Jacques Zylberg

Denise Ecke

Bilha Fischer
email: bfischer@mail.biu.ac.il.

Georg Reiser
email: georg.reiser@medizin.uni-magdeburg.de

Structure and ligand binding site characteristics of the human P2Y 11 nucleotide receptor deduced from computational modelling and mutational analysis

Keywords: Nucleotide receptor, P2Y receptor, Molecular Dynamics, Virtual Screening, Mutagenesis Enzyme-linked-Immunoassay, EL, extracellular loop, GFP, green fluorescent protein, GPCR, G-protein coupled receptor, hP2Y-R, human P2Y receptor, MD, molecular dynamics

The P2Y 11 -receptor (P2Y 11 -R) is a less explored drug target. We computed a hP2Y 11 -R homology model with two templates, bovine-rhodopsin (2.6Å resolution) and a hP2Y 1 :ATP complex model. The hP2Y 11 -R model was refined using molecular dynamics calculations and validated by virtual screening methods, with an enrichment factor of 5. Furthermore, mutational analyses of R106, E186, R268, R307 and A313 confirmed the adequacy of our hP2Y 11 -R model and the computed ligand recognition mode. The E186A and R268A mutants reduced the potency of ATP by one and three orders of magnitude, respectively. The R106A and R307A mutants were functionally inactive. We propose that residues R106, R268, R307 and E186 are involved in ionic interactions with the phosphate moiety of ATP. R307 is possibly also H-bonded to N 6 of ATP via the backbone carbonyl. Activity of ATP at the F109I mutant revealed that the proposed π-stacking of F109 with the adenine ring is a minor interaction. The mutation A313N, which is part of a hydrophobic pocket in the vicinity of the ATP-C2-position, partially explains the high activity of 2-MeS-ATP at P2Y 1 -R as compared to the negligible activity at the P2Y 11 -R. Inactivity of ATP at the Y261A mutant implies that Y261 acts as a molecular switch, as in other G-protein coupled receptors. Moreover, analysis of cAMP responses seen with the mutants showed that the efficacy of coupling of the P2Y 11 -R to G s is more variable than coupling to G q .

Our model also indicates that S206 forms an H-bond with Pγ and M310 interacts with the adenine moiety.

Introduction

P2 receptors are activated by extracellular nucleotides [START_REF] Burnstock | Purinergic nerves[END_REF]. These receptors are divided into two groups: ligand-gated cation channels, classified as P2X receptors [START_REF] Abbracchio | Purinoceptors: are there families of P2X and P2Y purinoceptors?[END_REF], and the P2Y receptors, coupled to G proteins [START_REF] Fredholm | Nomenclature and classification of purinoceptors[END_REF]. P2Y-Rs can be further subdivided into two phylogenetic groups. One group comprises the hP2Y 1,2,4,6,11 -Rs that are coupled mainly to phospholipase C. In the second group consisting of the hP2Y 12,13,14 -Rs, the inhibition of adenylyl cyclase is triggered. hP2Y 12,13 are classical nucleotide receptors, whereas hP2Y 14 is activated by UDP-glucose [START_REF] Chambers | A G protein-coupled receptor for UDPglucose[END_REF]. The hP2Y 11 -R, however, is coupled to stimulation of both the adenylyl cyclase, and the phospholipase C pathways [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF]. This is a unique feature amongst the P2Y-R family.

Out of the first P2Y-R subgroup, the hP2Y 1 -R and hP2Y 11 -R are exclusively activated by adenine nucleotides, ATP and ADP [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF][START_REF] Webb | Cloning and functional expression of a brain G-protein-coupled ATP receptor[END_REF], whereas the P2Y 4,6 -Rs prefer uracil nucleotides [START_REF] Jacobson | Purine and Pyrimidine (P2) Receptors as Drug Targets[END_REF]. Specifically, the hP2Y 6 -R is selectively activated by UDP, and the hP2Y 4 -R is activated mainly by UTP. The hP2Y 2 -R seems not to discriminate between purine or pyrimidine nucleotides and is stimulated equipotently by ATP and UTP (see refs. in [START_REF] Von Kugelgen | Pharmacological profiles of cloned mammalian P2Yreceptor subtypes[END_REF]).

Unlike the hP2Y 1 -R, which is the most thoroughly investigated hP2Y-R, its closest homologue, the hP2Y 11 -R, sharing 33% identity, has been far less studied [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF].

To date, no mutational analyses of the hP2Y 11 -Rs were reported. Likewise, the hP2Y 11 -R has been scarcely studied by computational methods [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF].

The hP2Y 1 -R was modelled extensively by Moro et al. [START_REF] Moro | Human P2Y 1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites[END_REF]11]. These computational studies, supported by mutational analyses, suggested the residues involved in ligand binding. Arg128, Tyr136, Lys280 and Arg310 were reported to interact with the ATP phosphate moiety, while Ser314 and Arg310 were believed to interact with the adenine moiety. These authors also proposed His132, His277 and Ser317 to be involved in H-bonds with the sugar moiety [START_REF] Moro | Human P2Y 1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites[END_REF]. The homology model of the hP2Y 1 -R described by Moro and co-workers was based on a bovine (b)-rhodopsin template and subsequently refined using a cross-docking approach [START_REF] Moro | Human P2Y 1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites[END_REF]. Different computational and optimization techniques were applied later by Major et al. for the modelling of the hP2Y 1 -R [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF][START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF]. Thus, we performed the optimization of the hP2Y 1 -R model in an explicitly hydrated lipid bilayer (triphasic system) [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF]. The binding-pocket in the hP2Y 1 -R model was then optimized using a Monte-Carlo and molecular dynamics (MD) protocol. In this way, we could explain not only the molecular recognition determinants of ATP and synthetic analogues, but we also provided an explanation for the hP2Y 1 -R diastereoselectivity [START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF].

The P2Y-R family is the subject of intense ongoing investigations because of its important influence in physiological processes and involvement in various diseases, varying from cystic fibrosis [START_REF] Buescher | P2Y 2 receptor polymorphisms and haplotypes in cystic fibrosis and their impact on Ca 2+ influx[END_REF] to platelet secretion and aggregation disorders [START_REF] Cattaneo | The P2 receptors and congenital platelet function defects[END_REF]. Specifically, the hP2Y 11 -R was reported to play roles in the treatment of neutropenia [START_REF] Boeynaems | Overview of P2Y receptors as therapeutic targets[END_REF], acute myocardial infarcts [START_REF] Amisten | Increased risk of acute myocardial infarction and elevated levels of C-reactive protein in carriers of the Thr87 variant of the ATP receptor P2Y 11[END_REF] and in the maturation process of dendritic cells [START_REF] Wilkin | The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells[END_REF].

In this study, we aimed to provide an accurate model of the hP2Y 11 -R supported by mutational analyses to open the possibility of designing specific ligands for this receptor. By means of these investigations, we provide an insight into the ligand binding determinants of the hP2Y 11 -R. The proposed ligand recognition mode is consistent with the pharmacological data.

Methods

Alignment

Multiple sequence alignment involving human(h)-P2Y 11 -R has been performed using the ClustalW software [START_REF] Thompson | The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]. The alignment included the following sequences originating from the NCBI database:

Modelling

Both b-rhodopsin crystallographic structure at 2.6Ǻ resolution [START_REF] Okada | Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography[END_REF], and the hP2Y 1 -R:ATP complex model [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF], constructed previously, were used as templates to construct the hP2Y 11 -R model. A set of 200 models with different energies were generated using the Modeler software [22], and the best one was selected. The models did not include any of the hP2Y 11 -R's loops. Structural features known to be conserved, in G-protein coupled receptors (GPCRs) in general or in the hP2Y-R subfamily in particular, have been introduced using constraints during the construction of the hP2Y 11 -R model. Thus, an ionic bridge, Asp196 and Arg275 in the hP2Y 11 -R [11] was constrained. In addition, the TM backbones were constrained as αhelices to remove helical kinks specific to b-rhodopsin. In addition, α-helical constraints were introduced near the extra-and intracellular sides of the helices. These constraints prevent the helices from unwinding during optimization procedures in the building protocol of Modeler.

Model refinement

The best model obtained from the homology modelling was used here as a starting coordinated set. The receptor model consists of the helical bundle and the ATP molecule. Harmonic constraints (factor 15) have been imposed on the dihedral angles of the peptide backbone. The dihedrals from the Pro residues in all TMs have not been included in these constraints. A distance constraint has been used in all molecular dynamics (MD) simulations, which prevented the ATP molecule from flipping or floating out of its binding-pocket. The helices' extremities have been capped using an acetyl group for C-terminals and methyl for the N-terminals.

Blocking method has been adopted to remove interactions between the helices' termini and between some residues and the ATP molecule. The model has first been subjected to an extensive minimization using a combination of the steepest descent and conjugated derivatives algorithm (with energy tolerance 0.0001 Kcal/mol).

All MD calculations performed here use the Velocity Verlet integration algorithm as implemented in CHARMM (developmental version 31b1). The minimized model was heated from 0˚ K up to 300˚ K for 9 ps with a 5˚ K increment every 100 time steps (time step = 0.0015 ps). It has then been subjected to a 150 ps simulation. The latter was concluded with a minimization using the steepest descent algorithm with a 0.001 Kcal/mol energy tolerance.

The receptor relaxation could be easily monitored when tracking the overall system energy. No trajectory post-processing has been done. The relaxed receptor, except the binding-pocket, was then constrained. The radius pattern surrounding the ATP molecule followed a 6Å-10Å-∞ scheme (free-constrained-fixed). The ligandreceptor complex has been heated from 0˚K to 900˚K for 27 ps with a 5˚K increment every 100 time steps (time step = 0.0015 ps) followed by 300 ps of simulation.

In order to retrieve relevant parameter values, the simulation has been quenched using a steepest descent minimization with 0.001 Kcal/mol energy tolerance for every recorded frame. Interaction energies between the ATP molecule and selected residues have been extracted as well as the overall energy. The interval of the investigated frames counted 600 integration steps.

