigated.

Loss of spatial-temporal information. Soluble proteins cannot be investigated. Performed in yeast.

MaMTH

Highly suitable

Easy to perform. Membrane anchored proteins can be investigated.

Performed in mammalian cells

Loss of spatial-temporal information. Soluble proteins cannot be investigated.

KISS Possible

Sensitive enough for studying interaction dynamic.

Both membrane and cytosolic proteins can be investigated.

Loss of spatial-temporal information Proteins involved in the STAT3 cascade cannot be investigated.

2.Biophysical

BRET/FRET Not suitable.

Precise spatial-temporal information. High sensitivity.

Possibility to study interactions in living cells.

Generation of fusion proteins.

Relies on the proximity and relative orientation between donor and acceptor.

PLA

Not suitable.

Precise spatial information (single molecule resolution). Possibility to perform in ex-vivo models.

Relies on antibodies. High cost. Not easy to scale up in large studies

BioID Suitable

Precise spatial information. Several interactions in parallel. Possibility to perform in living cells.

Not well suited for studying interaction dynamic (fluorescent signal is delayed).

!

Methods for the identification of GPCR-interacting proteins (1 to 3) and phosphorylated residues (4) Classification Method Screening Advantages Disadvantages

3.Proteomic

Co-IP Highly suitable Purification of protein complexes in living cells and tissues.

Rely on antibodies.

Loss of spatial-temporal information.

Lysis conditions might influence results.

Pull-down

Highly suitable Can prove direct interaction.

Loss of spatial-temporal information.

In vitro binding assays. Fusion of the receptor on the beads might alter receptor conformation.

BioID

Highly suitable Can detect weak and transient interactions in living cells.

Fusion of the biotin to the receptor might alter its targeting or functions.

4.Phosphorylation [ 32 P]

Suitable Very sensitive.

Radioactive method. Cannot give information on the number of phosphorylated residues nor their position.

LC-MS

Highly suitable Can pinpoint phosphorylated residues.

Can yield false negatives. Not quantitative unless combined with very expensive isotope tags. Hemagglutinin (HA)-tagged CXCR4 was transiently expressed in HEK293T cells. We then immunoprecipitated the receptor-associated complex with an agarose bead-conjugated anti-HA antibody and co-immunoprecipitated proteins were systematically identified by nano-flow liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Proteins identified at MS level in the CXCR4 co-precipitating complex in all three triplicated and absent in the three controls (Co-IP performed in HEK293T cells transfected with empty plasmid) are here reported. Protein name, gene name, Uniprot ID, normalized MS/MS count and average iBAQ are indicated. The normalized MS/MS was calculated dividing the average MS/MS count of the triplicate for the protein length ! !

Mutagenesis

  Le récepteur atypique ACKR3 et le récepteur CXCR4 sont des récepteurs couplés aux protéines G appartenant à la famille des récepteurs CXC des chimiokines. Ces deux récepteurs sont activés par la chimiokine CXCL12 et sont surexprimés dans de nombreux cancers comme les gliomes, dont ils favorisent la prolifération et le caractère invasif. Le récepteur CXCR4 active des voies de signalisation qui dépendent de la protéine Gi et des barrestines et s'associe à plusieurs protéines impliquées dans la transduction du signal, le trafic et la localisation cellulaire du récepteur. Par contre, les mécanismes de signalisation impliqués dans les effets d'ACKR3 restent mal connus. Le récepteur déclenche une signalisation dépendant des b-arrestines, mais son couplage aux protéines G dépend du type cellulaire ou se fait par un mécanisme indirect via son association au récepteur CXCR4. Le

  PHARMACOLOGY: BALANCED VS. BIASED AGONISM

Figure 2

 2 Figure 2 Balanced vs. biased agonism. "Balanced" agonists have equal efficacy to available downstream pathways associated with the GPCR activation. On the other hand, "biased" agonists preferentially activate only specific pathways, β-arrestin-dependent pathways in this case.

Figure 3

 3 Figure 3 Two binding site model. Site 1 present in the N-terminal domain of the receptor is needed for the binding of the chemokine to its receptor. After binding the receptor N-terminal wrap the chemokine. The flexible N-terminal domain of the chemokine (site 2) is then free to activate the receptor.

  and, finally, sustains T cell priming 102 . CXCR4 involvement in severe diseases including immunodeficiencies, autoimmunity and cancer has been largely confirmed. During HIV infection, CXCR4 functions as a co-receptor (together with CD4) for viral entry into T cells 103 . An heterozygous mutation of CXCR4, consisting in the truncation of the C-terminal domain and leading to a gain of function of the receptor, was identified as the genetic basis of the Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome 104 .

Figure 5

 5 Figure 5 ACKR3 signals trough β-arrestins and GRKs. It is well recognized that ACKR3 is able to interact with GRKs and β-arrestins leading to increase phosphorylation status or altered expression level of downstream proteins involved in the control of migration or cell proliferation.

  conserved extracellular and one cytoplasmic loops, four transmembrane domain and cytoplasmic N-and C-terminal domains 223 (Figure! 6). Six connexins

Figure 6 Figure 8

 68 Figure 6 General Cx schematic topology.

Figure 7

 7 Figure 7 Homomeric vs. heteromeric connexins. In blue and orange two different Cx subtypes are illustrated.

Figure 8 36 !Figure 9

 8369 Figure 8 Mechanisms of Cx closure. (A) Subunit rotation model. (B) Plu-gating model. (C) Loopgating model. (D) Particle-receptor model.

38 !

 38 clamped at a potential close to the average resting membrane potential (source VA and VB). A junctional current (Ij) is triggered by applying a step-wise increasing transjunctional voltage in one of the two cells (referred as cell C1 in the Figure 10). If the two cells are electrically coupled, a current (I2) is recorded in cell C2. It is of ! equal magnitude but opposite sign of the junctional current (Ij), in order to maintain constant the cell potential. The junctional conductance (Gj) can be calculated by simply dividing the junctional current by the amplitude of the voltage step applied to C1 247 . The main advantage of this technique is its exquisite sensitivity, which even allows the recording of activity down to a single channel level 248 . The main disadvantage is that analysis of electrical conductance is a time and labor-intensive technique.

Figure 10 39 !

 1039 Figure 10 Schematic representation of double whole-cell voltage clamp. VA & VB = source, I1 & I2 = electric current, C1 & C2 = cellule, Ij = junctional current.

! 40 !

 40 Three other techniques employing a dye for the investigation of gap junctional activity are the Gap-FRAP, the preloading assay and the LAMP (Local Activation of Molecular Fluorescent Probe) (Figure12). For all these assays cells are preloaded with the tracer.A. In the former, cells forming a confluent monolayer are equally loaded with the tracer. A cell is then photobleached by a laser. After photobleaching, the tracer of a non-bleached neighbouring cell diffuses into the bleached one through Gap junctions. Monitoring the redistribution of the tracer as a function of time will give the gap junctional activity of the cells 256 . B. In the preloading assay, different from the Gap-FRAP where all cells are equally preloaded with the tracer, only a portion of cells are preloaded with the tracer 257 . These cells are then plated with unloaded cells. Again the passage of tracer from pre-loaded to unloaded cells is used as surrogate for quantifying gap junctional activity. This method is well suited for studying gap junctional communication between homogenous cell populations.C. The LAMP is the most recent technique using a specific type of tracer defined as caged tracers 258 . These tracers have the characteristic to become fluorescent only upon irradiation with an UV beam. For this experiment cells are equally loaded with a caged tracer. Using an UV lamp the cage is removed and the tracer becomes fluorescent. The diffusion of the tracer is

Figure 11

 11 Figure 11 Tracer-based methods for the study of Cx activity. (A) Scrape loading method. (B) Microinjection. (C) Electroporation where the conductive surface is represented in dark grey.

Figure 12

 12 Figure 12 Preloaded tracer-based methods for the study of Cx activity. (A) GAP-FRAP method. (B) Pre-loading assay. (C) LAMP method.

  expression and Src kinase activity are regulated in a variety of cancer types. Src is activated by a multitude of mechanisms including interactions with receptor tyrosine kinases and integrin receptors 265 . As it will be discussed later, Src interacts with and phosphorylates Cx43, disrupting its interaction with ZO-1 and triggering Cx43 internalisation 266 .

Figure 13

 13 Figure 13 Cx43 amino acid sequence and schematic topology. The sequences used for the generation of the mimetic peptides are highlighted

45 !Figure 14

 4514 Figure 14 Schematic representation of Cx43 trafficking. Cx43 is synthesized as monomer. Hexameric channels are formed in Trans-Golgi apparatus. After oligomerization, Cx43 hemichannels are transported to the membrane with either a targeted localization (in proximity of tight junctions) or a random one. Cx43 hemichannels are free to diffuse along the cellular membrane. Clusters of Cx43 gap junctions form Cx43 plaques. Cx43 are constantly internalised and synthetized. Internally translated Cx43 also participates in the trafficking of Cx43 hemichannels.

Figure 15 52 !

 1552 Figure 15 Role of Cx43 in the regulation of BBB permeability and leukocyte entry into the brain parenchyma. The physiological condition is represented on the left hand (correct Aquaporin4 expression, intact DAPC complex and well formed tight junctions). Suppression of Cx43 expression leads to the suppression of Aquaporin4 and the disruption of the DAPC complex (right hand). In addition Cx43 suppression also causes an increase of CXCL12. These effects increase BBB permeability and leukocyte entry into the brain.

Figure 16

 16 Figure 16 Neuronal progenitor cell (NPC) migration in presence or absence of Cx43. On the left hand the correct migration of NPC (green) along radial glial cells (blue) is represented. Suppression of Cx43 expression, represented on the right hand, impairs migration of NPCs that do not reach the Cortical Plate (CP). In addition NPCs are randomly oriented and exhibit several, multidirectional processes.

  for invasion, the role of Cx43 in migration strongly depends on the type of cells that are coupled. In fact, inhibition of Gap junctional communication between glioma cells increase motility, whereas the inhibition of Gap junctional communication between glioma cells and surrounding astrocytes had opposite effects 387 . In addition, down-regulation of Gap junctional communication between glioma cells change the migratory pattern from collective to single cell migration 385

BOX 7

 7 COLLECTIVE MIGRATION Collective migration is the coordinate movement of cell groups 383 . This process relies on the ability of cells to integrate signals from the neighbouring ones. This cell-cell communication is mediated by several proteins including chemokine and their receptors 384 as well as connexins 385 . Collective migration is crucial for organ development and shaping as well as wound healing. It also plays a pivotal role in tumour progression 386 . This contrasts with single cell migration that occurs independently of the surrounding cells. ! 55!

  THE ACKR3 INTERACTOME IN HEK-293T CELLS BY AP-MS.

! 58 !

