Thierry Magnin
email: magnin@esbs.u-strasbg.fr

Cédric Fiez-Vandal

Noelle Potier-Holler

Aline Coquard

Isabelle Leray

Tania Steffan

Christel Logez

Fatima Alkhalfioui

Franc Pattus

Renaud Wagner

Noëlle Potier

A novel, generic and effective method for the rapid

Keywords: G protein-coupled receptor Pichia pastoris Solubilization Purification Cannabinoid Opioid Ligand GPCRs, G protein-coupled receptors, SAV, streptavidin, TEV, tobacco etch virus, PBS, phosphate buffer saline, CHS, cholesteryl hemisuccinate

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Considerable research is ongoing in the area of membrane protein structure and function, and in recent years many efforts have been, in particular, focused on G protein-coupled receptors (GPCRs). 1 These proteins share a conserved architecture based on seven transmembrane segments; they are the key players of signal transduction and constitute the major group of drug targets for the pharmaceutical industry, with already more than half of modern drugs and almost 25% of the top 200 best-selling drugs targeting them [START_REF] Fredriksson | The G-proteincoupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints[END_REF]. Despite these crucial biological implications, very little is known about the detailed molecular mechanisms by which these membrane proteins recognize their extracellular stimuli and transmit the associated messages. The speed of discovery of novel GPCR-active drugs is hampered by the lack of structural information about these proteins, which is reflected by the fact that, to date, only rhodopsin, the rod-cell GPCR involved in dim light vision and the b 2 -adrenergic receptor, which plays an important role in cardiovascular physiology, have been crystallized and their structures solved at 2.2 and 2.4 Å, respectively [START_REF] Okada | The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure[END_REF]3].

For several protein families, including most of helical membrane proteins and in particular GPCRs, structural studies are still limited by low expression levels in heterologous systems 58 and/or by difficulties in obtaining pure, homogeneous protein 59 preparations [START_REF] Lacapère | Determining 531 membrane protein structures: still a challenge![END_REF]. 60 During the last decade, the methylotrophic yeast Pichia pastoris 61 has been increasingly used for heterologous GPCR expression 62 [START_REF] Noguchi | Purification of human b 2 -adrenergic receptor expressed 534 in methylotrophic yeast Pichia pastoris[END_REF][START_REF] Weiß | Expression of an integral membrane 536 protein[END_REF][START_REF] Fraser | Expression of a glycosylation deficient version of the human 539 adenosine 2a receptor for structural studies[END_REF][START_REF] Sarramegna | Heterologous expression of 542 G-protein-coupled receptors: comparison of expression systems from the 543 standpoint of large-scale production and purification[END_REF]. This yeast is particularly suitable for these applications, 63 combining multiple advantages: (i) the relatively low costs related 64 to protein expression; (ii) the availability of protease deficient lulose membrane. The membrane was developed either with the anti-FLAG M2 antibody or with alkaline phosphatase conjugated streptavidin.

In vitro biotinylation

Membranes (50 lg) were mixed with 10 mM ATP, 10 mM MgOAc, 50 lM biotin, 50 mM bicine buffer (pH 8.3) with or without 12,500 U of biotin-protein ligase (BirA; Genecopoeia).

The suspensions were incubated at 30 °C for 40 min, then submitted to SDS-PAGE and Western blotting. Biotinylated proteins were detected as described above.

Solubilization

Detergents were obtained from Anatrace except for CHAPS (Euromedex). Membranes were diluted to obtain a protein concentration of 1 mg/ml in the solubilization buffer (final concentrations: 50 mM sodium phosphate pH 7.4; 10% glycerol; 400 mM NaCl). Then the detergent n-dodecyl b-D-maltopyranoside (DDM) was added to a final concentration of 1% and the solution was stirred at room temperature for 15 min. After ultracentrifugation (150,000g, 45 min), the supernatant was directly used for purification.

Purification with streptavidin beads

The supernatant obtained after solubilization was mixed with streptavidin-coated beads (see below) at various ratios. This preparation was kept overnight at 4 °C with orbital shaking; then the suspension was centrifuged at 1000g for 5 min, the supernatant was removed and the beads were incubated for 1 h at 30 °C in solubilization buffer supplemented with 0.1% DDM and endoglycosidase (PNGase F, 1000 U). The beads were then sequentially washed with (i) solubilization buffer supplemented with 1% CHAPS, (ii) solubilization buffer supplemented with 0.1% DDM, (iii) solubilization buffer supplemented with NaCl (1.5 M final concentration) and 0.1% DDM, (iv) solubilization buffer supplemented with 1 M NaSCN and 0.1% DDM, (v) three times with solubilization buffer supplemented with 2 mM DTT and 0.1% DDM (TEV buffer).