Average Structure

An average structure of the three most stable structures encountered during the simulation was calculated using the BLOCK facility in CHARMM [START_REF] Brooks | CHARMM: a program for macromolecular energy, minimization, and dynamics calculations[END_REF] and further minimized with an energy tolerance of 0.0001 Kcal/mol.

Model Validation

The calculated hP2Y 11 -R model was validated: A. by inspection of the geometrical parameters using the Ramachandran plot, B. by virtual screening.

A. The geometric parameters of the hP2Y 11 -R model have been revised using the Ramachandran plot generated by ProCheck software [START_REF] Laskowski | PROCHECK: a program to check the stereochemical quality of protein structures[END_REF]. More than 99 % of residues were classified in allowed regions, leaving one out of a total of 203 residues in a disallowed region.

B. The compound library created for the virtual screening included the following: seven known hP2Y 11 -R agonists (Fig. 2) constructed and further optimized at AM1 level using the Gaussian98 [START_REF] Frisch | Gaussian, Inc[END_REF]; a set of 42 nucleotides downloaded at random from the ChemBank online database [26] (Supplementary Material); the druglike compounds library proposed by Accelrys [27], which contains 970 molecules.

The volume of the hP2Y 11 -R binding pocket has been defined by the ATP molecule calculated based on the previously reported hP2Y 1 -R:ATP complex model [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF]. The model has been subjected to Surflex [START_REF] Jain | Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine[END_REF] routines in order to characterize the binding-pocket (creating the "protomol" using default parameters). The entire compound library has been docked in the hP2Y 11 -R model, using the Surflex software [START_REF] Jain | Surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine[END_REF]. The resulting output has been processed using advised parameter values (1.0 polarity penalty threshold; -3.0 penetration penalty threshold; 100 maximum allowed rotatable bonds, as proposed by the Surflex documentation). The aptitude of the virtual screening was expressed in terms of enrichment factor (EF). The EF is the ratio of the database percentage containing the target molecules before (100%) and after ranking (DB%). EF = 100% / DB% .

Mutagenesis analysis

Site-directed mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA, USA). The DNA sequence of the hP2Y 11 -R (GenBankTM/EBI accession number AF030335) was kindly provided by Dr. D.

Communi and placed between the EcoRI/BamHI restriction sites of the eGFPN1 vector (Clontech, Heidelberg). The mutations were introduced using customized oligonucleotides (Quiagen, Hilden) and confirmed by DNA sequencing. The P2Y 11 -GFP (green fluorescent protein) receptor DNA and DNA carrying the respective mutation were used to transfect 1321N1 human astrocytoma cells. Cells were grown at 37°C in 10% CO 2 in high-glucose DMEM medium supplemented with 5% FCS, 100 U/ml penicillin, 100 IU/ml streptomycin (Seromed, Biochrom, Berlin) and transfected with the recombinant plasmids using FuGENE TM 6 transfection reagent (ratio Fugene:DNA 3:2) as given in the manufacturer's protocol (Roche, Mannheim, Germany). Transfected cells were selected with 0.5 mg/ml G418 (Calbiochem, La Jolla, CA, USA) for stable expression of the wild type and mutant receptors.

Stably transfected cells were plated on glass coverslips (Ø = 22 mm, OmniLab, Bremen, Germany), and single cell measurement was done after 3 days, when the cells were 30-50% confluent. The changes in free intracellular Ca 2+ concentration ([Ca 2+ ] i ) were measured, as described before [START_REF] Ubl | Co-existence of two types of [Ca 2+ ] i -inducing protease-activated receptors (PAR-1 and PAR-2) in rat astrocytes and C6 glioma cells[END_REF] using the calcium indicator fura-2AM (Biomol, Hamburg/Molecular Probes) and recording the change in fluorescence intensity after stimulation with various agonists (Sigma, Deisenhofen, Germany). The cells were imaged with a system from TILL Photonics (München) using a X40 oil immersion objective and a flow rate of 1 ml/min in a recording chamber containing 0.2 ml [START_REF] Vöhringer | A chimeric rat brain P2Y 1 receptor tagged with green-fluorescent protein: high-affinity ligand recognition of adenosine diphosphates and triphosphates and selectivity identical to that of the wild-type receptor[END_REF]. Calcium data were analyzed with the Excel program applying basal deduction to the calcium traces and calculating the peak height for each cell. Concentration-response data obtained with average values from 40 to 70 single cells were further analyzed to derive EC 50 values (half-maximal response) using the SigmaPlot program (Systat, Erkrath, Germany). Calculation of the EC 50 values and curve fitting were performed using the following equation with a standard slope:

( ) x EC R R R y - + - + = 50 log min max min 10 1
, where the maximal response (R max ) was adjusted to the top plateau of the ATP curve.

For cAMP measurements, stably transfected cells were seeded in 6-well plates at a density of 100.000 cells per well and grown for 2 days till 80% confluency was reached. Prior to stimulation, the medium was aspirated and replaced by Na-HBS buffer (HEPES buffered saline: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 25 mM glucose and 20 mM HEPES/ Tris pH 7.4) containing 0.5 M IBMX (isobutyl-methyl-xanthine) and 1 µM DPSPX (1,3-dipropyl-8-sulfophenyl-xanthine) an A 1 /A 2 receptor antagonist. After 30 min incubation at 37°C in this buffer, cells were stimulated with ATP for 10 min. After stimulation, the buffer was aspirated and cells were washed with PBS and lysed with 0.1 M HCl containing 0.05 % Triton X-100. Lysates were harvested and a 100 µl aliquot kept for protein estimation. Tubes were centrifuged at 1000 x g for 10 min and supernatants were directly used for the assay. Determination of intracellular cAMP was done using the Direct cAMP EIA (Enzyme-linked-Immunoassay) kit (assay designs, Ann Arbor, Michigan, USA). Data were analyzed using Graph Pad Prism.

The expression levels of wild type and mutant receptors were analyzed by flow cytometry using a FACS LSR (BD Biosciences, Heidelberg). Cells were grown in 5 cm culture dishes (Nunc, Wiesbaden, Germany) to 80% confluency, harvested, and resuspended in culture medium. The expression levels of 10.000 cells were analyzed by determining the mean intensity (geometric median) of the GFP fluorescence per cell using the Flow Jo software.

Results and Discussion

Similarity and identity of hP2Y 11 -R and hP2Y 1 -R, and modelling of the hP2Y 11 -R

The human (h)P2Y 11 -R is characterized by considerably larger second and third extracellular loops (EL2 and EL3, respectively) as compared to other hP2Y-R subtypes and bovine (b)-rhodopsin (e.g., [START_REF] Gales | Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor[END_REF] amino acids (aa) vs. 25 aa for EL2, and 31 aa vs. 22 aa for EL3, as compared to the hP2Y 1 -R). b-rhodopsin and the hP2Y 11 -R share 21% sequence identity and 52% similarity in the transmembrane (TM) domains, whereas the hP2Y 1 -R shares 38% identity and 72% similarity with the hP2Y 11 -R in the TM domains (Table 1). The amino acid sequence of the hP2Y 11 -R exhibits 33% overall amino acid identity with the hP2Y 1 receptor, its closest homologue.

To model the hP2Y 11 -R we first aligned the hP2Y 11 -R TM regions with multiple sequences: b-Rhodopsin and the hP2Y 1,2,4,6 -Rs. The alignment in the TM domains has no gaps except for a single residue gap that was introduced in the beginning of TM6 (Fig. 1). This negligible gap is far away from the putative binding site (see below) and is located at the flexible TM extremity. All the conserved residues of the GPCR family A, as mentioned by Mirzadegan et al. [START_REF] Mirzadegan | Sequence Analyses of G-Protein-Coupled Receptors: Similarities to Rhodopsin[END_REF], were aligned correctly. This includes highly conserved residues as well as residues related to the known GPCR patterns such as (E/D)RY in TM3 and NPxxY in TM7 (Fig. 1) [START_REF] Mirzadegan | Sequence Analyses of G-Protein-Coupled Receptors: Similarities to Rhodopsin[END_REF].

The model computed here for the hP2Y 11 -R did not include any loops, as opposed to our previously reported hP2Y 1 -R model [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF]. This is due to the very flexible nature of the loops that prevents their accurate modelling. Furthermore, EL2 and EL3 in the hP2Y 11 -R are significantly longer (9 and 19 amino acids, respectively) than those of b-rhodopsin and many related GPCRs [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF]. Therefore, alignment and modeling of these loops is certainly unreliable. Computational study of only the helical bundle of GPCRs has become a well established procedure [32]. b-rhodopsin at 2.6Å resolution [START_REF] Okada | Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography[END_REF] was used as a template to calculate the hP2Y 11 -R model, despite the relatively low overall identity (<20%). Moreover, the hP2Y 1 -R:ATP complex model [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF] was used as an additional template, because of the high homology of the hP2Y 1 -R with the hP2Y 11 -R. The need for this model as a template arises as the experimental data (e.g. mutational analysis and pharmacological data) are scarce for the hP2Y 11 -R, unlike for the hP2Y 1 -R. Furthermore, the hP2Y 11 -R-bound ATP was modelled based on hP2Y 1 -R-bound ATP positioning [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF].

For obtaining sufficiently accurate GPCR models, extensive optimization protocols are required. Therefore, our model was further subjected to a two-stage MD optimization protocol. At first, we optimized the helical bundle of the model by subjecting it to MD simulation without constraining the structure. The MD simulation lasted 150 ps. In the first 70 ps a full relaxation of the model by 300 Kcal/mol occurred. Previously reported MD simulation of the hP2Y 11 -R [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF] lasted only 50 ps, which we have found insufficient for a full relaxation of the receptor. The structural nuances which distinguish even closely related proteins are of utmost importance when e.g. subtype receptor selectivity has to be considered, which is the case here.