 58 Among the 151 ACKR3 interacting proteins, we also identified Gap Junction Alpha-1 protein (GJA1, also called Connexin 43 -Cx43), one of the proteins involved in Gap Junctional Intercellular Communication (GJIC) connecting two adjacent cells.Increasing evidence has been showing that numerous proteins that are physically or functionally connected with Cx43 regulate GJIC. Among those proteins, Dynactin (DCTN1)288 and the Desmosomal cadherin desmoglein 2 (DSG2)398 were shown to affect the localization of Cx43. Ubiquillin-4 (UBQLN4) 286,399 interacts and promotes the degradation of Cx43. Cytochrome P450 oxidoreductase (CYPOR) 400 downregulation triggers transcriptional repression and inhibition of Cx43. The solute carrier family 1 member 5 (SLC1A5) 336 interacts with Cx43 to stimulate cytotrophoblast fusion, whereas the beta-subunit of the electron-transfer protein (ETFB) 401 interacts with Cx43 to regulate mitochondrial respiration and reactive oxygen species (ROS)

Figure 17 ACKR3

 17 Figure 17 ACKR3 interacting proteins identified in HEK-293T cells and their relative abundance in immunoprecipitates from ACKR3-expressing cells vs. mock cells. HA coimmunoprecipitation followed by nanoLC-MS/MS was performed in three distinct biological replicates in HEK-293T cells transiently expressing HA-tagged ACKR3 and cells transfected with empty plasmid (Mock). Log transformed intensities, obtained by Label Free Quantification (LFQ), of proteins identified in all three biological replicates in at least one set of cultured cells were than used for the comparison. The volcano plot was obtained plotting the differences of LFQ values between ACKR3 and mock cells (X axis) vs. -log of P value (Y axis). The upper part of the graph includes proteins with a high inter-replicate reproducibility whereas the right or left parts include plotted proteins with large differences in abundance between the two conditions. Specifically, on the right there are proteins enriched in the ACKR3 condition. The proteins were considered statistically significant using a T-test conducted on both sides setting the number of randomization at 250 the False Discovery Rate at 0.01 and the S0 at 0.1.Therefore, all proteins "above" the dotted lines are significantly enriched. In dark blue the bait (ACKR3) is represented. In light blue its known interacting partners are depicted. The proteins known to physically or functionally interact with GJA1 (Cx43) are highlighted in orange, whereas Cx43 is in red.

Figure 18

 18 and consistent with LC-MS/MS analyses, endogenously expressed Cx43 co-precipitates with ACKR3 in HEK-239T cells, but not in Mock cells. A much lower Cx43 amount was also detected in CXCR4 immunoprecipitate, but Cx43 abundance in CXCR4 immunoprecipitates was not significantly different from that measured in precipitates obtained from Mock cells (p = 0.081, n = 3) (Figure 18B).

Figure 18

 18 Figure 18 Cx43 co-immunoprecipitates with ACKR3. (A) Immunoprecipitation (IP) of HA-ACKR3 and HA-CXCR4 transiently expressed in HEK-293Tcells compared to Mock cells (transfected with an empty plasmid). (B) Cx-43 co-immunoprecipitation (Co-IP) with HA-ACKR3 vs. HA-CXCR4 and Mock cells. In both (A) and (B) representative blots of the three independent replicates are shown. (C) Average Input and Co-IP Cx43 immunoreactive signal quantified in three independent experiments (± SEM). Values were normalized to those measured in ACKR3 precipitates. Two-way Anova with Turkey's post-hoc test was used (**** P≤0.0001).

Figure 19 BRETFigure 21

 1921 Figure 19 BRET analysis of Cx43 interaction with ACKR3 and CXCR4 in living HEK-293T cells. (A) Titration curves of the Cx43-YFP-ACKR3-NLuc and Cx43-YFP-CXCR4-NLuc interaction in HEK-293T cells. Three biological replicates per conditions are plotted. Points represent the average of the technical triplicates within a biological replicate. BRET values were normalized to the maximum BRET obtained in each replicate. Prism was let decided between fitting a One-site total line with background constraint to 0 and a line through origin. P value of fitting for a One-site total line for ACKR3 is < 0.001. The same curve did not fit for CXCR4. On the other hand, the line trough the origin fits the CXCR4 point with an R 2 =0.926. (B) Quantification of the BRET signal between YFP-Cx43 and NLuc ACKR3 expressed in HEK-293T cells with and without ACKR3 activation by CXCL12 (10 -8 M). The YFP/NLuc ratio used was the one giving the BRET50 in saturation curve. The histogram represents averages of three biologically independent replicates (± SEM). Unpaired t-test was used for the difference.

Figure 20 ACKR3

 20 Figure 20 ACKR3 and Cx43 co-expression in astrocytes in the sub-ventricular zone and surrounding blood vessels. Confocal images of 50 μM brain slices obtained from 8 week-old BAC-EGFP-ACKR3 mice. EGFP signal was amplified using an anti-GFP antibody. Scale bar = 100 μm.

!Figure 22 ACKR3 71 !Figure 23

 227123 Figure 22 ACKR3 activation inhibits Cx43-mediated GJIC through β-arrestin2-dependent Cx43 internalisation in primary cultures of mouse astrocytes. (A) Representative pictures of the scrape loading assay, performed in confluent primary astrocytes culture obtained from embryonic WT or β-arrestin2 -/-mice (E15.5), taken ten minutes after the scrape in the presence of LY. All compounds (CXCL12 (10 -8 M), AMD3100 (10 -6 M), CXCL11 (10 -7 M), NB-74330 (10 -6 M), Dyn=Dynasore (80 μM)) were applied for 30min but CBX (50μM) was applied overnight. Scale Bar = 100 μm. (B) and (C) Quantification of the LY diffusion by calculation of the distance from the scrape where LY intensity is halved. Values were normalized to LY diffusion in vehicle-treated astrocytes (Control). Average values (± SEM) from at least three independent experiments performed in triplicate are represented. For comparison with control (indicated by *) one-way Anova with Dunnett's post-hoc test was used. For the other comparisons the one-way Anova with Sidak's post-hoc test was used (****P≤0.001, ***P≤0.001, * P≤0.05).

Figure 24 CXCL12 73 !

 2473 Figure 24 CXCL12 increases membrane resistance. Coupled astrocytes were challenged with either a saline solution, or CXCL12 (10 -8 M), or CBX (100 μM). Resistance values of each cell measured before and after treatment are plotted. Paired Wilcoxon test was used for the analysis *P≤0.05, **P≤0.01 vs. before treatment.

Figure 25 CXCL12

 25 Figure 25 CXCL12 reduces the electrical coupling between secondary mouse astrocytes. (A) Time-course of the effect of CXCL12 (10 -8 M), CBX (100μM) or saline on the coupling ratio. Depolarizing voltage steps (50 mV) were applied every 30 s to cell 1 and coupling ratio was computed as Ij/I1. Perfusion of the recorded pairs with the drug started at the time indicated by the dotted line. Data are expressed as a percentage of coupling ratio (normalized to the average ratio recorded during the first 4 minutes). Values are the average ± SEM of six pairs. (4)=**** P≤0.0001, (3)=*** P≤0.001, (2)=** P≤0.01, (1)=* P≤0.05 vs. saline. Two-way Anova with Tukey's post-hoc test was used for the point-to-point comparison. (B) Coupling ratios before and after treatment for each cell pair recorded in the different conditions are plotted. Paired Wilcoxon test was used for the analysis *P≤0.05 vs. before treatment.

! 75 !

 75 Both inhibition of dynamin by Dynasore and β-arrestin2 suppression prevented the ability of CXCL12 and CXCL11 to promote Cx43 internalisation. In fact, in both conditions Cx43 was mainly detected at the plasma membrane even in CXCL12-and CXCL11-treated cultures (Figure26). Therefore ACKR3 activation by CXCL12 or CXCL11 inhibits GJIC by triggering dynamin and β-arrestin2-dependent Cx43 internalisation in primary culture of mouse astrocytes.

Figure 26

 26 Figure 26 Agonist stimulation of ACKR3 promotes Cx43 internalisation in primary astrocyte cultures. Representative confocal pictures of confluent primary astrocytes cultures obtained from embryonic WT or β-arrestin2-/mice (E15.5) are illustrated. Cx43 is stained in red. Astrocytes were exposed to either vehicle or CXCL12 (10-8 M) or CXCL11 (10-7 M) in absence or presence of Dynasore (Dyn, 80 μM) for 30 min. Scale bar = 10 μm.

Figure 27 ACKR3

 27 Figure 27 ACKR3 activation increases membrane expression of Cx43 hemichannels.Representative blots of the total and surface Cx43 hemichannel expression in control and either CXCL12(10-8 M) or CXCL11 (10-7 M) treated (30 min) confluent primary astrocytes culture obtained from embryonic mice (E15.5).

Figure 28 ACKR3

 28 Figure 28 ACKR3 activation by CXCL12 and CXCL11 inhibits Cx43 hemichannel internalisation. (A) Representative blots of the total and internalised Cx43 hemichannel expression in astrocytes kept at 4°C (minimal internalisation) at 37°C (control) or exposed to either CXCL12 (10-8 M) or CXCL11 (10-7 M) for 30 min. (B) Quantification of Cx43 chemiluminescent signal of three biological independent experiments. Average values ± SEM are plotted. Values were normalized to the signal obtained at 37°C. Two-way Anova with Sidak's post-hoc test was used for the analysis comparing values to the 37°C condition. **** P≤0.0001, *** P≤0.01.

Figure 29

 29 Figure 29 Dynasore impairs CXCL12-mediated inhibition of Cx43 hemichannel internalisation. (A) Representative blots of the total and internalised Cx43 hemichannel expression in astrocytes treated with Dynasore (80μM) and kept at 4°C at 37°C (control) or exposed to CXCL12 (10 -8 M) for 30 min. (B) Quantification of Cx43 chemiluminescent signal of three biological independent experiments. Average and SEM are plotted. Values were normalized comparing the signal to the one obtained at 37°C. Two way Anova with Sidak's post-hoc test was used for the analysis comparing values to the 37°C condition.

Figure 30 ACKR3

 30 Figure 30 ACKR3 but not CXCR4 co-localizes with Cx43 in a glioma initiating cell line isolated from human glioblastoma. (A) Representative 3D reconstruction of the signal obtained from confocal images of R633 cells using the Imaris software. The overlapping volume has been calculated as described in the material and methods section. (B) Quantification of the % of Cx43 intensities present in the co-localizing volume in each cell type. Average values collected from at least four cells (50 z-stacks for cell) are plotted. (C) Quantification of the % of receptor (ACKR3 or CXCR4) intensities present in the co-localizing volume in each cell type. Average values collected from at least four cells (50 z-stacks for cell) are plotted ±SEM. Two-way Anova with the Sidak's posthoc test were used for statistics (**** P≤0.0001, ** P≤0.001, * P≤0.05). Scale bar = 3μm.

  7 cells when CXCR4transfected cells were microinjected (n = 10), indicating that CXCR4 expression does not affect GJIC (p=0.24 vs. mock). On the other hand, LY diffused into 2 ± 0.43 cells from ACKR3 transfected cells (n = 19). Likewise, Carbenexolone (80 μM) strongly reduced LY diffusion from microinjected, empty vector-transfected cells (0.2 ± 0.2 LY-stained cells, n = 5). Collectively, these results indicate that ACKR3 expression constitutively inhibits GJIC to a comparable extend as Carbenexolone (CBX), a GJIC chemical inhibitor in HEK-293T cells.

Figure 31 ACKR3

 31 Figure 31 ACKR3 expression inhibits GJIC in HEK-293T cells. (A) Representative pictures obtained after LY microinjection in single HEK-293T cells transiently expressing mCherry targeted to cell membrane (mock and CBX) as described in 439 , mCherry-tagged ACKR3 and mCherry CXCR4. Scale bar = 10 μm (B) Quantification of neighbouring cells receiving LY. Individual values and SD are represented. One-way Anova with Tukey's post-hoc test was used for the analysis. **** P≤0.0001 vs. mock, $$$$ P≤0.0001.

Figure 32 ACKR3 83 !

 3283 Figure 32 ACKR3 constitutively and ligand dependently internalises in HEK-293T cells. (A) Constitutive internalisation of SNAP-tagged ACKR3 and CXCR4 transiently expressed in HEK293T over time measured using a DERET assay. Average of three independent replicates ± SEM are plotted. Values were normalized to the maximal internalisation. (B) Representative pictures taken from HEK-293T cells transiently expressing HA-tagged ACKR3 and CXCR4 stained with anti-HA antibodies. (C) SNAP-ACKR3 internalisation upon CXCL12 stimulation in transfected HEK-293T cells. Average of three independent replicates ± SEM are plotted. Values were normalized to the ACKR3 internalisation at the highest CXCL12 concentration.

Figure 33

 33 Figure 33 Dynasore does not reverse ligand-independent inhibition of GJIC mediated by ACKR3 in HEK-293T cells. (A) Representative pictures obtained after LY microinjection in single HEK-293T cells transiently expressing mCherry targeted to cell membrane (mock) as described in 439 and mCherry tagged ACKR3 treated or not with dynasore (80 μM) for 30min. (B) Quantification of neighbouring cells receiving LY. Individual values and SD are represented. One-way Anova with Tukey's post-hoc test was used for the analysis. **** P≤0.0001 vs. mock, $$$$ P≤0.0001.

  is a major site mediating protein-protein interactions, we generated ACKR3 mutants partially or totally truncated of the C-terminal domains (ACKR3Δ16 and ACKR3ΔCt) and examined the ability of the truncated receptors to interact with Cx43 in HEK-293T cells by co-immunoprecipitation (Figure 34A & B). Both mutants retained unaltered ability to interact with Cx43 (p = 0.84, n = 3 and p = 0.34, n = 3 for ACKR3

Figure 34 Figure 35 Figure 36

 343536 Figure 34 The C-terminal domain of ACKR3 does not mediate its interaction with Cx43. (A) Immunoprecipitation (IP) of HA-ACKR3, HA-ACKR3Δ16, HA-ACKR3ΔCt and HA-CXCR4 transiently expressed in HEK-293Tcells compared to mock cells. (B) Cx43 co-immunoprecipitation in the same conditions as (A). In both (A) and (B) representative blots of the three independent replicates are shown. (C) Average Input and Co-IP Cx43 chemiluminescence quantified in three independent experiments (± SEM). Values were normalized comparing them to ACKR3. Two-way Anova with Tukey's post-hoc test was used for the comparisons. **** P≤0.0001 vs. ACKR3.

Figure 37 ACKR3

 37 Figure 37 ACKR3 activation by CXCL12 and 11 inhibits cAMP production in primary cultured astrocytes. Quantification of endogenous cAMP was performed after stimulation of cAMP production with the adenylyl cyclase activator forskolin (FSK, 10 -6 M) for 5 min and treatment of cells with CXCL12 or CXCL12 or saline solution for 5 min. In the case of AMD3100-treated cells, the antagonist (10 -6 M) was added for 30 min before the CXCL12 challenge. Values represented are the average of three independent replicates ± SEM. Two-way Anova with Dunnett's post-hoc test was used for the analysis. **** P≤0.0001, * P≤0.05 vs. Fsk

Figure 38 ACKR3

 38 Figure 38 ACKR3 constitutively interacts with Gαi3 in living HEK-293Tcells. (A) Titration curves of the ACKR3-YFP-Gαi3-RLuc interaction in HEK-293T cells. Values represented are the average of three independent replicates ± SEM. BRET values were normalized to the maximum BRET obtained in each replicate. Again, the one site total curve was fitted as described in Figure 3. (B) Quantification of the BRET signal between YFP-ACKR3 and RLuc-Gαi3 expressed in HEK-293T cells with and without ACKR3 activation by CXCL12 (10 -8 M) for 5 min and PTX (100 ng/ml, 18 h). YFP/RLuc ratio used was the one giving the BRET50 in saturation curve. Graph represents average of three biologically independent replicates (±SEM). Two-way Anova with Sidak's post-hoc test was used for the statistical analysis.

Figure 39 ACKR3 91 !

 3991 Figure 39 ACKR3 does not activate Gαi/o proteins in HEK-293T. (A) cAMP production in HEK-293T cells transiently transfected with empty plasmid (mock) or cDNAs encoding ACKR3 or CXCR4. cAMP production was stimulated by addition of forskolin (Fsk, 10-6 M) in cells pre-treated or not with PTX (100 ng/ml, 18 h). Values represented are the average of three independent replicates ± SEM. Two-way Anova with Tukey's post-hoc test was used for the analysis. **** P≤0.0001 vs. Fsk mock, $$ P≤0.01. (B) Quantification of neighbouring cells receiving LY in three independent biological replicates after microinjection of LY in HEK-293T cells, pre-treated or not overnight with PTX (100 ng/ml), expressing mCherry-tagged ACKR3 or mCherry targeted to cell membrane (Mock). Individual values and SD are represented. One-way Anova with Turkey's posthoc test was used for the analysis. **** P≤0.0001 vs. mock, $$$$ P≤0.0001.

Figure 40

 40 Figure 40 Only mouse CXCR4 but not ACKR3 activates G proteins in HEK-293T cells. HEK-293T transiently expressing RLluc-Gαi1 protein, Venus-γ2 and β2 alone (mock) or with mouse ACKR3 (mACKR) or mouse CXCR4 (mCXCR4) were used. G protein activation was quantified by measuring BRET decrease upon CXCL12 stimulation. Values represented the average of three independent biological replicates ± SEM. Two-way Anova with Dunnett's post-hoc test was used for the analysis *** P≤0.001, * P≤0.05 vs. staring BRET value in each condition.

Figure 41 CXCR4

 41 Figure 41 CXCR4 interacting proteins identified in HEK-293Tcells and their relative abundance in immunoprecipitates from CXCR4-expressing cells vs. mock cells. HA coimmunoprecipitation followed by nanoLC-MS/MS was performed in three distinct biological replicates in HEK-293T cells transiently expressing HA-tagged CXCR4 and cells transfected with empty plasmid (Mock). Log transformed intensities, obtained by Label Free Quantification (LFQ), of proteins identified in all three biological replicates in at least one set of cultured cells were than used for the comparison. The volcano plot was obtained plotting the differences of LFQ values between CXCR4 and mock cells (X axis) vs. -log of P value (Y axis). The upper part of the graph includes proteins with a high inter-replicate reproducibility whereas the right or left parts include plotted proteins with large differences in abundance between the two conditions. Specifically, on the right there are proteins enriched in the CXCR4 condition. The proteins were considered statistically significant using a T-test conducted on both sides setting the number of randomization at 250 the False Discovery Rate at 0.01 and the S0 at 0.1.Therefore, all proteins "above" the dotted lines are significantly enriched. The bait protein (CXCR4) is illustrated in blue; proteins already known to interact with CXCR4 (at least functionally) are in light blue.

Figure 42

 42 Figure 42 EphrinB1 co-immunoprecipitates with CXCR4 but not ACKR3 in HEK-293T cells. On the left immunoprecipitation (IP) of HA-ACKR3 and HA-CXCR4 transiently expressed in HEK-293T cells compared to mock cells (transfected with empty plasmid). On the right Ephrin-B1 coimmunoprecipitation (Co-IP) with HA-ACKR3 and HA-CXCR4 transiently expressed in HEK-293T cells compared to mock cells.

99 !

 99 was among the 151 proteins identified as potential ACKR3interacting proteins in our proteomic screen. Cx43 is a 43kDa protein that after synthesis oligomerizes in hexameric channels (hemichannels). Hemichannels present on adjacent cells dock head-to-head together forming gap junctions permeable to ions and small molecules, which ensure Gap Junctional Intercellular Communication (GJIC)224 .Only a few studies suggested a possible functional link between gap junctions and the CXCL12/CXCR4/ACKR3 axis. Two of them focused on the CXCL12/CXCR4 axis. In fact, in a large phosphoproteomic screen aimed at identifying proteins phosphorylated upon CXCL12 stimulation, CXCR4 triggered phosphorylation of Cx43 at Ser 279 and Ser 282 in breast cancer cells419 . In line with these findings, an other study conducted on breast cancer showed that low CXCL12 concentrations influence Cx43 phosphorylation states and GJIC 420 . Specifically, CXCL12 through activation of CXCR4 induces PKC activation and increases GJIC. However, the data reported in this study are fragmentary and sometimes contradictory. In fact, the data illustrated on Fig 2C showed that high CXCL12 concentrations reduce Cx43 expression in confluent cells. However, this result was not reproduced in Fig 4A.This contradiction could originate from the fact that the experiments might have been conducted only once (number of replicates is not indicated). In addition, the authors attributed the effect solely to CXCR4 and not ACKR3 based on the evidence that pre-treatment with AMD3100 reduced CXCL12 dependent Cx43 phosphorylation. However, the effect of AMD3100 on GJIC was not investigated.Schajnovitz and colleagues elegantly showed a dual regulatory control of connexin 43 in CXCL12 production and release in bone marrow-derived mesenchymal stem cells (schematically represented in Figure43). Specifically, Cx43 involved in gap junctions controls CXCL12 release whereas Cx43 that is not involved in gap junctions regulates Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1 421 (Figure43). In line with previous results indicating that Cx43 suppression leads to the inhibition of CXCL12 expression in adult brains 422 , they showed that Cx43 and Cx45 expression levels correlated with the CXCL12 one.Yet, inhibition of Cx43 activity by CBX and the mimetic peptide Gap27 inhibited CXCL12 secretion. The authors also observed that the propagation of Ca 2+ waves ! between coupled cells was necessary for the release of the chemokine into the extracellular space. A possible Cx43-mediated release of CXCL12 is also suggested in an other paper where CXCL12 was found co-localizing with Cx43 plaques in radial glial cells 423 .