The MD relaxation protocol lets those nuances come into play as we have demonstrated for the closely related hP2Y 1 -R and hP2Y 11 -R.

In the next step, the model was fixed while only the binding pocket area with a bound ATP molecule was subjected to a quenched dynamics simulation. The simulation revealed that the residues involved in ATP recognition have favourable interaction energies. Out of this simulation an average hP2Y 11 -R:ATP structure was extracted.

To validate the calculated hP2Y 11 -R models, they were subjected to a virtual screening study. This study used hP2Y 11 -R agonists [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF] (Fig. 2) as target molecules and a random nucleotides library (Supplementary Material) as well as a random druglike set of molecules as decoy molecules [27]. When testing the agonist discrimination capabilities of the homology structure, the enrichment factor was 4.

The average hP2Y 11 -R structure model, calculated from the MD refinement protocols, had an improved agonist discrimination, which was quantified by an enrichment factor of approximately 5 (Fig. 3), thus validating the calculated hP2Y 11 -R model.

Characteristics of the hP2Y 11 -R helical bundle

A multitude of positively charged amino acid residues in the ELs and at the ends of the TM helices are apparently unique for the hP2Y-R family (Fig. 4).

Together, the positively charged residues in the ELs may play a role in guiding the negatively charged nucleotide ligand into the binding site, as suggested by Moro et al. Unlike this conserved Ser residue, Pro311 is not involved in ligand recognition.

The conserved proline residues of GPCRs in TMs 6 and 7 are present in both the hP2Y 1 -R and the hP2Y 11 -R (Fig. 1). The induced kink by the highly conserved proline in TM6 has a pivotal role in GPCR activation and is involved in TM6 conformational change [START_REF] Visiers | Structural motifs as functional microdomains in Gprotein-coupled receptors: energetic considerations in the mechanism of activation of the serotonin 5-HT2A receptor by disruption of the ionic lock of the arginine cage[END_REF].

One of the most conserved patterns in GPCRs is the TM7 (N/D)PxxY motif present at the intracellular end. The mutation of the Asp or the Asn residue was shown to affect the activation of the phospholipase C and the adenylyl cyclase pathway. For instance, the mutation Asn322Ala in the β 2 adrenergic receptor resulted in complete uncoupling of the receptor [START_REF] Barak | The conserved seven-transmembrane sequence NP(X)2,3Y of the Gprotein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor[END_REF]. Furthermore, Gales et al. demonstrated for the cholecystokinin-B GPCR that the Asn391 residue of the NPxxY motif plays an essential role in the activation of the Gα protein [START_REF] Gales | Mutation of Asn-391 within the conserved NPXXY motif of the cholecystokinin B receptor abolishes Gq protein activation without affecting its association with the receptor[END_REF].

The pattern occurring in hP2Y 1,2,4,6 -Rs is DPxxY. Interestingly enough, in the hP2Y 11 -R this pattern has not been conserved. Instead, one finds the segment HPxxY. This unique motif may imply an alternative G protein activation mechanism and thus requires further investigation.

The hP2Y 11 -R binding pocket and ATP binding-mode

The hP2Y 11 -R amino acid residues interacting with the ATP molecule include Arg106, Phe109, Ser206, Arg268, Arg307 and Met310 (Table 2, Fig. 5). The three positively charged residues probably interact electrostatically with the triphosphate moiety, while Ser206 may form an H-bond with P γ . The adenine moiety possibly interacts with Phe109 via π-stacking interactions, as was observed for the hP2Y 1 -R:ATP complex model. A suspected unique interaction of the hP2Y 11 -R with the adenine moiety involves Met310. Divalent sulfur atoms, such as in methionine residues, have an electrophilic character and interact with electron rich rings [START_REF] Zauhar | Evidence for a strong sulfur-aromatic interaction derived from crystallographic data[END_REF][START_REF] Pal | Non-hydrogen bond interactions involving the methionine sulfur atom[END_REF][START_REF] Tatko | Investigation of the nature of the methionine-p interaction in b-hairpin peptide model systems[END_REF].

Met310 is aligned to Ala313 in the hP2Y 1 -R, which is not involved in ligand binding.

However, an important H-bonding interaction involving N1 and N 6 -adenine positions and Ser residues (e.g. Ser314 in the hP2Y 1 -R) in hP2Y-Rs complexes, is missing in the hP2Y 11 -R. In the latter receptor, instead of a Ser residue, Pro311 is present, which does not form any specific interactions with the adenine ring. No alternative residue was found to interact with the adenine N1 position in the hP2Y 11 -R:ATP complex. In this way, an important binding interaction is lost in the hP2Y 11 -R as compared to the hP2Y 1 -R. In addition, no specific interactions of hP2Y 11 -R residues were observed with the ribose ring.

In general, the EL2 is believed to be part of the GPCR binding pocket [START_REF] Palczewski | Crystal structure of rhodopsin: A G protein-coupled receptor[END_REF], and thus involved in ligand recognition. The Asp204 (in EL2) in hP2Y 1 -R has been found to be a critical residue [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF]. Major et al. proposed that the ATP phosphate chain coordinates with a Mg 2+ ion, which is in turn coordinated with Asp204 [START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF].

The EL2 in the hP2Y 11 -R is considerably longer than that of the hP2Y 1 -R (35 vs. 25 amino acid residues, respectively). Attempts to model this very long EL2 were unsuccessful. Hence, the role of a residue in hP2Y 11 -R (tentatively Glu186), corresponding to Asp204 in the hP2Y 1 -R, could not be investigated computationally.

Recognition of C2-substituted ATP analogues

C2-substituted ATP analogues, such as 2-MeS-ADP or 2-MeS-ATP, proved to be exceptionally potent at the hP2Y 1 -R [START_REF] Palmer | Agonist action of adenosine triphosphates at the human P2Y 1 receptor[END_REF]. Yet, at the hP2Y 11 -R these analogues proved to be extremely poor agonists [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF]. However, AR-C67085 (2-PrS-β,γdichloromethylene-D-ATP, 1 Fig. 2), was shown to be a potent agonist at the hP2Y 11 -R [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF]. These observations prompted us to explore a tentative binding pocket for the C2-substituents of ATP. In both the hP2Y 1 -R and the hP2Y 11 -R models, a hydrophobic pocket is located in the vicinity of the ATP C2 position (Fig. 6). That pocket in the hP2Y 11 -R is situated between TM2 and TM7, and is confined by residues Leu82 (TM2), Phe109 (TM3), Leu113 (TM3), Pro311 (TM7) and Ala313 (TM7). In our virtual screening study, C2-thioether substitutions such as MeS, EtS, PrS, BuS fitted into the hP2Y 11 -R hydrophobic pocket. However, bulkier substitutions such as 2-tBuS and 2-neopentylS did not fit. Indeed, the 2-neopentylS-ATP analogue which was subsequently tested at the hP2Y 11 -GFP receptor to stimulate intracellular calcium release, showed no significant increase in the calcium level at concentrations up to 10 µM. However, the reduced potency of 2-MeS-ATP at the hP2Y 11 -R as compared to ATP remains unresolved by virtual screening study and might be due to other reasons than poor fitting.

Site-directed mutagenesis of the hP2Y 11 -R

Based on the hP2Y 11 -R model proposed here, we next tested the binding mode hypothesis by mutating amino acid residues Arg106 (TM3), Phe109 (TM3), Glu186 (EL2), Arg268 (TM6) and Arg307 (TM7). 1321N1 cells were used to stably express the wild type and the mutant receptors, respectively. All receptors were constructed as GFP fusion proteins. The expression level was analyzed by flow cytometry detecting the GFP fluorescence intensity per cell. Both, wild type and mutant receptors displayed comparable fluorescence intensities. Some mutants (E186A, R268A) showed even a higher expression and other mutants (R106A, Y261A, and R307A) had around 80% expression level as compared to the wild type receptor (Table 3). The subcellular localization of the mutant receptors was examined by confocal microscopy. It was found to be comparable to that of the unmutated receptor for the E186A, R268A, R268Q, A313N mutant receptors. Although the F109I mutant receptor was only partially located at the plasma membrane, it exhibited a significant potency for ATP. Similar observations were made in a different mutagenesis approach, where even a 90% reduction in surface expression levels of the wild type hP2Y 1 -R had no significant influence on the EC 50 values of the investigated agonist [START_REF] Hoffmann | The role of amino acids in extracellular loops of the human P2Y 1 receptor in surface expression and activation processes[END_REF]. Therefore, the potency of ATP found at the F109I mutant as well as the potency at the mutants with a similar subcellular localization (R106A, Y261A, and R307A) likely reflects the intrinsic activity of these constructs.

Functional activity of the receptors was determined, firstly, by monitoring the intracellular [Ca 2+ ] i release in the stably transfected cells and, secondly, by measuring cAMP accumulation induced after agonist stimulation. We employed the calcium indicator fura-2 and a cAMP-EIA, as described in Methods.

The two arginine residues in TM3 (Arg106) and TM7 (Arg307) were found to be most critical for hP2Y 11 -R activation. After mutation to alanine, the ability of ATP to trigger a calcium signal at these receptors was almost abolished. Only at concentrations ≥10 mM an increase in calcium levels could be observed (Table 3). This confirms the hypothesis that these arginine residues stabilize the bound ATP through electrostatic interaction with ATP-P α and P γ of the phosphate moiety (Fig. 5).

Besides the interaction with ATP-P α , Arg307 is also believed to take part in an Hbond through its backbone carbonyl with N 6 . The corresponding residues in the hP2Y 1 receptor (Arg128, Arg310) are similarly essential for ligand recognition. Their mutation resulted in functionally inactive receptors [START_REF] Jiang | A mutational analysis of residues essential for ligand recognition at the human P2Y 1 receptor[END_REF]. A model of the hP2Y 6 receptor showed the involvement of these conserved arginine residues in binding of the phosphate moiety of the nucleotide to the receptor [START_REF] Costanzi | Human P2Y 6 receptor: molecular modeling leads to the rational design of a novel agonist based on a unique conformational preference[END_REF]. Surprisingly, at the hP2Y 2 receptor only the mutation of the corresponding arginine in TM7 (Arg292) resulted in loss of function of the receptor, but mutation of the arginine in TM3 (Arg110) to leucine had little effect on the potency of the agonists [START_REF] Erb | Site-directed mutagenesis of P2U purinoceptors. Positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity[END_REF]. The arginine in TM7 is part of a conserved motif (Q/KxxR) within the G q -coupled subgroup of P2Y receptors. This motif is considered important for receptor activation.