Figure 43

 43 Figure 43 Schematic representation of Cx43 influence on CXCL12 secretion and production. Cx43 not involved in gap junctions stimulates the translocation of the Sp1 transcription factor into the nucleus. Sp1 then promotes the transcription of the Cxcl12 gene. Gap junctional-dependent Ca2+ mobilization stimulates adenylyl cyclase activity, which leads to increase in cAMP levels. cAMP activates the small GTPase RalA proteins (catalyzing the GDP-GTP exchange) via a PKA-dependent mechanism, which in turns mediate CXCL12 secretion via exocytosis.

  ACTIVATES GαI/O PROTEINS IN MOUSE PRIMARY ASTROCYTES BUT NOT IN HEK-293T CELLS Since a few studies have shown that ACKR3 recruits and activates G proteins in specific cell types, including astrocytes 150,153 , and that Cx43 is regulated by several GPCRs through pathways involving the activation of G proteins 299 , we decided to investigate if Gαi/o protein activation is involved in the observed ACKR3-dependent GJIC inhibition. Inhibition of Gαi/o protein activation by PTX treatment completely reversed the inhibition of GJIC induced by agonist stimulation of ACKR3 in primary cultured astrocytes. Interestingly, we observed that PTX increases basal GJIC. In line with previous works showing that activation of G proteins inhibits GJIC 299 and that GPCR are able to constitutively activate G proteins 427 , PTX treatment might impair constitutive activation of Gαi/o proteins by endogenously expressed GPCRs, thereby increasing GJIC.

Figure 44

 44 Figure 44 Proposed mechanism for the ACKR3-mediated Cx43 regulation. (1-5) Integrating our results with the canonical time course of GPCR activation the following series of molecular events starting with the activation of ACKR3 and concluding with Cx43 internalisation and GJIC inhibition can be proposed. (1) ACKR3 stimulation triggers G protein activation and dissociation of the Gα subunit from the βγ complex. (2) Dissociation of the G proteins allows the recruitment and activation of β-arrestin2. (3) ACKR3 activation leads to the β-arrestin2and dynamin-dependent inhibition of GJIC and concomitant Cx43 internalisation. (4) We suggest that ACKR3 and Cx43 are co-internalising as ACKR3/Cx43 complex. (5) ACKR3 activation inhibits dynamin-dependent Cx43 hemichannels internalisation, increasing their plasma membrane expression.

  -immunoprecipitation. HEK293T cells expressing either HA-ACKR3 or HA-CXCR4 or ACKR3-mutants or mock cells were lysed in ice cold Lysis Buffer (LB) ! 115! containing 1% n-Dodecyl-β-D-Maltopyranosid (DDM) purchased from Antrace (D310), 20mM TRIS (pH=7.5), 100mM NaCl, 2.5mM CaCl2, 10mM, phosphatase inhibitors (NaF, 10 mM; Na + -vanadate, 2 mM; Na + -pyrophosphate, 1 mM; and βglycerophosphate, 50 mM) protease inhibitor cocktail (Roche) for 1 h at 4 °C. Lysate was mixed for 1hr at 4°C. afterwards, samples were centrifuged at 15,000g for 15 min at 4 °C. Soluble proteins were quantified by bicinchoninic acid (BCA) assay from Sigma (B29643 and C2284)) and equal protein amount per each condition was incubated with the agarose-conjugated anti-HA antibody (Sigma A2095) overnight at 4 °C. Beads were then washed two times with an ice cold PBS solution containing 0.5M of NaCl and phosphatase inhibitors and two times with an ice cold PBS solution containing 0.150M of NaCl and phosphatase inhibitors. Immunoprecipitated proteins were then eluted in Laemmli sample buffer by shaking them at 37°C for 2hrs.Mass spectrometry. Proteins were separated on SDS-PAGE gels (12 % polyacrylamide, Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad, Hercules USA) and stained with Protein Staining Solution (Euromedex, Souffelweyersheim France). Gel lanes were cut into7 gel pieces and destained with 50 mM TriEthylAmmonium BiCarbonate (TEABC) and three washes in 100% acetonitrile. After protein reduction (with 10 mM dithiothreitol in 50mM TEABC at 60 °C for 30 min) and alkylation (55mM iodoacetamide TEABC at room temperature for 60 min) proteins were digested in-gel using trypsin (500 ng/band, Gold, Promega, Madison USA) as previously described431 . Digest products were dehydrated in a vacuum centrifuge and reduced to 3 μL. The generated peptides were analysed online by nano-flowHPLCnanoelectrospray ionization using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham USA) coupled to an Ultimate 3000 HPLC (Thermo Fisher Scientific). Desalting and pre-concentration of samples were performed on-line on a Pepmap® pre-column (0.3 mm × 10 mm, Dionex). A gradient consisting of 0-40% B for 60 min and 80% B for 15 min (A = 0.1% formic acid, 2% acetonitrile in water; B = 0.1 % formic acid in acetonitrile) at 300 nL/min was used to elute peptides from the capillary reverse-phase column (0.075 mm × 150 mm, Acclaim Pepmap 100® C18, Thermo Fisher Scientific). Eluted peptides were electrosprayed online at a voltage of 1.8 kV into an Oribtrap Elite mass spectrometer. A cycle of one full-scan mass spectrum (400-2,000 m/z) at a resolution of 120,000 (at 400 m/z), followed by 20 data-dependent MS/MS spectra was repeated continuously throughout the nanoLC separation. All MS/MS spectra were recorded using normalised collision energy (33%, activation Q 0.25 and activation time 10 ms) with an isolation window of 2 m/z. Data were acquired using the Xcalibur software (v 2.2). For all full scan ! measurements with the Orbitrap detector a lock-mass ion from ambient air (m/z 445.120024) was used as an internal calibrant as described 432 . Analysis of MS data was performed using MaxQuant software package (v 1.5.5.1) as described by J Cox and M Mann 433 . Tandem mass spectra (MS/MS) were searched by the Andromeda search engine 434 against the UniProtKB Reference proteome UP000005640

1 .

 1 MS data. Co-IP data were analysed and compared to mock cells using Perseus v1.5.6.0. Proteins identified only by site and labelled as contaminant were eliminated. Log2(LFQ intensities) were used for the analysis and only proteins identified in all three biological replicates in at least one group (ACKR3/CXCR4 or mock) were maintained. For the statistical analysis missing values were substitute with the minimum LFQ value quantified in the remaining list. The proteins were ! considered statistically significant using a T-test conducted on both sides setting the number of randomization at 250 the False Discovery Rate at 0.01 and the S0 436 at 0.Western blotting. Proteins were electrophoretically transferred to nitrocellulose membranes (Bio-Rad) after resolution onto 10% polyacrylamide gels. After blockage with a 5% milk solution, membranes were immunoblotted with primary antibodies dissolved in a 5% Bovine Serum Albumin (from Sigma A2153) solution overnight at 4°C. Then membranes were immunoblotted with either anti-mouse (Sigma-Aldrich GENA931V) or anti-rat (Jackson ImmunoResearch 112-035-003) or anti-rabbit (Sigma-Aldrich GENA934V) horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1 in 5000 in a 5% milk solution for 2hrs at room temperature.Immunoreactivity was detected with an enhanced chemiluminescence method (Western lightning ® Plus-ECL,Perkin Elmer) on a ChemiDoc Tm Touch Imaging System (Bio-Rad). Quantification was performed using the dedicated Bio-rad software Image Lab.BRET. 50,000 transfected HEK293T were seeded per well in white and black 96 well plates (Greiner). For saturation BRET an increasing amount of acceptor (YFPtagged) was transfected with a constant amount of donor (RLuc or NanoLuc). For ligand induced BRET the amount of plasmids (donor and acceptor) needed for generating the BRET50 signal in the saturation curve were used. After 24hrs cells were gently washed two times with PBS. Coelenterazine was added at a final concentration of 5 μM in the white plate. Readings were then immediately performed after the addition of different ligands or PBS at 37°C using the Mithras LB 940 plate reader (Berthold Biotechnologies, Bad Wildbad, Germany) that allows the sequential integration of light signals detected with two filter settings (Rluc/NLuc filter, 485 ± 20 nm; and YFP filter, 530 ± 25 nm). Data were collected using the MicroWin2000 software (Berthold Biotechnologies). BRET for saturation curve was expressed as % of net BRET compared to the maximal BRET. For the ligand induced BRET the BRET was expressed as % of BRET compared to the non stimulated. YFP used for calculating the NLuc or RLuc/YFP ratio was measured from the black plate using the INFINITE500 plate reader (TECAN) setting the excitation filter at 485±20nm and the emission one at 520±10nm. When needed PTX (100 ng/mL) was added overnight before the experiment.Immunofluorescence was performed on paraformaldehyde fixed cells. Specifically, cells were fixed with a 4% solution of PFA in PBS for 10min. Excess of PFA was ! 118! quenched washing three times with a 0.1M solution of glycine in PBS. Cells were permeabilized and blocked with a PBS solution of 5% heat inactivated goat serum (Vector Laboratories S-100) and 0.1 % Triton X-100 for 20min. Primary antibodies were then incubated overnight at 4°C in a 2.5 and 0.05% PBS solution of goat serum and Triton X-100, respectively. After extensive PBS washings cells were incubated for 2hrs, at room temperature and protected from light, with a PBS solution with 2.5% goat serum and 0.05% Triton X-100 PBS solution containing the appropriate secondary antibody and Hoechst 33342 (2 μM, Thermo Scientific Pierce). Antigen retrieval was performed heating up cells to 80°C for 20min in a citrate buffer solution (pH=6) containing Tween 0.05%. After cooling down to a room temperature, cells were washed three times and IF performed.Co-localization. TG1 and R633 cells were stained using the general protocol described earlier. Antibody 2, 3, 4 and 5 were used for staining Cx43, ACKR3 and CXCR4. Alexa Fluor 594-conjugated anti-mouse and Alexa Fluor 488-conjugated anti-rabbit were used. Pictures were taken with a Leica SP8-UV confocal microscope imaging between 20 and 50 different confocal plans for each cells. 3D pictures were then reconstructed using the Imaris software (Bitplane). Overlapping volume was defined firstly identifying the center of mass of each object belonging to group A (Cx43) or B (ACKR3/CXCR4). Then, two objects were considered co-localized if the center of one falls within the area of the other 408 . Following a non-biased approach the different objects were defined setting an automatic threshold 409 . The intensity of Cx43 or ACKR3/CXCR4 signal coming from the overlapping volume was quantified.

  Specifically, fluorescence was plotted against the distance from the cut. The average fluorescence of the 50 most distant pixels from the cut was considered as background and subtracted from all values. Fluorescence was then normalized setting the highest value as 100%. One exponential decay was then interpolated using Prism (v. 7.0,GraphPad Software Inc.) constraining the Y0 at 100 and the plateau at 0. Half-life (distance from the cut where the fluorescence is 50% of the maximum) of the interpolate curves where used for comparison. For each experiment the half-life was normalized setting the half-life of the control as 100%. At least three technical replicates for each condition were repeated in every biological replicate.Doublepatch clamp. To assess the effect of CXLC12 on gap-junction-mediated electrical coupling between astrocytes, junctional currents (Ij) were recorded in astrocyte pairs using the dual voltage-clamp technique 246 . Overnight cultured secondary cultures of cortical astrocytes were used for this experiments. For testing, coverslips were transferred to a recording chamber attached to the stage of an upright microscope (Axioskop FS; Zeiss, Le Pecq, France) and continuously superfused with Ringer's saline (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 12 glucose and buffered to pH 7.4. The saline was maintained at 32°C and continuously bubbled with carbogen (95% O2 / 5% C02). Patch pipettes were pulled to a resistance of 4-5 M from borosilicate glass (1.5 mm ! 120! outer diameter; 1.17 mm inner diameter) and filled with the following internal solution (in mM): 140 potassium-gluconate, 2 MgCl2, 1.1 EGTA, 5 Hepes, that was titrated to pH 7.2 with KOH. Electrical signals were acquired with an EPC-9 dual patch-clamp amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) in cell pairs voltage-clamped at -50 mV and were filtered at 3 kHz, as previously reported 437 . Membrane resistance of the recorded cells was calculated from an hyperpolarizing voltage step (-10 mV, 50 ms duration). Cells were challenged with depolarizing voltage steps (40 mV amplitude, 300 ms duration, a step every 30 s) and the amplitude of resulting Ij in the non-stimulated cell was continuously monitored for 15-20 min. Results are expressed as the coupling ratio, which corresponds to the amplitude of Ij over the total current amplitude recorded in the stimulated cell. Consistent with the presence of weakly and robustly coupled astrocyte pairs, the coupling ratio exhibited disparate values between pairs, ranging from 0.07 to 0.91 (n = 42 cell pairs). Pairs with a coupling ratio <0.25 were discarded. CXCL12-or carbenoxolone (CBX)-containing solutions

1 ACKR3, atypical chemokine receptor 3 AIP4, 1 GOLT1B 2 PECAM- 1 ,PKC

 13121 after transfection coverslips were transferred to the recording chamber of a patch clamp setup equipped with the Zeiss Axiovert 5100TV fluorescence inverse microscope having the required filters. Cells were then maintained in a solution of NaCl 140mM, CaCl2 2mM, KCl 3mM, Hepes 10mM, D-glucose 10mM pH 7,4, 320mosm. LY was dissolved, to a concentration of 5mg/ml, in a recording solution composed of CsCl 140mM, CaCl2 0,5mM, EGTA 20mM, Hepes 10mM, D-glucose 10mM, ATP-Na2 2mM, pH 7,2, 300mosm. LY was then microinjected in a single cell using a glass patch pipette. With the perfusion activated cells were microinjected for 5min. After 5min perfusion was stopped and patch pipette removed. Neighboring cells receiving the LY were manually counted after other 5min. For representative pictures coverslips were fixed in PFA and picture were taken using an AxioImagerZ1 microscope equipped with epifluorescence (Zeiss). Dynasore treated cell were ! 122! pretreated for 30min with the chemical before microinjection. Dynasore was kept in the recording chamber during the whole recording. PTX(100 ng/mL) and CBX(50μM) treated cells were treated overnight. CBX was kept in the recording chamber during the whole recording.DERET. DERET internalisation assay was performed as described in 410 . Briefly, 48 after transfection of cDNA encoding SNAP tagged receptor culture medium was substituted with 50 μl of 100 nM SNAP-Lumi4-Tb diluted in Tag-lite labeling medium.Cells were then incubated for 1 h at 4°C. After wasing the excess SNAP-Lumi4-Tb internalisation experiments were carried on by incubating cells at 37°C with Tag-lite labeling medium, either alone or containing CXCL12, in the presence of fluorescein (48μM). Singal emitted at at 620 and 520nm were collected using a PHERAstar FS. Ratio 620/520 was obtained dividing the donor signal by the acceptor signal and multyopling this value for 10,000. Data are expressed as % of Maximal internalisation after subtraction of the Internalisation at time 0. E3 ubiquitin ligase atrophin Interacting protein 4; AMFR, E3 ubiquitin-protein ligase AMFR AP-MS, affinity purification coupled with mass spectrometry ARIH1, E3 ubiquitin-protein ligase ARIH1 AUP1, ancient ubiquitous protein 1 BiFC, bimolecular fluorescent complementation assay BioID, proximity-dependent biotin identification BIRC6, baculoviral IAP repeat-containing protein 6 BRET, bioluminescence resonance energy transfer growth factor receptor ERK, extracellular signal-regulated kinases ETFA, electron transfer flavoprotein subunit alpha ETFB, electron transfer flavoprotein subunit beta FRET, fluorescence resonance energy transfer GET4, Golgi to ER traffic protein 4 homolog GIP, G protein-coupled receptor interacting protein GLRX3, glutaredoxin-3 GNAI, guanine nucleotide-binding protein G(i) subunit alphachannel subfamily K member 1 KISS, kinase substrate sensor LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry ! LYN, tyrosine-protein kinase Lyn MaMTH, mammalian membrane two-hybrid assay MAP2K2, dual specificity mitogen-activated protein kinase kinase 2 mDIA2, diaphanous-related formin-2 MIF, macrophage migration inhibitory factor MYBL2, myb-related protein B MYTH, membrane yeast two-hybrid assay NMMHC, motor protein non-muscle myosin H chain NR2F2, COUP transcription factor platelet endothelial cell adhesion molecule PI3Kg, PI3-kinase isoform p110g X-C chemokine receptor type 4 CXCR4 and the atypical chemokine receptor 3 (ACKR3/CXCR7) are class A G protein-coupled receptors (GPCRs). Accumulating evidence indicates that GPCR sub-cellular localization, trafficking, transduction properties and, ultimately, their pathophysiological functions are regulated by both interacting proteins and post translational modifications. This has encouraged the development of novel techniques to characterize the GPCR interactome and to identify residues subjected to post-transitional modifications, with a special focus on phosphorylation. This review first describes state-of-art methods for the identification of GPCR-interacting proteins and GPCR phosphorylated sites.

  . Phosphorylation of CXCR4 by different GRKs can elicit several molecular responses, such as fluctuations in intracellular calcium concentration and extracellular signal-regulated kinases (ERK) 1 and 2 phosphorylation, leading to integrated cellular responses. In HEK293 cells, calcium mobilisation is negatively regulated by GRK2, GRK6, and b-arrestin2. On the other hand, GRK3 and 6 together with b-arrestins act as positive regulators of ERK1/2 (Busillo, 2010). Overall, these studies show non-overlapping roles of the different GRKs in the regulation of CXCR4 signalling. These differential roles may explain distinct cell type-dependent responses to CXCL12. However, what dictates the specific GRK subtype recruitment still needs to be investigated. Changes in the normal CXCR4 phosphorylation pattern as a result of receptor mutations or altered GRK activity can lead to abnormal receptor expression and/or responsiveness, promoting aberrant cell signalling and thus can contribute to several pathologies. Deletion of Ser 346/347 leads to a gain-of-CXCR4-function and decreases receptor internalisation and subsequent desensitisation, indicating that mutations in the far C-terminus affect CXCR4-mediated signalling

  Several techniques are nowadays available to decipher GPCR interactome and phosphorylation profile. These techniques have been successfully applied to CXCR4 revealing important interacting proteins as well as key residues involved in the regulation of receptor-mediated signal transduction. Using an AP-MS strategy we identified novel potential CXCR4 interacting proteins that might reveal new mechanisms of CXCR4-dependent signalling. Systematic studies of the ACKR3 interactome and its key phosphorylated residues might likewise open new avenues in the understanding of ACKR3 pathophysiological functions and the underlying molecular mechanisms.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. CXCR4 and ACKR3 residues potentially subjected to post-transitional modification.

  129 , normal endometrial stromal cells 130 , marmoset and human testes 131 , human umbilical cord venous endothelial cells 132 and mouse limb muscles 133 .

		1.2.2.2.1.1 ACKR3 EXPRESSION IN THE CENTRAL NERVOUS SYSTEM
		After the pioneering study that detected ACKR3 in astrocytes 117 , additional groups of
		investigators identified the receptor in several areas of the Central Nervous System
		(CNS) both during development and in adulthood, suggesting an important role of
		this receptor for the development and functions of the CNS (see Table! 3 for detailed
		information on ACKR3 expression in different species and different ages). ACKR3
		expression was found to increase between E14 to E18 in rat and then to dramatically
		decrease after birth 134 . Different groups have consensually found ACKR3 in several
		cellular populations, namely neurons, astrocytes, endothelial, neuronal and
		oligodentrocyte progenitor cells 134-137 . Regarding the neuronal sub-populations Cajal-
	Species	Retzuis neurons, GABA-ergic neurons, interneurons and olfactory tubercle neuron Stage Quantification Structures Cell Type	Ref
				Dentate gyrus, CA3 pyramidal cell layer, cortex, ventral striatum thalamus	Neurons (pyramidal and GABAergic)
				hypotalamaus
		Adult	mRNA	Blood vessels	Endothelial cells
				Choroid plexus, wall lateral ventricle	Non neuronal (GLAST-positive astrocytes)
		E14-E18	mRNA	Marginal zone	Cajal-Retzius neurons
		E15-E17	mRNA	Appearance in the cortical plate (lateral and medial part)	Cortical plate neurons
	Rat	E18	mRNA	Appearance in the ventricular and subventricular zone
			mRNA	caudate putamen) Telencephalon (germinative zone, medial ganglionic eminences and	Migrating GABA-ergic precursors
		Late-embryonic and	mRNA	Scattered throughout brain. Intense staining in the subventricular zone, granular layer of the dentate gyrus and hippocampal subfield CA3 Corpus callosum, cortex, striatum.	GABAergic (high expression) and pyramidal (low expression) neurons. GLAST-positive astrocytes
		postnatal		Cerebellum	Bergmann glial cells
		E11.5	mRNA	ganglionic eminence (ventral part) Medial ganglionic eminence (ventricular and subventricular zone), lateral	Cajal-Retzius neurons
		E12.5	mRNA	Ganglionic eminence and ventral pial surface	Cortical interneurons and olfactory tubercle neuron precursors (Cajal-Retzius and subplate)
			mRNA	Subventricular an marginal zone	Immigrating cortical interneurons
		Postnatal	mRNA	Striatal subventricular zone
	Mouse		Protein	Blood vessels	Endothelial cells
					Neurons, astrocytes and neuronal stem cells
		Adult
			Protein	Subventricular zone	Cells morphologically resembling migrating oligodendrocyte progenitors
	Human	Adult	Protein	Cortex and hippocampus	89% (MAP2-positive neurons), 11% remaining not investigated
	!					22!
		!			21!

precursors express ACKR3. GLAST-positive as well as GFAP-positive and Bergmann glial cells were also found to express ACKR3. Immunohistochemistry performed on human brain slices showed that 89% of the cells positive for ACKR3 in both cortex and hippocampus were mature neurons 138 . However, the remaining 11% of the population was not investigated.