The aromatic amino acid in TM3 (Phe109) that was thought to possibly interact with the adenine moiety via π-stacking interaction seems to be not very critical for receptor activation. The EC 50 value was increased only by a factor of 4 after mutation of phenylalanine to isoleucine. Thus, there was no major effect on the potency of ATP at the receptor (Table 3, Fig. 7). However, this phenylalanine is highly conserved throughout the P2Y receptor family (Fig. 1). In the hP2Y 1 receptor, mutation of this residue to alanine caused a loss in potency for 2-MeS-ADP of about one order of magnitude, but it was still less critical for ligand recognition than other sites of the receptor [START_REF] Moro | Human P2Y 1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites[END_REF][START_REF] Jiang | A mutational analysis of residues essential for ligand recognition at the human P2Y 1 receptor[END_REF]. Moreover, this phenylalanine is possibly involved in hydrophobic interactions with the uracil ring of docked UDP in a molecular model of the hP2Y 6 receptor [START_REF] Costanzi | Human P2Y 6 receptor: molecular modeling leads to the rational design of a novel agonist based on a unique conformational preference[END_REF]. The mutation of Phe109 to isoleucine in the hP2Y 11 receptor did probably not much disturb the recognition of ATP at the receptor, since isoleucine is also a bulky, hydrophobic amino acid and therefore loss in potency was only 4-fold.

Thus, the prediction of π-stacking between the adenine and the phenyl ring could not be verified experimentally.

The role of the ELs for nucleotide binding by P2Y-Rs has been suggested previously [START_REF] Hoffmann | The role of amino acids in extracellular loops of the human P2Y 1 receptor in surface expression and activation processes[END_REF]. Furthermore, the importance of the EL2 Asp204 residue in ligand recognition has been already shown for the hP2Y 1 -R [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF][START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF]. Glu186 was a residue in the EL2 of the hP2Y 11 -R predicted to be involved in ligand recognition. Mutation of this glutamate to alanine resulted in a decreased potency of ATP at the receptor. The shift was more than one order of magnitude (Table 3, Fig. 7), consistent with the finding at the hP2Y 1 -R. However, for the more potent hP2Y 11 -R agonist ATPγS the shift in potency was only 5-fold, compared to the wild type receptor. This implies that Glu186 interacts with phosphates P α and P β of the triphosphate moiety, probably also via coordination of a Mg 2+ ion as proposed for the corresponding residue in the hP2Y 1 -R [START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF]. The relatively small shift in potency for agonists at the Glu186 mutant implies a modulatory function of this residue in receptor functionality, as proposed for the corresponding residue in the hP2Y 1 -R [11,[START_REF] Hoffmann | The role of amino acids in extracellular loops of the human P2Y 1 receptor in surface expression and activation processes[END_REF]. The Arg268 residue that was thought to be involved in ATP-P β recognition is part of a conserved motif in TM6. For members belonging to the G q -coupled subgroup of P2Y receptors the motif is HxxR/K, the G i -coupled receptors have all arginine and not lysine. P1 receptors lack these arginine/ lysine residues, indicating the role of the positively charged amino acids in coordination of the phosphate moiety.

The Arg268Ala mutant receptor displayed a clearly reduced potency for ATP, compared to the wild type receptor (Table 3, Fig. 7), indicating the significance of an intact motif in TM6. When this arginine was substituted by glutamine, the potency of ATP at this receptor could be partially rescued (Table 3, Fig. 7). These findings are consistent with the interpretation of a partial recovery of the Arg268 interaction with ATP-P β , which results in the partially restored activity. However, we cannot ignore the importance of a conserved pattern in TM6. Indeed, it has been suggested that at least one mechanism of GPCR activation originates in TM6, being the "aromatic zipper" [START_REF] Rosenkilde | Activation of the CXCR3 chemokine receptor through anchoring of a small molecule chelator ligand between TM-III, -IV and -VI[END_REF]. Although in cases where that mechanism of activation is clearly missing, we cannot rule out the existence of alternative mechanisms, which could involve Arg268. For both mutant receptors the change in potency for ATPγS compared to the wild type receptor was not as drastic as for ATP, indicating that the major interaction is with P β and a favourable interaction with P γ -S, as compared to P γ -O, still remains in the mutant. This favourable interaction may be due to a tighter fit of the larger P γ -S moiety, compared to the phosphate moiety. The corresponding residues in the hP2Y 1 -R (Lys280) and the hP2Y 2 -R (Arg265) were also found to be essential for activation at low ATP concentrations, because a clear decrease in potency was found by substitution of Lys280 or Arg265 by uncharged amino acids [START_REF] Jiang | A mutational analysis of residues essential for ligand recognition at the human P2Y 1 receptor[END_REF][START_REF] Erb | Site-directed mutagenesis of P2U purinoceptors. Positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity[END_REF]. In the hP2Y 6 -R model, this position (Lys259) was part of a positively charged subpocket that bound the phosphate moiety of docked UDP [START_REF] Costanzi | Human P2Y 6 receptor: molecular modeling leads to the rational design of a novel agonist based on a unique conformational preference[END_REF], again highlighting the importance of this residue. For a member of the G i -coupled subgroup of P2Y receptors the hP2Y 12 -R, the significance of this arginine residue in TM6 was also confirmed [START_REF] Cattaneo | The P2 receptors and congenital platelet function defects[END_REF][START_REF] Cattaneo | Molecular bases of defective signal transduction in the platelet P2Y 12 receptor of a patient with congenital bleeding[END_REF][START_REF] Hoffmann | Evidence for the involvement of basic amino acid residues in transmembrane regions 6 and 7 of the human platelet P2Y 12 -receptor in ligand recognition[END_REF].

The importance of a tyrosine residue in TM6 for ligand recognition was also investigated by means of mutagenesis. Tyr261 was not found to be directly interacting with the ATP molecule docked in our hP2Y 11 -R model. However, it has been shown that a comparable residue (Tyr273) in the hP2Y 1 -R located at the same position (6.48) seems to act as a molecular switch for receptor activation [START_REF] Costanzi | Architecture of P2Y nucleotide receptors: Structural comparison based on sequence analysis, mutagenesis, and homology modeling[END_REF]. A Tyr273Ala mutation led to a functionally inactive receptor that was still able to bind agonist/antagonist with the same affinity as the wild type receptor. This is in accordance with the "aromatic zipper" theorem proposing a probable mechanism of activation. As it was suggested that this residue might also be important for other GPCRs, we generated a Tyr261Ala mutant hP2Y 11 -R. The mutant receptor was incapable of being activated by ATP at concentrations ≤10 mM. Therefore, this tyrosine seems to play an important role in hP2Y 11 -R activation. We assume that Tyr261 is solely involved in receptor activation, since it was not found to be binding the ATP significantly in any of our models. Due to the lack of a selective radioligand at the hP2Y 11 -R we were not able to directly prove this suggestion.

At the entrance to the hydrophobic pocket located in the vicinity of the C2 position of ATP in the hP2Y 11 -R model (Fig. 6A) an alanine residue (Ala313) is situated. This Ala313 is a unique feature of the hP2Y 11 -R, as all other hP2Y receptors have an asparagine amino acid at this position. We expected that this Ala313 might have an impact on the reduction in potency for C2-substituted ATP derivatives at the hP2Y 11 -R [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF][START_REF] Ecke | Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5'-O-(alpha-boranotriphosphate) analogues[END_REF]. At the Ala313Asn mutant receptor, ATP was 2-fold less potent (Table 3, Fig. 7). However, 2-MeS-ATP (EC 50 = 8.37±2.05 µM, n=8) displayed a gain in potency as compared to the wild type receptor (EC 50 = 11.1±6.30 µM, n=3). This finding supports our hypothesis of this residue being a key player in the interactions involving ATP-C2 substitutions.

The hP2Y 11 -R is also coupled to the activation of the adenylyl cyclase, besides induction of intracellular calcium release. Thus, we also investigated the ability of the receptor mutants to induce cAMP production upon stimulation. The mutants that showed no calcium responses were not considered.

The hP2Y 11 -R expressed in 1321N1 cells was found to have a low efficacy in coupling to G s [START_REF] Communi | Pharmacological characterization of the human P2Y 11 receptor[END_REF]. Stimulation with ATP promoted cAMP accumulation with a 15fold lower potency than IP 3 accumulation in these cells [START_REF] Qi | Differential coupling of the human P2Y 11 receptor to phospholipase C and adenylyl cyclase[END_REF]. Therefore, we investigated the induction of agonist-induced [cAMP] increase for two relevant ATP concentrations (Fig. 8). The unmutated hP2Y 11 -R caused a significant [cAMP] increase already at 100 µM ATP that was 80% of the maximum response seen at 1 mM ATP. There, the response reaches already the plateau-phase [START_REF] Qi | Differential coupling of the human P2Y 11 receptor to phospholipase C and adenylyl cyclase[END_REF]. For the F109I and A313N receptor mutants we obtained similar results, supporting the notion of a minor influence of these residues on the potency of ATP, as also observed in the calcium measurements. However, the maximal increase in intracellular [cAMP] was much lower than in cells expressing the wild type receptor, suggesting that the phenylalanine residue in TM3 and the alanine residue in TM7 are important for efficient coupling of the receptor to G s . A strong influence on the cAMP response maxima was also found for a point-mutation in the glucagon receptor. Mutation of a phenylalanine to alanine in TM2 of the glucagon receptor lead to a reduced maximal response without affecting the EC 50 value of the agonist [START_REF] Strudwick | Evidence to support a spectrum of active states for the glucagon receptor[END_REF].

In contrast, the glutamate in EL2 of the hP2Y 11 -R seems to play a role in regulating the agonist potency and activity of the receptor. After mutation to alanine the agonist-induced [cAMP] increase was much stronger as compared to the rise with the wild type receptor. Stimulation with 1 mM ATP resulted in a 3-fold higher cAMP content than in cells expressing the unmutated hP2Y 11 -R. The influence of the E186A mutation on the potency of ATP could not be easily quantified in the [cAMP] measurements. It was not clear, whether at 1 mM ATP the maximum response was reached. Attempts of using higher concentrations were unsuccessful because at 10 mM ATP an interference with the cAMP response of the hP2Y 11 -R by other mechanisms took place (data not shown). However, the E186A mutant seems to display a reduced potency in activating adenylyl cyclase.

Similar observations were made when the cAMP accumulation after ATP stimulation was investigated in 1321N1 cells expressing the R268A receptor mutant.

At 100 µM ATP no increase in the cAMP content was found compared to basal levels whereas at 1 mM cells responded like control cells. This reflects the drastically reduced potency of ATP at this receptor mutant as found in the calcium measurements described above. The cells expressing the R268Q receptor mutant showed even higher

[cAMP] values after stimulation with 1 mM ATP than the wild type hP2Y 11 -R-1321N1 cells. For both, the R268A and R268Q receptor mutants, maximal stimulation could not be established, as already described.

Thus, the E186A and R268A,Q receptor mutants seem to be more efficiently coupled to the activation of adenylyl cyclase compared to the wild type hP2Y 11 -R. A similar phenomenon was observed in other GPCR mutants. Mutation of polar residues to alanine in TM6 and TM7 of the glucagon receptor resulted in increased response maxima to glucagon-NH 2 [START_REF] Strudwick | Evidence to support a spectrum of active states for the glucagon receptor[END_REF]. The β 1 receptor carrying a point-mutation in TM2 and the TSH receptor with a naturally occurring mutation in TM1 displayed both a more pronounced basal activity and higher cAMP accumulation after agonist stimulation, compared to the respective wild type receptors [START_REF] Ahmed | Beta-blockers show inverse agonism to a novel constitutively active mutant of beta1-adrenoceptor[END_REF][START_REF] Biebermann | The first activating TSH receptor mutation in transmembrane domain 1 identified in a family with nonautoimmune hyperthyroidism[END_REF].

Taken together, the analysis of G s coupling reveals similar changes in agonist potency in the mutated hP2Y 11 -Rs as compared to G q coupling. Moreover, specific residues in the hP2Y 11 -R seem to be important for controlling the stimulatory extent of cAMP-dependent processes.

Conclusions

The hP2Y 11 -R model calculated here has proven to be adequate for the investigation of the molecular recognition of this receptor. The computed models, which were extensively refined by MD simulation protocols, were not only able to reproduce experimental data but also to predict the affinity of previously untested ligands. We have found that the binding pockets of the hP2Y 1 -R and the hP2Y 11 -R are very similar. We have established that in the hP2Y 11 -R the residues involved in ligand binding are Arg106, Phe109, Ser206, Arg268, Arg307 and Met310. The involvement of Arg106, Tyr261, Arg268, Arg307 and Ala313 in ligand recognition was confirmed by a mutational study. Glu186 in the EL2 of the hP2Y 11 -R, aligned with the critical Asp204 in the hP2Y 1 -R, also proved significant for ligand recognition. Furthermore, mutation of Phe109, Glu186, Arg268, and Ala313 influences coupling of the hP2Y 11 -R to G s , whereas the extent of coupling to G q remains unaffected.

Only minor amino acid residue variations were observed in the binding site of hP2Y 11 -R as compared to hP2Y 1 -R (e.g. Pro311 vs. Ser314, in P2Y 11 -vs. P2Y 1 -R). In the hP2Y 11 -R, His317 appears instead of Asp in the conserved DPxxY motif in TM7, a typical motif for all other P2Y-Rs. This new motif which may trigger a different mechanism of activation, is worth further investigations.

Although the hP2Y 11 -R has a hydrophobic binding-pocket at the vicinity of the C2-position of ATP, similar to that of hP2Y 1 -R (Fig. 6), the significantly lower activity of 2-MeS-ATP at hP2Y 11 -R may be due to the lack of H-binding interactions which are present in the hP2Y 1 -R. Specifically, the Ser314 in the hP2Y 1 -R is probably involved in H-bonding interactions with the sulfur atom of 2-MeS-ATP [START_REF] Major | Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y 1 -receptor[END_REF][START_REF] Major | Molecular recognition in purinergic receptors. 2. Diastereoselectivity of the h-P2Y 1 -receptor[END_REF].

Therefore, we propose that the natural mutation of Ser314 as well as Asn316 in the

Tables

  hP2Y 1 -R (gi:4505557), hP2Y 2 -R (gi:28872720), hP2Y 4 -R (gi:4505561), hP2Y 6 -R (gi:14424758), hP2Y 11 -R (gi:21263830) and b-rhodopsin (gi:129204) [20].

[

  11]. The hP2Y 11 -R contains 27 Pro residues in total and 11 of them are located in the TM regions. TM3 is the only TM domain without a Pro residue. The hP2Y 11 -Runique Pro311 residue replaces a highly conserved Ser residue in the other hP2Y-Rs.

hP2Y 1 -

 1 R to Pro311 and A313 in the hP2Y 11 -R, as compared to other hP2Y-Rs, could be responsible for the lower potency of 2-MeS-ATP as compared to ATP at the hP2Y 11 -R.

Figure Legends:Fig. 1 :

 1 Figure Legends:

Fig. 2 :

 2 Fig. 2: A list of known hP2Y 11 -R agonists with respective EC 50 values for IP 3 elevation given in theTable, as reported by Communi et al. [5].

Fig. 3 :

 3 Fig. 3: Results of virtual screening of the drug-like compounds, random nucleotides and hP2Y 11 -R agonists data sets on the hP2Y 11 -R model. An enrichment factor of 5 was obtained, thus validating the calculated hP2Y 11 -R model. Code: random ligand recovery -black line, enrichment obtained with the hP2Y 11 -R model -grey line.

Fig. 4 :

 4 Fig. 4: A: hP2Y 11 -R model, B: hP2Y 1 -R model, C: b-rhodopsin. Residue color code: Blue -Arg, Lys; Red -Asp, Glu; Yellow -Pro. Loops of the hP2Y 11 -R were not modelled accurately.

Fig. 5 :Fig. 6 :

 56 Fig. 5:Binding mode of ATP at the hP2Y 11 -R. Major binding interactions include ionic interactions with the triphosphate chain -Arg106, Arg268 and Arg307; Hbonding interaction with P γ -Ser206; π-stacking interaction with the adenine ring -Phe109, and interaction with Met310. (See alsoTable 2)

Fig. 7 :

 7 Fig. 7: Concentration-response curves for ATP in inducing intracellular [Ca 2+ ] i rise in 1321N1 cells stably expressing the wild type and mutant P2Y 11 -GFP receptors. Cells

Fig. 8 :

 8 Fig. 8:Intracellular cAMP content determined at basal level and after stimulation with ATP (100 µM, 1 mM) in 1321N1 cells stably transfected with the wild type hP2Y 11 -R, and mutant receptors, respectively. cAMP was determined after 10 min incubation with ATP in cellular extracts by a cAMP-EIA. Bar graphs show the mean and standard error from four separate experiments.

Figure 2 37

 2 Figure 2

Figure 3 Figure 4 Figure 5

 345 Figure 3

  Figure 6

Table 1

 1 Identity (upper triangle) and similarity (lower triangle) percentages between various hP2Y-Rs (number n) and b-Rhodopsin for the TM domains only.

	hP2Y n hP2Y n	14	13	12	11	6	4	2	1	b-Rh
	b-Rh	16	21	20	21	19	20	22	23	100
	1	31	29	26	38	44	49	45

Table 3

 3 Intracellular calcium release induced by stimulation of mutant hP2Y 11 -GFP receptors and expression levels of mutant receptors. Data represent mean EC 50 values (µM) ± s.e.m., obtained from n (numbers) concentration response curves of 1321N1 cells stably expressing the wild type or mutated receptor. The receptor expression level was derived from n experiments.

	hP2Y 11 -R construct	residue	EC 50 value (µM) ATP ATPγS	Receptor expression level (%) (n)
	wt		2.37±0.88 (4) 1.04±0.38 (4)	100
	R106A	3.29	a	n.d.	76.1±4.7 (4)
	F109I	3.32	10.7±2.29 (5)	n.d.	102±13	(7)
	E186A	EL2	32.8±21.1 (5) 4.92±1.07 (4)	111±10	(9)
	Y261A	6.48	a	n.d.	77.4±3.9 (4)
	R268A	6.55	1806±354 (3) 247±178 (8)	143±23	(3)
	R268Q	6.55	102±20 (5) 10.3±2.59 (3)	95.6±8.0 (7)
	R307A	7.39	a	n.d.	87.5±5.9 (4)
	A313N	7.45	4.52±0.99 (7)	n.d.	97.6±11.9 (4)

a ATP induced no significant increase in intracellular calcium up to concentrations of 10 mM. n.d. -not determined. a ref 5.

Biochemical Journal Immediate Publication. Published on 6 Mar 2007 as manuscript BJ20061728 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Dr. T. Hanck and Dr. F. Sedehizadeh for helpful suggestions in the mutagenesis experiments and Ms. D. Terhardt for technical help in the complete experimental study. We thank Dr. R. Hartig (Institute of Immunology, Medical Faculty Magdeburg) for providing the facilities to carry out flow cytometry analysis. BF and JZ thank Mr. M. Amitai (Department of Pharmaceutical Chemistry, School of Pharmacy, Hebrew University, Jerusalem) for helpful discussions.