Protein

Ventral striatum (nucleus accumbens), basal ganglia (globus pallidus), cerebral cortex (layer IV-V of the parietal cortex), hippocampus (subgranular layer, molecular layer, pyramidal layer, oriens layer and the hilus), hypothalamus (ventromedial hypothalamic and supraoptic nucleus), cerebellum (Purkinje cell layer).

Table 3 Table summarizing the ACKR3 expression profile in the CNS.

 3

	!

and Figure 4 ACKR3 as decoy receptor. ACKR3

  was originally described as decoy receptor, able only to bind CXCL12 and CXCL11 and internalise without activating any intracellular signalling cascade.

	11 in transfected HEK-293 cells. In the same study down-regulation of either β-
	arrestin-1 or 2 inhibited CXCL12 and CXCL11 induced ACKR3-dependent migration.
	Since ACKR3 was unable to trigger calcium mobilization, as in the aforementioned
	studies, it was concluded that ACKR3 signals only through β-arrestins and not G
	proteins. In later studies it has been shown that β-arrestins are recruited to the C-
	terminal domain of the receptor 140-142 that is de-ubiquinated upon stimulation 142 .
	BOX 3 ACKR3 internalisation
	ACKR3 C-terminal domain was shown to be involved in its internalisation since two C-terminal
	truncating mutants, lacking the last 16 and 40 AA, were characterized by lower constitutive and
	ligand-induced internalisation 141 . In addition, expression of chimera ACKR3 whose C-terminal domain
	is switched with the CXCR4's one diminished internalisation 140 . Internalisation that is dynamin-
	dependent since ACKR3 does not internalise in HEK-293T cells expressing dominant negative K44A
	dynamin 141 or cells treated with the dynamin inhibitor Dynasore 143 . In addition, both mutants also
	displayed less interaction with β-arrestin2. Another article from the same group showed that ACKR3
	internalisation is dependent on β-arrestin2 and not β-arrestin1 143 . In the same article ACKR3 co-
	localized with Rab7 and lysosomes after 30 min challenges with CXCL12, indicating that the receptor
	is trafficked to late endosomes, leading to its subsequent degradation 143 . The pivotal role played by β-
	arrestins in ACKR3 internalisation was confirmed by a work from a different group where
	simultaneous suppression of both β-arrestin1 and 2 completely inhibited CXCL12-mediated ACKR3
	internalisation 142 . Inhibition of clathrin with sucrose also inhibited ACKR3 internalisation 142,143 . Both
	phosphorylation 140 and de-ubiquitination 142 of the receptor regulate ACKR3 internalisation.
	Consistent with engagement of β-arrestin-dependent signalling by ACKR3,
	subsequent studies showed ACKR3 and β-arrestin-dependent ERK1/2
	phosphorylation upon receptor activation in melanocytes 144 , transfected HEK-293
	cells 141,142 and microglia 145 .
	CXCL12 dose-dependently stimulates AKT and ERK1/2 phosphorylation as well as
	PKCζ/λ in primary cultures of postnatal rat cortical astrocytes 146 . On the other hand, it
	fails to activate p38 and PKCα/β. Inhibition of either CXCR4 (by AMD3100) and
	ACKR3 (by CCX754) completely impairs engagement of all these pathways
	suggesting that both receptors are involved in the CXCL12 responses. Furthermore,
	This agonist-dependent internalisation would result in a lower extracellular chemokine concentration. invalidation of ACKR3 using RNA interference abrogates CXCL12 signalling.
	Interestingly, CXCL12 triggers only phosphorylation of AKT and ERK1/2 in
	1.2.2.2.2.2 ACKR3 AS ATYPICAL CHEMOKINE RECEPTOR embryonic mouse primary cultures, whereas PKCs were not activated. Since ACKR3
	was expressed at comparable levels to that measured in rat cultures, it suggests
	inter-species or age differences in ACKR3 signalling. In addition, ACRK3 activation
	was linked to higher expression of the proliferative marker Ki67 in both cultures.
	! !	24! 25!

The simplistic view of ACKR3 as a silent receptor was overcome with the discovery that the receptor signals through β-arrestins 139 . Using co-localization techniques ACKR3 was found co-localizing with β-arrestin-2 upon stimulation with CXCL12

BOX 4 Pertussis Toxin (PTX)

	was provided in 2007 by FRET and co-precipitation techniques performed in the 54S ribosomal protein L4, mitochondrial MRPL4, different ATP synthases
	Pertussis toxin (PTX) is the toxin produced by the transfected HEK-293 cells. The distinct signalling profile of the dimer was evidenced (ATP5H, ATP5B, ATP5A1, ATP50), ACKR3 itself, the caspase Separin ESPL1 and
	whooping cough causing bacterium Bordetella by the fact that it evokes a larger Ca 2+ increase and a slower ERK phosphorylation, the Putative E3 ubiquitin-protein ligase UBR7 97 .
	pertussis. It is used in the investigation of Gαi/o-compared with CXCR4 alone 119 . These first pieces of evidence of heterodimerisation
	dependent signalling pathways engaged by GPCRs due to its ability of irreversible ADP-ribosylate the Gαi/o were elegantly confirmed 150 using BRET approaches in transfected HEK-293 cells 1.2.2.2.3 ACKR3 PHYSOLOGICAL ROLES
	-/-mouse primary astrocytes increased [ 35 S]-GTPγS binding. This increase was lost after the inhibition of ACKR3 expression by siRNA. CXCL12 induced a subunits 152 . This does not preclude the coupling of G proteins with the receptor 150 but it avoids the dissociation of the Gαi/o subunit from the βγ complex and therefore the activation of the G protein. where ACKR3 forms constitutive and ligand-modulated heterodimer with CXCR4 as The paramount importance of ACKR3 in life became clear with the discovery that well as a homo-dimer with itself. In addition, although ACKR3 on itself is unable to 95% of mice ACKR3 -/-die only one day after birth 119 . Consistent with its expression activate calcium mobilization in HEK-293 cells, it reduces the potency of CXCL12 to pattern, ACKR3 has been recognized to participate to the cardiovascular, trigger calcium mobilization upon activation of CXCR4. Accordingly, ACKR3 expression modifies the ability of CXCR4 to recruit and activate Gαi proteins. The reproductive, renal and neuron physiology 158 .
	Calcium mobilization that was inhibited by CCX771, ACKR3 invalidation by RNA possible cross talk between ACKR3 and CXCR4 to activate G protein was confirmed In addition to the aforementioned role of ACKR3 in the migration of primordial germ
	interference, Pertussis Toxin (BOX 4) and the PLC inhibitor U73122 suggesting that by experiments showing that ACKR3 expression reduces the potency of CXCL12 to cell 49 ACKR3 also influences the migration of T cells 65 , vascular smooth muscle cells
	ACKR3 activation by CXCL12 induces a Gαi/0 and PLC-dependent cytosolic Ca 2+ inhibit cAMP production 154 . In addition, the ACKR3/CXCR4 heterodimer interacts (in response to CXCL11) 139 , human epidermal melanocytes (NHEM) 144 , B-cells 159 ,
	increase. Inhibition of Gαi/0 proteins also hindered the ACKR3-mediated more efficiently with β-arrestin (with or without CXCL12 stimulation) than ACKR3 microglia (in Experimental autoimmune encephalomyelitis) 145 and neurons. In 2011,
	phosphorylation of AKT and ERK1/2. CXCL12 mediates ACKR3-dependent alone. Corroborating these findings, the co-expression of CXCR4 and ACKR3 leads two articles showed that ACKR3 expressed by migrating interneurons plays a pivotal
	migration, proliferation and internalisation that are also dependent of Gαi/o proteins. to more sustained ERK, Stress-activated protein kinase and p38 constitutive role in their migration during embryonic brain development. Wang and colleagues 160
	Although CXCL11 was unable to activate G proteins, it triggers β-arrestin2-phosphorylation (only ERK phosphorylation was induced by CXCL12) compared to
	dependent AKT and ERK1/2 phosphorylation via ACKR3 and not CXCR3 in rat that measured in cells expressing each receptor alone. Silencing of β-arrestin2
	cortical astrocytes 153 , suggesting that ACKR3 can engage different signal expression reversed this effect. Although the existence of ACKR3/CXCR4
	transduction mechanisms upon activation by its cognate chemokine agonists heterodimers is well proven in transfected cell lines, their identification in cells or
	CXCL12 and CXCL11. tissues endogenously expressing the two receptors is still lacking.
	These results do not pretend to overlook all the literature classifying ACKR3 as an CXCR4 is not the only protein able to interact with ACKR3. Using PLA and co-
	atypical chemokine receptor in most of the cellular contexts. However, they raise the localization strategies, ACKR3 was shown interacting with the Epithelial Growth
	interesting possibility that in specific cellular contexts, where not yet identified Factor Receptor (EGFR) in breast cancer cell lines but not in normal tissues 155 . β-
	specific and necessary partners are expressed, ACKR3 might activate G proteins. arrestin2 is involved in this interaction. ACKR3 phosphorylates EGFR and
	participates in the EGFR mediated ERK phosphorylation via a β-arrestin2-dependent
	1.2.2.2.2.4 ACKR3 AS A TALKATIVE RECEPTOR mechanism. A subsequent study showed that β-arrestin2 is a negative regulator of
	Discovered as a silent receptor unable to activate any intracellular signalling, ACKR3 ACKR3-mediated EGFR activation and nuclear translocation 156 .
	later became an atypical chemokine receptor unable to activate G proteins but EGFR is not the only receptor interacting with ACKR3 as also the MIF receptor CD74
	capable of recruiting β-arrestins and activating their downstream signalling cascades. weakly interacts with ACKR3 112 . Moreover, ACKR3 co-localizes with PECAM-1, the
	The evolution continued when ACKR3 was shown to interact with other proteins cell adhesion molecule required for leukocyte transendothelial migration in human
	including the other CXCL12 chemokine receptor CXCR4. coronary artery endothelial cells 157 . Using a Membrane Yeast two Hybrid assay
	(MYTH) screen, ATP13A2 was identified as a putative ACKR3 interacting protein 95 . ACKR3 and CXCR4 interconnection might not only be limited to the sharing of In a study aimed at characterizing the human interactome of 1,125 GFP-tagged CXCL12 binding but also involves physical interaction between both receptors. First proteins by Co-IP followed by MS analysis, ACKR3 was found to interact with the evidence of a possible constitutive hetero-dimerization between the two receptors gap junction beta-2 protein GJB2, the probable E3 ubiquitin-protein ligase HECTD2,
	! !	28!