Robert Wattiaux
email: robert.wattiaux@fundp.ac.be

Simone Wattiaux-De Coninck

Jacqueline Thirion

M C Gasingirwa

Michel Jadot

Lysosomes and Fas mediated liver cell death

Keywords: Apoptosis, lysosomes, Fas, liver, Jurkat cells

A number of studies, mostly performed « ex vivo », suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol owed to a fragilisation of the membrane of these organelles. These enzymes could then be contributing to the permeabilization of the outer mitochondrial membrane, they could also activate effector caspases. The work presented here aims at testing if the membrane of liver lysosomes is disrupted during Fas-mediated cell-death of hepatocytes « in vivo », a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with antiFas antibody (aFas). The state of the lysosomes was assessed by determining the proportion of lysosomal enzymes (ß-galactosidase, ß-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, obtained by high speed centrifugation and in some cases, by analyzing the behaviour in differential and isopycnic centrifugation of ß-galactosidase taken as reference enzyme of these organelles. Apoptosis was monitored by measuring caspase-3 activity with a specific tetrapeptide substrate (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the intermembrane space of mitochondria. Results show that an injection of 10 µg aFas causes a rapid and large increase of DEVDase activity and of unsedimentable sulfite cytochrome c reductase. This neither modifies the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behavior of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5µg) which ensures a longer lifespan of the animals indicate that lysosomal hydrolase unsedimentable activity increases in the

INTRODUCTION

The involvement of lysosomes in apoptosis as suggested by several studies is still highly debatable (For a review, see 1). Lysosome mediated apoptosis would result from a destabilization of the lysosomal membrane causing a release of cathepsins into the cytosol. These proteases would stimulate the mitochondrial pathway of apoptosis by contributing to the permeabilization of the outer mitochondrial membrane, perhaps as a result of Bid cleavage by these enzymes [START_REF] Cirma | Selective disruption of lysosomes in Hela cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins[END_REF]. They could also trigger the cytosolic pathway, by activating effector caspases. However, several caspases, caspase-3 for example, seem to be poor substrates for lysosomal cathepsins «in vitro» [START_REF] Ferri | Organelle-specific initiation of cell death pathways[END_REF]. The aim of our work was to investigate if lysosomes are involved in apoptosis of hepatocytes «in vivo» due to Fas ligation. The Fas apoptotic pathway was selected, because it has been extensively investigated as a model system for mammalian apoptosis. Moreover, Fas mediated cell death of hepatocytes is observed in several severe liver pathologies. Knowing whether lysosomal enzymes are involved «in vivo» in Fas apoptotic pathway may be of some help to explore specific therapeutic targets for cell death inhibition in these pathologies [START_REF] Green | Pharmacological manipulation of cell death: clinical applications in sight ?[END_REF][START_REF] Kroemer | Caspase-independent cell-death[END_REF][START_REF] Czaja | TNF toxicity-Death from caspase or cathepsin, that is the question[END_REF]. Until now, results published on the possible involvement of lysosomes in Fas induced apoptosis have been mostly obtained «ex vivo» and are controversial.

While some suggest that lysosomes are involved [START_REF] Brunk | Oxidative stress, growth factor starvation and Fas activation may all cause apoptosis through lysosomal leak[END_REF][START_REF] Brunk | Lysosomal involvement in apoptosis[END_REF][START_REF] Neuzil | α-Tocopheryl succinate -induced apoptosis in Jurkat T cells involves caspase-3 activation and both lysosomal and mitochondrial destabilisation[END_REF][START_REF] Tardy | Mannose 6phosphorylated proteins are required for Tumor Necrosis Factor-α-induced apoptosis[END_REF] others suggest that they are not involved [START_REF] Guicciardi | Cathepsin B contributes to TNFa-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c[END_REF][START_REF] Werneburg | Tumor necrosis factorα-associated lysosomal permeabilization is cathepsin B dependent[END_REF] in apoptotic death caused by agonistic antiFas. The work presented here aims at testing whether after an injection of anti Fas antibody (aFas) the lysosomal membrane is disrupted in mouse liver. Our method is simple and it involves determining the proportion of lysosomal enzymes found in high-speed supernatants of liver homogenates originating from controls and from animals injected with aFas. If lysosomes are involved in apoptosis after injection of aFas, one would expect that lysosomal enzymes would be released into the cytosol as a result of the injection and that an increase in proportion of unsedimentable lysosomal hydrolases in homogenates should definitely be observed, as it has been seen for example in ischaemic liver necrosis [START_REF] Wattiaux | Effect of a transitory ischemia on the structure-linked latency of rat-liver acid phosphatase and ß-galactosidase[END_REF][START_REF] Wattiaux | Effects of ischaemia on lysosomes[END_REF]. Hepatocyte apoptosis was assessed by measuring the activity of the major executioner caspase, caspase-3, with a sitespecific tetrapeptide substrate (DEVDase activity) and by determining the percentage of sulfite oxidase released in the high-speed supernatant of the homogenate. This enzyme is located in the intermembrane space of mitochondria [START_REF] Wattiaux-De Coninck | Subcellular distribution of sulfite cytochrome c reductase in rat-liver tissue[END_REF] and is co-released with cytochrome c into the cytosol during apoptosis [START_REF] Kluck | The proapoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol[END_REF]. The measurement of this enzymatic activity with cytochrome c as electron acceptor (sulfite cytochrome c reductase) allows quantification of the proportion of mitochondria whose outer membrane is permeabilized. This is easier than the determination of immunoprecipitated cytochrome c. In a limited number of experiments, the state of lysosomes was assessed by analyzing their behaviour in differential and isopycnic centrifugation. Experiments were first performed by injecting 10 µg aFas to animals. In these conditions, a rapid and massive apotosis of the liver takes place as assessed by morphological and biochemical observations [START_REF] Woo | In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes[END_REF][START_REF] Mootha | A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c[END_REF][START_REF] Rodriguez | Systemic injection of tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death[END_REF] and causes the death of most animal after one to two hours. Our results show that in that case, aFas injection causes a large and rapid increase of DEVDase activity and of unsedimentable sulfite cytochrome c reductase without modifying the proportion of unsedimentable activity of lysosomal enzymes. If the amount of aFas injected is lower (5 µg), the development of apoptosis is slower, the peak of DEVDase activity and of unsedimentable sulfite cytochrome c reductase is observed later and the animal can be kept in life for several hours. In these conditions, the percentage of unsedimentable lysosomal enzymes begins to increase, 2 to 3 hours after injection, indicating a progressive damage of lysosomes with time. For a sake of comparison, similar experiments were performed «ex vivo» with Jurkat cells. In these cells, involvement of lysosomes in Fas mediated apoptosis has been described [START_REF] Neuzil | α-Tocopheryl succinate -induced apoptosis in Jurkat T cells involves caspase-3 activation and both lysosomal and mitochondrial destabilisation[END_REF]. Our results show that aFas treatment of these cells causes an increase of the unsedimentable activity of lysosomal enzymes but that takes place with a marked delay with respect to caspase-3 activation (DEVDase). Such increase almost coincides with a release into the culture medium of dipeptidylpeptidase III and DEVDase, two cytosolic enzymes.

EXPERIMENTAL « In vivo » experiments

«In vivo» experiments were performed with female NMRI mice weighing 20-25 g. Animals were cared for using protocols approved by the FUNDP Commission d'éthique et en expérimentation animale. Mice were injected intravenously via tail vein with anti-Fas antibody (BD Biosciences Pharmingen) in a volume of 200 µl saline or with 200 µl saline for control experiments. Mice were sacrificed after selected times, the liver was removed and homogenized in ice-cold 0.25 M sucrose with a Potter & Elvehjem type homogenizer consisting of a smooth -walled glass tube fitted with a Teflon pestle (Arthur Thomas Co, Philadelphia, USA), rotating at 3000 rpm. Part of the homogenate was centrifuged at 35 000 rpm for 40 min in a 40 Beckman rotor to obtain an high-speed supernatant for determination of unsedimentable enzyme activity. Fractionation of the homogenate by differential centrifugation was achieved using the method of de Duve et al. [START_REF] De Duve | Tissue fractionation studies. VI. Intracellular distribution patterns of enzymes in rat-liver tissue[END_REF]. A nuclear fraction N, a heavy mitochondrial fraction M, a light mitochondrial fraction L, a microsomal fraction P and a soluble fraction S were successively isolated by integrated forces, respectively of 10 000, 33 000, 250 000 and 3 000 000 g.min using the fractionation scheme described by these authors [START_REF] Kluck | The proapoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol[END_REF].

For isopycnic centrifugation, preformed linear density gradients of Percoll (27%-90%) with 0.25 M sucrose as a solvent were used and centrifugation was performed at 25 000 rpm in the SW 65 Beckman rotor for 60 min.