Table 4 Table summarizing the connexin genes identified. The genes are identified starting with

 4

  280 , heart 281 and brain. Neurons express several others

	connexins including Cx26, Cx30.2, Cx36, Cx45, and Cx57; with Cx36 being the
	principal one. Microglial cells express Cx43, Cx36 and Cx32. Oligodendrocytes
	express Cx32, Cx47, and Cx29 in vivo. Neuronal precursors express Cx26, Cx30,
	Cx40 and Cx43. Finally astrocytes express Cx43, Cx26, Cx30, Cx40, Cx45, and
	Cx46 with Cx43 being the most abundant one 234 . In astrocytes, the expression

pattern of Cx43 changes during development. Before spreading throughout all the brain, Cx43 appears around E12 in radial glial cells. In adults, Cx43 is expressed uniformly in all astrocytes with higher abundance in chemical synapses, nodes of ! 44!

  and Tyr 265 . Epidermal growth factor (EGF) rapidly inhibits gap junctional activity triggering ERK1/2-mediated phosphorylation of Cx43 at Ser 255 , Ser 279 and Ser 282

	Phosphorylation site	Kinases involved	Effect on gap junctional communication
	Y247	Src, Tyk2	Decrease
	S255	CDK1, ERK1/2	Decrease
	S262	CDK, ERK1/2	Decrease
	Y265	Src, Tyk2	Decrease
	S279	ERK1/2	Decrease
	S282	ERK1/2	Decrease
	S325	CK1	Increase
	S328	CK1	Increase
	S330	CK1	Increase
	S364	PKA	Increase
	S368	PKC	Decrease
	S369	AKT	Increase
	S373	AKT	Increase

301

. ERK1/2 was also found to phosphorylate Cx43 at Ser 262 . In addition to phosphorylation, SUMOylation 302 , Ubiquitination 303 , and Acetylation 304 regulate Cx43 trafficking. Interestingly, Cx43 trafficking can also be regulated by Cx43

Table 5 Cx43 phosphorylated residues with associated kinases and consequence of their phosphorylation on gap junctional activity of Cx43

 5

. Adapted from 438 ! 47!

Table 6 Non-exhaustive list of known Cx43 interacting proteins

 6

. The biological role of the interaction is also reported.

! 49!

4.6 CX43 IN THE PHYSIOLOGY OF THE CNS Multiple

  roles have been attributed to Cx43 in the CNS, most often involving glial cells, consistent with its cellular expression pattern. Cx43 expressed on glial cells participates in the buffering of K + ions during neuronal activity 352 and provides metabolic sustenance to neurons. In fact, astrocytes end feet surrounding blood vessels are strategically placed for taking up glucose from the blood flow. Glucose is then diffused, from one astrocyte to another, through Cx43 gap junctions and delivered to neurons far away from the energy source 353 . In addition, Cx43 contributes to the propagation of Ca 2+ waves in the brain (BOX 6).

	• The bioactive lysophospholipids sphingosine-1-phosphate (S1P) and
	lysophosphatidic acid (LPA) activate GPCRs belonging to the
	lysophospholipid (LPL) receptor gene family. S1P and LPA transiently inhibit
	gap junctional communication. Both PTX and inhibition of the Rho/ROCK
	pathways partially reversed the S1P-induced inhibition of Cx43, suggesting
	that Gαi/o and Gα12/13 proteins are involved in the observed effect 251 .
	• Expression of a GTPase-deficient Gαq in Rat-1 fibroblasts inhibits Cx43 gap
	junctional activity through the activation of PLCβ3 351 .

1 mediated inhibition of Cx43 gap junctional communication is dependent on Gαi/o proteins but independent of PKC, MAPK and Rho/ROCK 299 . ! 50! 1.3.

Table 7 List of proteins that specifically co-immunoprecipitate with ACKR3 in HEK-293T cells

 7 !

	Protein names	UniProtID	Gene names	Difference	P value
	Atypical chemokine receptor 3	P25106	ACKR3	11.93	3.07
	UDP-glucose:glycoprotein glucosyltransferase 1	Q9NYU2	UGGT1	8.85	4.91
	26S proteasome non-ATPase regulatory subunit 1	Q99460	PSMD1	8.67	3.82
	E3 ubiquitin-protein ligase HUWE1	Q7Z6Z7	HUWE1	8.31	3.09
	Protein sel-1 homolog 1	Q9UBV2	SEL1L	7.62	6.88
	Proteasome subunit beta type-4	P28070	PSMB4	7.04	3.77
	E3 ubiquitin-protein ligase HECTD1	Q9ULT8	HECTD1	7.01	4.25
	Proteasome subunit beta type-5	P28074	PSMB5	6.90	3.44
	Cleft lip and palate transmembrane protein 1	O96005	CLPTM1	6.88	3.94
	Nuclear pore membrane glycoprotein 210	Q8TEM1	NUP210	6.88	2.82
	Proteasome subunit beta type-6	P28072	PSMB6	6.80	3.20
	Proteasome subunit alpha type-5	P28066	PSMA5	6.64	3.12
	Proteasome subunit beta type-1	P20618	PSMB1	6.61	2.80
	Proteasome subunit alpha type-1	P25786	PSMA1	6.61	3.30
	ATPase WRNIP1	Q96S55	WRNIP1	6.60	2.89
	Rab5 GDP/GTP exchange factor	Q9UJ41	RABGEF1	6.60	5.69
	26S proteasome non-ATPase regulatory subunit 13	Q9UNM6	PSMD13	6.54	3.37
	Calmegin	O14967	CLGN	6.47	4.19
	Ankyrin repeat domain-containing protein 13A	Q8IZ07	ANKRD13A	6.35	5.49
	Large proline-rich protein BAG6	P46379	BAG6	6.28	2.53
	Ubiquilin-1	Q9UMX0	UBQLN1	6.24	4.04
	Serine/threonine-protein phosphatase 6 regulatory subunit 1	Q9UPN7	PPP6R1	6.11	4.22
	GTPase-activating protein and VPS9 domain-containing protein 1	Q14C86	GAPVD1	6.10	2.46
	Importin-9	Q96P70	IPO9	6.06	3.42
	E3 ubiquitin-protein ligase AMFR	Q9UKV5	AMFR	5.84	3.26
	26S proteasome non-ATPase regulatory subunit 7	P51665	PSMD7	5.81	3.83
	Nodal modulator 1	Q5JPE7	NOMO1	5.69	3.48
	Carboxypeptidase D	O75976	CPD	5.68	3.38
	Nuclear pore complex protein Nup133	Q8WUM0	NUP133	5.64	4.14
	V-type proton ATPase subunit S1	Q15904	ATP6AP1	5.58	4.43
	Transmembrane and ubiquitin-like domain-containing protein 1	Q9BVT8	TMUB1	5.55	2.90
	Transmembrane protein 9	Q9P0T7	TMEM9	5.54	3.40
	Pyridoxal-dependent decarboxylase domain-containing protein 1	Q6P996	PDXDC1	5.54	4.09

. Proteins statistically enriched according to their LFQ level in the ACKR3 complex compared to Mock cells are reported. Protein name, Uniprot ID, gene name, LFQ difference between ACKR3 and mock cell (Difference) and thelog P values (P value) are indicated. The statistical analysis was performed using the Perseus software as detailed in the "Materials and Methods" section. Proteins are ranked based on their difference in abundance in immunoprecipitates from ACK3-expressing cells vs. Mock cells. The bait ACKR3 is shown in blue, its known interacting proteins in light blue and the accessory proteins of Rab and Ubiquitin complexes as well as MAP2K2 are depicted in green. GJA1 (Cx43) is highlighted in red and the proteins already known to interact with Cx43 (at least functionally) in orange.

2 CX43 INTERACTS PREFERENTIALLY WITH ACKR3 COMPARED TO CXCR4.

Table 8 List of proteins that specifically co-immunoprecipitate with CXCR4 in HEK-293T cells. Proteins

 8

	Protein names	UniProtID	Gene names	Difference	P value
	C-X-C chemokine receptor type 4	P61073	CXCR4	8.13412	4.81752
	Ephrin-B1	P98172	EFNB1	7.29111	4.95875
	Speckle targeted PIP5K1A-regulated poly(A) polymerase	Q9H6E5	TUT1	7.05343	4.7274
	Glutaredoxin-3	O76003	GLRX3	5.96408	4.23064
	Minor histocompatibility antigen H13	Q8TCT9	HM13	5.81814	3.8084
	26S proteasome non-ATPase regulatory subunit 14	O00487	PSMD14	5.69013	5.6534
	Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial	P45954	ACADSB	5.65867	3.56233
	Nuclear pore membrane glycoprotein 210	Q8TEM1	NUP210	5.40417	5.33476
	Electron transfer flavoprotein subunit beta	P38117	ETFB	4.97812	3.59581
	Beta-1,4-glucuronyltransferase 1	O43505	B4GAT1	4.82992	3.82343
	Baculoviral IAP repeat-containing protein 6	Q9NR09	BIRC6	4.74226	3.81439
	NEDD8-activating enzyme E1 regulatory subunit	Q13564	NAE1	4.72246	4.79763
	Nucleoporin NDC1	Q9BTX1	NDC1	4.69831	5.36129
	FH1/FH2 domain-containing protein 1	Q9Y613	FHOD1	4.6166	6.25869
	Inositol-3-phosphate synthase 1	Q9NPH2	ISYNA1	4.14048	4.05474
	ER membrane protein complex subunit 4	Q5J8M3	EMC4	4.04863	3.42766
	COUP transcription factor 2	P24468	NR2F2	3.53283	5.77999
	Origin recognition complex subunit 4	O43929	ORC4	3.38933	4.20258
	26S protease regulatory subunit 6B	P43686	PSMC4	2.43658	4.02678

statistically enriched according to their LFQ level in the CXCR4 complex compared to Mock cells are reported. Protein name, Uniprot ID, gene name, LFQ difference between CXCR4 and mock cell (Difference) and thelog P values (P value) are indicated. The statistical analysis was performed using the Perseus software as detailed in the "Materials and Methods" section. Proteins are ranked based on their difference in abundance in immunoprecipitates from CXCR4-expressing cells vs. Mock cells.

  In brief, for the internalisation assay, overnight starved astrocyte primary cultures were washed twice with ice cold PBS and incubate for 45min with biotine at 4°C on a plate shaker. Biotin was then quenched and cells washed twice again with ice cold PBS. Cells were then incubate either with ice cold

	manufacturer's instructions. DMEM, 37°C DMEM or the appropriate agonist for 30min. In the case of dynasore
	treated cells, cells were pre-trated for 30min with dynasore prior to agonist
	stimulation. After the 30min surface biotin was cleaved by two incubations with
	MESNA (50mM) of 20 at 4°C. Cells were then washed twice with ice cold PBS and
	lysed in LB. Lysates were then mixed for 2hrs at 4°C and centrifuged for 10min at
	15,000g. Before overnight incubation at 4°C with NeutrAvidin Agarose beads a
	portion of lysate was saved for using as total control. The next day, beads were
	washed twice with the lysis buffer and one with PBS. Protein were eluted in Laemmli
	sample buffer with DTT (50mM) by shaking them at 37°C for 2hrs.
	For quantifying the amount of Cx43 at the cell surface, overnight starved astrocyte
	primary cultures were washed twice with PBS and incubated at 37°C with DMEM or
	the appropriate agonist for 30min. Cells were then washed twice with ice cold PBS
	and incubate for 45min with biotine at 4°C on a plate shaker. Cells were then washed
	twice with ice cold PBS and lysed in LB. Lysates were then mixed for 2hrs at 4°C and
	centrifuged for 10min at 15,000g. Before overnight incubation at 4°C with NeutrAvidin
	Agarose beads a portion of lysate was saved for using as total control. The next day,
	beads were washed twice with the lysis buffer and one with PBS. Protein were eluted
	in Laemmli sample buffer with DTT (50mM) by shaking them at 37°C for 2hrs.
	Microinjection. 75,000 HEK293T/well were seeded in a 24 well plate containing
	glass coverslips. The day after seeding cells were transfected with PEI. Two days
	!	121!

Table 1 . Principal methods used to identify GPCR-interacting proteins and phosphorylated residues.

 1 Y2H, yeast two-hybrid assay; MYTH, membrane yeast two-hybrid assay; MaMTH, mammalian membrane two-hybrid assay; KISS, kinase substrate sensor; PLA, proximity ligation assay; BiFC, bimolecular fluorescent complementation assay; BioID, proximity-dependent biotin identification.

			Indirect method.
			Mutagenesis of the C-terminus can impair
		Cheap and easy.	expression and/or localization of the
	Suitable	Based on functional data in living cells.	receptor.
		Can pinpoint phosphorylated residues.	Labor intensive in case of multiple
			phosphosites.
			Not quantitative.

Table 2 . CXCR4 interacting proteins described in the literature.

 2 TLR2, toll-like receptor 2; AIP4, E3 ubiquitin ligase atrophin Interacting protein 4; RTN3, reticulon3, NMMHC, motor protein non-muscle myosin H chain; CD164, endolyn; mDIA2, diaphanous-related formin-2; PI3Kg, PI3-kinase isoform p110g; PECAM-1, platelet endothelial cell adhesion molecule; MYBL2, Myb-related protein B; KCNK1, potassium channel subfamily K member 1; TMEM63B, CSC1-like protein 2; GOLT1B, vesicle transport protein GOT1B.

	!

Cysteine-rich and transmembrane domain-containing protein 11

	Protein name	Gene name	UniProt ID	Normalized MS/MS count	Average iBAQ CXCR4
	Evolutionarily conserved signalling intermediate in Toll pathway	ECSIT	Q9BQ95	0.01160	586,114
	E3 ubiquitin-protein ligase RNF5	RNF5	Q99942	0.01111	178630
	Dynactin subunit 5	DCTN5	Q9BTE1	0.01099	120954
	Calpain small subunit 1	CAPNS1	P04632	0.00995	193850
	Guanine nucleotide-binding protein G(i) subunit alpha-1	GNAI1	P63096	0.00942	237333
	Integral membrane protein 2B	ITM2B	Q9Y287	0.00877	130,189
	Inositol-3-phosphate synthase 1	ISYNA1	Q9NPH2	0.00836	137,886
	Dihydrolipoyl dehydrogenase, mitochondrial	DLD	P09622	0.00786	258,572
	Ancient ubiquitous protein 1	AUP1	Q9Y679	0.00770	278,788
	Synembryn-A	RIC8A	Q9NPQ8	0.00753	203,420
	Proteasome activator complex subunit 2	PSME2	Q9UL46	0.00697	217,121
	Ubiquitin-conjugating enzyme E2 K	UBE2K	P61086	0.00667	197,517
	Nucleoporin p54	CYSTM1 NUP54	Q9H1C7 Q7Z3B4	0.01375 0.00657	658,329 149,909
	Proteasome subunit alpha type-3 Proteasome subunit alpha type-1	PSMA3 PSMA1	P25788 P25786	0.01307 0.00634	324,119 114,455
	Golgi to ER traffic protein 4 homolog	GET4	Q7L5D6	0.01223	345,230
	Speckle targeted PIP5K1A-regulated poly(A) polymerase	TUT1	Q9H6E5	0.01220	597,661
					!

Dual specificity mitogen-activated protein kinase kinase 2

	MAP2K2	P36507	0.00583	139,521

Zinc finger CCCH-type antiviral protein 1-like

	Protein name Protein name Protein name	Gene name Gene name Gene name	UniProt ID UniProt ID UniProt ID	Normalized MS/MS count Normalized MS/MS count Normalized MS/MS count	Average iBAQ CXCR4 Average iBAQ CXCR4 Average iBAQ CXCR4
	N-terminal kinase-like protein Nuclear pore complex protein Nup107 C-terminal-binding protein 1	SCYL1 NUP107 CTBP1	Q96KG9 P57740 Q13363	0.00536 0.00396 0.00303	186,696 96,797 108,421
	E3 ubiquitin-protein ligase KCMF1 Peptidase M20 domain-containing protein 2 COP9 signalosome complex subunit 5	KCMF1 PM20D2 COPS5	Q9P0J7 Q8IYS1 Q92905	0.00525 0.00382 0.00299	197,300 101,885 108,293
	E3 ubiquitin-protein ligase HUWE1 Acyl-CoA dehydrogenase family member 9, mitochondrial Transmembrane 9 superfamily member 3	HUWE1 ACAD9 TM9SF3	Q7Z6Z7 Q9H845 Q9HD45	0.00518 0.00376 0.00283	181,922 251,019 50,251
	Protein FAM134C Equilibrative nucleoside transporter 1 Putative HLA class I histocompatibility antigen, alpha chain H	FAM134C SLC29A1 HLA-H	Q86VR2 Q99808 P01893	0.00501 0.00365 0.00276	203,073 206,126 148,707
	Integrator complex subunit 11 Proteasome subunit beta type-7 Nuclear pore membrane glycoprotein 210	CPSF3L PSMB7 NUP210	Q5TA45 Q99436 Q8TEM1	0.00500 0.00361 0.00247	102,079 116,231 203,550
	Rap1 GTPase-GDP dissociation stimulator 1 TraB domain-containing protein MAGUK p55 subfamily member 6	RAP1GDS1 TRABD MPP6	P52306 Q9H4I3 Q9NZW5	0.00494 0.00355 0.00247	105,848 130,584 109,651
	COUP transcription factor 2 Transmembrane 9 superfamily member 2 Nuclear envelope pore membrane protein POM 121C	NR2F2 TM9SF2 POM121C	P24468 Q99805 A8CG34	0.00483 0.00352 0.00217	111,176 73,594 104,673
	E3 ubiquitin-protein ligase ARIH1 Serine/threonine-protein phosphatase 6 regulatory subunit 1 E3 ubiquitin-protein ligase AMFR	ARIH1 PPP6R1 AMFR	Q9Y4X5 Q9UPN7 Q9UKV5	0.00479 0.00341 0.00207	124,675 93,490 124,862
	Basic leucine zipper and W2 domain-containing protein 1 GTPase-activating protein and VPS9 domain-containing protein 1 Nuclear pore complex protein Nup133	BZW1 GAPVD1 NUP133	Q7L1Q6 Q14C86 Q8WUM0	0.00477 0.00338 0.00202	119,855 205,182 92,921
	NEDD4 family-interacting protein 1 Fascin Clustered mitochondria protein homolog	NDFIP1 FSCN1 CLUH	Q9BT67 Q16658 O75153	0.00452 0.00338 0.00153	136,055 187,190 74,561
	Ribonuclease H2 subunit A Spermatogenesis-associated protein 5 DNA polymerase delta catalytic subunit	RNASEH2A SPATA5 POLD1	O75792 Q8NB90 P28340	0.00446 0.00336 0.00151	325,037 68,425 49,692
	Monofunctional C1-tetrahydrofolate synthase, mitochondrial Condensin complex subunit 1 Baculoviral IAP repeat-containing protein 6	MTHFD1L NCAPD2 BIRC6	Q6UB35 Q15021 Q9NR09	0.00443 0.00333 0.00103	374,534 82,016 44,825
	Exportin-7 Probable methyltransferase-like protein 15 Acetyl-CoA carboxylase 1	XPO7 METTL15 ACACA	Q9UIA9 A6NJ78 Q13085	0.00429 0.00328 0.00085	212,689 42,614 38,883
	Dimethyladenosine transferase 2, mitochondrial Tyrosine-protein kinase Lyn Deubiquitinating protein VCIP135	TFB2M LYN VCPIP1	Q9H5Q4 P07948 Q96JH7	0.00421 0.00326 0.00082	122,863 67,845 30,284
	Transmembrane protein 209 Sideroflexin-3	TMEM209 SFXN3	Q96SK2 Q9BWM7	0.00416 0.00312	73,409 127,637
	Protein FAM8A1 Serine/threonine-protein kinase Chk2	ZC3HAV1L FAM8A1 CHEK2	Q96H79 Q9UBU6 O96017	0.00556 0.00404 0.00307	106,885 839,75 87,680
					! ! ! !

Table 3 . List of proteins that specifically co-immunoprecipitate with CXCR4 in HEK293T cells.

 3

Acknowledgments:

We thank all our colleagues from the ONCORNET consortium for the continuous scientific discussions and insights.

Author contributions: AF Conducted experiments, Performed data analysis, Wrote or contributed to the writing of the manuscript. AZ Wrote or contributed to the writing of the manuscript. MN Wrote or contributed to the writing of the manuscript. BC Wrote or contributed to the writing of the manuscript. SH contributed to the writing of the manuscript.

FM contributed to the writing of the manuscript. MS contributed to the writing of the manuscript. PM Wrote or contributed to the writing of the manuscript, Participated in research design.