«Ex vivo» experiments

«Ex vivo» experiments were carried out with Jurkat cells (clone E 6-1) cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 10 mM Hepes, penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamine (0.03%). For the experiments, incubation was performed at a density of 2.10 6 cells /ml, in absence or in presence of human aFas (clone CH-11, Bio Connect, Huissen, The Netherlands). For the preparation of homogenates, cells were collected by centrifugation, suspended in 0.25 M sucrose and sonicated for 5 sec (SONIC Vibra Cells, amplitude 40). The efficiency of homogenisation was checked by phase contrast microscopy. In addition, the percentage of dipeptidyl peptidase III, a cytosolic enzyme, recovered in high speed supernatants of homogenates was regularly measured; it was always greater than 90%.of the homogenate total activity , indicating that most of the cells are disrupted by the homogenisation procedure. On the other hand, as shown in the results, only 10 to 15% of lysosomal enzyme activities were found in high speed supernatant of homogenates originating from control cells, illustrating that homogenisation did not disrupt the majority of normal lysosomes. High-speed supernatants were prepared as described for the liver

Enzyme measurements

To measure caspase-3 like activity (DEVDase activity), aliquots of homogenates were incubated at 37°C with 50 µM Ac-DEVD-AMC (N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin), 50 mM Tris HCl buffer, pH 7.5, 0.1 M NaCl, 0.1% Triton X-100 and 10 mM dithiothreitol in a volume of 0.2 ml. The reaction was stopped by adding 1.3 ml of 50 mM glycine buffer, pH 10.5, containing 5 mM EDTA and 0.5% Triton X 100. To assay ß-glucuronidase activity, the medium contained 1 mM 4-methylumbelliferyl-ß-D-glucuronide, 20 mM acetate buffer pH 5, 0.05% Triton X-100 in a volume of 0.2 ml. Incubation was performed at 37°C and the reaction stopped by adding 1.3 ml of 50 mM glycine buffer pH 10.5, containing 5 mM EDTA and 0.5% Triton X-100. Medium for the determination of cathepsin B contained 250 µM Z-Arg-Arg-7-amido-4methylcoumarin, 0.1 M phosphate buffer pH 6, 5 mM aminoethanethiol, 1 mM EDTA and 0.05% Triton X-100 in a volume of 0.5 ml. The reaction was stopped by adding 1.3 ml of 50 mM glycine buffer pH 10.5, containing 5 mM EDTA and 0.5% Triton X-100. For dipeptidylpeptidase III, incubation was carried out in a medium containing 0.25 mM Arg-Arg-naphthylamide and 25 mM Tris buffer, pH 8 in a volume of 0.2 ml. Reaction was stopped by adding 1.3 ml of 50 mM glycine buffer, pH 10.5, containing 5 mM EDTA and 0.5% Triton X 100. In each determination, the released fluorogenic group (aminomethyl-coumarin , 4-methyl umbelliferone or naphthylamine) was determined with a Versa Fluor Fluorometer, (Bio-Rad, Nazareth-Eke, Belgium), excitation filter 170-2420, emission filter 170-2421. ß-galactosidase and cathepsin C were measured as described by Lecocq et al. [START_REF] Lecocq | Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume[END_REF], sulfite cytochrome c reductase by the method of Wattiaux-De Coninck and Wattiaux [START_REF] Wattiaux-De Coninck | Subcellular distribution of sulfite cytochrome c reductase in rat-liver tissue[END_REF] and cytochrome oxidase as described by de Duve et al. [START_REF] De Duve | Tissue fractionation studies. VI. Intracellular distribution patterns of enzymes in rat-liver tissue[END_REF].

RESULTS

Initially, experiments were performed by injectig 10 µg of aFas. Such an injection causes a massive apoptosis of hepatocytes as assessed by biochemical and morphological criteria [START_REF] Woo | In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes[END_REF][START_REF] Mootha | A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c[END_REF][START_REF] Rodriguez | Systemic injection of tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death[END_REF] and the death of the animals 90 to 120 min after injection. To monitor liver apoptosis, we examined the chronological changes of DEVDase activity and of unsedimentable sulfite cytochrome c reductase taking place after aFas injection. The unsedimentable activity of ß-galactosidase, a reference hydrolase of lysosomes was estimated to see whether there is a possible rupture of these organelles' membrane. As shown in Figure 1, injection of aFas causes first a rapid increase of homogenate DEVDase activity up to a maximum 60 to 80 min after injection. With time, the percentage of unsedimentable sulfite cytochrome c reductase increases and by about 80 min, it is up to 95% of homogenate activity after 80 min, indicating that at that time most of the mitochondria have a permeabilized outer membrane. The percentage of unsedimentable ß-galactosidase is low before aFas injection (± 10%) and is not modified by such an injection. Even when apoptosis caused by aFas injection is stimulated by a fructose injection [START_REF] Latta | Metabolic depletion of ATP by fructose inversely controls CD95 and tumor necrosis factor receptor 1-mediated hepatic apoptosis[END_REF], one does not observe an increase of unsedimentable ß-galactosidase activity. The total and unsedimentable activities of three other lysosomal hydrolases, ß-glucuronidase, cathepsin C and cathepsin B were measured in the control homogenates and in the homogenates obtained 80 min after aFas injection. As shown in Figure 2A the proportion of these enzyme activities recovered in the homogenate supernatants is not increased by aFas injection, as observed for ß-galactosidase. Moreover, the total liver activities of the four lysosomal hydrolases are not significantly decreased after the injection (Figure 2 B). This indicates that the low proportion of their unsedimentable activity does not result from a loss or an inhibition of the hydrolases after they would have been released into the cytosol of apoptotic cells.

The previous results strongly suggest that liver lysosomes remain intact until the animal death, during apoptosis resulting from an injection 10 µg of aFas. To obtain additional information on the state of lysosomes under these conditions, we have determined the behaviour of these organelles when they are subjected to differential and isopycnic centrifugation in isoosmotic sucrose. Such an experimental approach allows to determine if the size and the density of the particles are modified after aFas injection. Figure 3 illustrates the distribution of ß-galactosidase taken as a reference enzyme for lysosomes, after differential centrifugation in 0.25 M sucrose performed according to de Duve et al [START_REF] De Duve | Tissue fractionation studies. VI. Intracellular distribution patterns of enzymes in rat-liver tissue[END_REF]. This was done on a liver homogenate originating from a control mouse and from a mouse injected with aFas sacrificed 60 min after injection. Obviously, both distribution patterns are similar, ß-galactosidase is mainly recovered in M and L fractions and present in low amount in the S fraction. This is in contrast to the distribution of sulfite cytochrome c reductase, as aFas injection strongly increases the percentage of that enzyme associated with the unsedimentable S fraction to the detriment of the N and M sedimentable fractions. These results indicate that aFas injection does not modify the average sedimentation coefficient of lysosomes in isoosmotic sucrose and therefore does probably not cause a swelling of these organelles as it is seen in the change from apoptosis to necrosis during cell death [START_REF] Ono | Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor a induced cell death[END_REF]. That the liver lysosomes are not swollen after aFas injection is supported by the results of isopycnic centrifugation reported in Figure 4. M+L fractions isolated from the liver of a control and from an aFas injected mouse sacrificed 60 min after injection, were subjected to an isopycnic centrifugation in a Percoll gradient with 0.25 M sucrose as a solvent. This procedure allows one to determine the density of a particle under iso-osmotic conditions. The distribution curve of ß-galactosidase is the same whether the M+L fraction originates from the control or the aFas injected mouse, indicating that the lysosome mean density in isoosmotic conditions, is not affected by the injection. This is contrary to the behavior of mitochondria as ascertained by the distribution curve of reference enzyme cytochrome oxidase. Such curve is markedly shifted towards the low-density zones of the gradient after aFas injection, probably as a result of the swelling of the mitochondria that takes place during apoptosis [START_REF] Kluck | The proapoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol[END_REF]. It is also worthwhile to mention that these results show that sedimentable lysosomal enzymes, present in the liver homogenates after aFas injection, are truly associated with lysosomes. They could not be an artefact arising due to readsorption after their release on sedimentable structures such as mitochondria, fragments of endoplasmic reticulum, Golgi or plasma membrane. In summary, our results show that an injection of 10 µg aFas to mice that causes a massive apoptosis of the liver leading to a quick death of the animal does not cause a leakage of lysosomes.

In a second set of experiments, we investigated the possible influence of the agonist dose on liver apoptosis caused by aFas injection.We found that apoptosis was delayed when the amount of injected aFas was decreased along with a increase in the duration of life of treated animals. As illustrated in Fig 5, after an injection of 5 µg of aFas, the increase of DEVDase and of unsedimentable sulfite cytochrome c reductase with time is slower than after a 10 µg injection, a maximum value is obtained only after 4 h and is followed by a marked decrease in activity for both enzymes. Unsedimentable activity of the lysosomal hydrolases, ß-galactosidase, cathepsin C and ß glucuronidase do not change until about two hours after injection. Later, a steady increase occurs indicating a progressive leakage of lysosomes.

The cell death pathway triggered by Fas ligation in Jurkat cells is a prototypic model of apoptosis «ex vivo». An involvement of lysosomes has been described in that process [START_REF] Neuzil | α-Tocopheryl succinate -induced apoptosis in Jurkat T cells involves caspase-3 activation and both lysosomal and mitochondrial destabilisation[END_REF]. This is why we were interested in seeing if a leakage of lysosomes can be observed with our methodology in Jurkat cells treated with aFas. We used homogenates originating from Jurkat cells that had been cultured in presence of aFas (50 ng/ml) for increasing times. Apoptosis was monitored by measuring DEVDase, leakage of lysosomes was assessed by determining the percentage of unsedimentable ß-galactosidase and cathepsin C. In addition, permeabilisation of plasma membrane was appraised by measuring in the culture medium, two cytosolic enzymes dipeptidyl peptidase III [START_REF] Parsons | Separation of rat muscle aminopeptidases[END_REF] and DEVDase [25]. Results are presented in Fig 6 . An increase of DEVDase activity is observed after aFas addition to the medium and about two hours are required to obtain a maximum value. On the other hand, an increase of unsedimentable lysosomal hydrolases occurs in the homogenate but with a delay with respect to DEVDase increase. Interestingly, in parallel, an increase of dipeptidyl peptidase III activity is seen in the culture medium. The same is true for DEVDase, an enzymatic activity that can only originate from cells in which caspase-3 activation -therefore apoptosis-occured. These results strongly suggest that the leakage of lysosomes we observed after aFas treatment is associated with a permeabilisation of the plasma membrane. Similar results (not shown) were obtained in a more limited number of experiments by treating Jurkat cells with aFas at a concentration of 20 ng and 200 ng/ml.

DISCUSSION

Our results show that if the injection to mice of aFas (10 µg/ animal) induces a fast and massive apoptosis of the liver leading to a rapid death of the animals, liver lysosomes remain intact, their enzymes are not required to cause death of hepatocytes. If the dose of aFas is lower (5 µg/ animal) and leads to a slower development of apoptosis aswell as a prolonged survival of animals, an increase of unsedimentable activity of lysosomal enzymes in the homogenates can be observed 2 or 3 h after the injection, indicating a disruption of lysosomes inside the cells or the organelles becoming more fragile making them more susceptible to homogenisation procedure. On the other hand, after 4h, DEVDase activity decreases. That could result from an inactivation of the enzyme in dead cells [START_REF] Vercammen | Dual signaling of the Fas receptor : Initiation of both apoptotic and necrotic cell death pathways[END_REF] or due to a release from liver cells owing to a plasma membrane leakage. A similar explanation could be used to explain the decrease of unsedimentable sulfite cytochrome c reductase activity. In Jurkat cells, aFas causes an increase of the unsedimentable activity of lysosomal hydrolases but that begins later than the caspase-3 activation (DEVDase).

Our results, «in vivo» and «ex vivo» could be explained by supposing that aFas can agress cells by two processes , one that does not involve lysosomes and begins relatively early and a second one that takes place later and imply lysosomes. According to previous biochemical and morphological observations [START_REF] Woo | In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of hepatocytes[END_REF][START_REF] Mootha | A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c[END_REF][START_REF] Rodriguez | Systemic injection of tripeptide inhibits the intracellular activation of CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death[END_REF] and our observations, it is probable that the first process corresponds to apoptosis. In Jurkat cells, the second process goes with a rupture of plasma membrane as illustrated by the release of cytosolic enzymes into the medium. That strongly suggests that it is a necrotic one and could explain the leakage of lysosomes, a phenomenom frequently observed in cell necrosis [START_REF] Guicciardi | Lysosomes in cell death[END_REF][START_REF] Wattiaux | Effect of a transitory ischemia on the structure-linked latency of rat-liver acid phosphatase and ß-galactosidase[END_REF][START_REF] Wattiaux | Effects of ischaemia on lysosomes[END_REF][START_REF] Ono | Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor a induced cell death[END_REF][START_REF] De Duve | Functions of lysosomes[END_REF] The striking similarity that we observe between the time courses of cytosolic enzyme release and the lysosome leakage is in agreement with the recent observations that plasma membrane disruption and necrosis are linked to the susceptibility of lysosomes to rupture (23) A same reasoning can probably be applied to liver. Leist et al. [START_REF] Leist | The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure[END_REF] have shown that after aFas injection to mice, apoptosis of hepatocytes, characterized by DNA fragmentation, largely precedes lysis of these cells, monitored by serum alanine aminotransferase level. Our interpretation is strongly supported by several observations suggesting the presence of a necrotic death pathway in Fas receptor signalling. Matsumura et al [START_REF] Matsumura | Necrotic death pathway in Fas receptor signaling[END_REF] have reported that in Jurkat cells, both apoptotic and necrotic death pathways are activated through the Fas receptor. As shown by Vercammen et al. [START_REF] Vercammen | Dual signaling of the Fas receptor : Initiation of both apoptotic and necrotic cell death pathways[END_REF], Fas triggering in fibrosarcoma cells leads first to a rapid and strong apoptotic response followed after 3h by a necrotic process, these two death pathways co-existing in the same cell. According to Kawahara et al [30], Fas associated protein with death domain (FADD) can cause a necrotic death of Jurkat cells.

Leist et al. [START_REF] Leist | The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure[END_REF] have shown that activation of Fas «in vivo» and «ex vivo» causes first apoptosis of hepatocytes followed by their necrosis. That we do not observe a leakage of lysosomes in liver after an injection of 10 µg aFas is probably explained by the fact that in these conditions, the massive apoptosis of hepatocytes induces a rapid death of the animal due to a fulminant liver failure, before the development of the slower necrotic process could occur. It is to be noted that the increase of undedimentable lysosomal enzyme after aFas treatment is relatively modest. The value found in the control liver and Jurkat cells homogenates is more or less 10 to 12 % of the total activity. After aFas treatment, it reaches 30 to 35%. If we refer to the postulate of biochemical homogeneity of subcellular structures [START_REF] De Duve | Tissue fractionation studies. VI. Intracellular distribution patterns of enzymes in rat-liver tissue[END_REF], that would mean that 20 to 25% of the lysosomes (on a mass basis) become fragile with the treatment. This could originate from the fact that on an average 20-25% of lysosomes are leaky in each cell or that all the lysosomes in 20-25% of the cells have become fragile What causes the leakage of lysosomes induced by Fas triggering ? There are probably several factors that are involved. First, activated caspases could destabilize lysosomes by proteolysis of their membrane. Many years ago, it was shown that proteolytic enzymes could disrupt lysosomal membrane «in vitro» [START_REF] Beaufay | Tissue fractionation studies. 9. Enzymic release of bound hydrolases[END_REF]. More recently, it has been reported that incubation of lysosomes with caspase-8 makes the lysosomal membrane more fragile [START_REF] Guicciardi | Cathepsin B contributes to TNFa-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c[END_REF]. A second possibility is that reactive oxygen radicals that accumulate in cells after stimulation of Fas receptor [START_REF] Vercammen | Dual signaling of the Fas receptor : Initiation of both apoptotic and necrotic cell death pathways[END_REF] disrupt lysosomal membrane as observed «in vitro» [START_REF] Decharneux | Effect of various flavonoids on lysosomes subjected to oxidative and osmotic stress[END_REF]. A third factor that could play a role is the strong decrease of cellular ATP concentration. aFas treatment brings about a progressive depletion of cytochrome c in mitochondria with consequently a mitochondrial respiratory dysfunction [START_REF] Mootha | A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c[END_REF] and a decrease of ATP production. As shown by Leist et al. [START_REF] Leist | LEGENDS Figure1 Caspase-3-like protease (DEVDase) activation (A) and unsedimentable activities (B) of sulfite cytochrome c reductase (open squares) and ß-galactosidase (open triangles) at increasing time after injection of anti Fas antibody (10 µg/animal). Determinations were performed on total homogenate for DEVDase, on total homogenate and on high speed supernatant (35 000rpm for 40 min, Beckman rotor type 40) for sulfite cytochrome c reductase and ß-galactosidase. DEVDase activity was given in nmoles methylumbelliferone released/ min/ liver. The unsedimentable activity was expressed as percentage of the total activity present in the homogenate. Error bars represent the standard error of the mean for at least three animals. Closed symbols give the values obtained after Caspase-3-like protease (DEVDase) activation (A) and unsedimentable activities of sulfite cytochrome c reductase (B), ß-galactosidase (C), cathepsin C (D) and ß-glucuronidase (E) at increasing time after injection of anti Fas antibody (5 µg/ animal). Determinations were performed on total homogenate for DEVDase, on total homogenate and on high speed supernatant (35 000rpm for 40 min, Beckman rotor type 40)[END_REF], ATP depletion induces a change from apoptosis to necrosis. Such depletion is associated with an increase of lysosome size, making these organelles fragile and favouring their rupture [START_REF] Ono | Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor a induced cell death[END_REF] Most of the studies aimed at investigating wether lysosomes are involved in Fas mediated apoptosis have been done on isolated cells. Our observations allow to compare results obtained «in stimulation of apoptosis by an intraperitoneal injection of fructose (250 mg) 15 min before aFas injection . but not for unsedimentable lysosomal hydrolases ( P> 0.05). To uniformize the presentation, total activity measured in the aFas injected mice liver homogenates is given as percentage of the activity found in control mice liver homogenates supposed to be equal to 100. Error bars represent the standard error of the mean for at least three animals. Values found in homogenates of injected and control mice do not significantly differ (P > 0.05).

Figure 2 Unsedimentable

 2 Figure 2 Unsedimentable (A) and total activities (B) of sulfite cytochrome c reductase (a), ß-galactosidase (b), ß-glucuronidase (c), cathepsin C (d) and cathepsin B (e) present in liver homogenates originating from mice injected with saline (open bars) or with antiFas antibody (10 µg/animal) (closed bars) and sacrificed 80 min after injection. Unsedimentable activity was expressed as percentage of the activity present in the homogenate. For sulfite cytochrome c reductase and ßgalactosidase, values indicated in Figure 1 have been used. Values for unsedimentable activity of sulfite cytochrome c reductase of treated versus control mice are significantly different ( P <0.01)

Figure 3 Distribution

 3 Figure 3 Distribution of ß-galactosidase (A) and of sulfite cytochrome c reductase (B) after differential centrifugation in 0.25M sucrose. N, nuclear fraction, M, heavy mitochondrial fraction, L, light mitochondrial fraction, P, microsomal fraction, S , soluble fraction. Liver originates from a saline injected mouse (open bars) or from a aFas injected mouse killed 60 min after injection (closed bars) DEVDase activities of homogenates were respectively of 0.1 nmole/min/liver for the control and 90.1 nmoles/min/liver for aFas injected mouse.

Figure 4 Distribution

 4 Figure 4Distribution of ß-galactosidase (A) and cytochrome oxidase (B) after isopycnic centrifugation in a Percoll gradient, with 0.25 M sucrose as a solvent, of a total mitochondrial fraction (M+L) isolated from the liver of a saline injected mouse (open squares) or of a aFas injected mouse killed 60 min after injection (closed squares). M+L fraction was layered at the top of the gradient. Centrifugation was performed at 25 000 rpm for 60 min in the Beckman rotor SW 65. Fractions are numbered from the top to the bottom of the gradient. DEVDase activities of homogenates were respectively of 0 nmoles/min/liver for the contrôle and 0 nmoles/min/liver and 66.1 nmoles /min/liver for aFas injected mouse

Biochemical Journal Immediate Publication. Published on 27 Nov 2006 as manuscript BJ20061738 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 27 Nov 2006 as manuscript BJ20061738

ACKNOWLEDGEMENTS

This work was supported by the Fonds de la Recherche Fondamentale Collective (FRFC). MC Gasingirwa holds a fellowship from Coopération Universitaire au Développement (CUD). Authors are indebted to Sandra Misquith for helping in the preparation of the manuscript and Michel Savels for the artwork.