All washing steps were performed at room temperature with gentle orbital shaking. Immobilized receptor were then cleaved off their tags and therefore released from the beads by addition of TEV protease (1 lg of protease for 20 mg of starting membrane proteins) and shaking at 25 °C for 0.5-16 h.

Preparation of streptavidin, streptavidin-coated agarose beads (SAV beads) and TEV protease

Streptavidin was overproduced inE. coli and purified as described [START_REF] Gallizia | 550 Arosio, Production of a soluble and functional recombinant streptavidin in 551 Escherichia coli[END_REF]. The protein was dialyzed against 0.1 M NaHCO 3 pH 8.3 containing 0.5 M NaCl and then concentrated on a vivaspin concentrator (30 kDa cut-off). The protein (0.8 mg/ml final concentration) was then coupled to cyanogen bromide-activated sepharose 4B (Sigma) according to the manufacturer's instructions, except that the sepharose beads were pre-hydrolyzed for 2 h at pH 8.3 before adding streptavidin. The beads were kept at 4 °C in PBS supplemented with 0.02% sodium azide (four volumes of PBS/azide added to one volume of wet beads), and no decrease in the GPCR capture efficiency was noticed after 6 months of storage. In some experiments, used streptavidin beads were recycled by denaturation with 8 M guanidine-HCl in 50 mM citrate pH 3.0 followed by renaturation with a decreasing guanidine concentration gradient. The capacity of these recycled beads to immobilize receptors was decreased by 40-60%, when compared with unused beads.

TEV protease was overexpressed in E. coli BL21(DE3)pLysS (overnight induction in LB at 23 °C with 0.5 mM IPTG) and then

U N C O R R E C T E D P R O O F

Results

GPCRs were overexpressed in P. pastoris as previously described [START_REF] André | Enhancing functional production of G 547 protein-coupled receptors in Pichia pastoris to levels required for structural 548 studies via a single expression screen[END_REF]. The recombinant proteins comprise an N-terminal a-factor signal sequence from Saccharomyces cerevisiae followed by a Flag tag, a deca-histidine-tag, a tobacco etch virus (TEV) protease site, the GPCR open reading frame, a second TEV site and finally the Propionibacterium shermanii biotinylation domain at the C-terminus.

Our earlier purification schemes relied on the presence of the N-terminal deca-histidine and Flag tags, but this strategy gave unsatisfactory results because of the poor specificity and efficiency of these tags. In these recombinant proteins, the C-terminal transcarboxylase biotinylation domain had been added mainly to detect the protein throughout the purification steps and to maintain its stability [START_REF] Lundstrom | Structural genomics on membrane proteins: comparison of more than 558 100 GPCRs in 3 expression systems[END_REF][START_REF] Grünewald | Production of 560 the human D2S receptor in the methylotrophic yeast P. Pastoris[END_REF]. As an alternative to the first purification strategy, we decided to take advantage of the biotinylation domain to efficiently capture the GPCRs on SAV-agarose beads. The protein is immobilized on the beads through high affinity interaction, and can undergo subsequent washing treatment and might also be reconditioned. The final step relies on exploiting the TEV protease sites flanking the receptor to release the purified protein from the beads (Fig. 1).

In vivo biotinylation of recombinant GPCRs expressed in Pichia pastoris

A prerequisite for the development of such a purification method is that the receptors are efficiently biotinylated in vivo.

Several studies conducted on membrane proteins fused with biotinylation tags and expressed in P. pastoris have shown that this yeast indeed possesses a biotin ligase (ortholog of the E. coli BirA protein) which biotinylates not only several low abundance Pichia proteins but also heterologous proteins [START_REF] Weiß | Comparative biochemical and 563 pharmacological characterization of the mouse 5HT 5A 5-hydroxytryptamine receptor and the human b 2 -adrenergic receptor produced in the methylotrophic yeast Pichia pastoris[END_REF][START_REF] Julien | Simple purification of highly active biotinylated P-glycoprotein: enantiomer-specific modulation of drugstimulated ATPase activity[END_REF]. Thus, we first verified that the Pichia strains used in this study effectively contained a biotin ligase activity and that the level of in vivo biotinylation was sufficient to directly undertake purification on streptavidin-agarose. For this purpose, yeast cells overexpressing four different GPCRs or yeast cells transformed with an empty vector were grown in induction conditions. Then, membrane proteins were extracted, submitted to SDS-PAGE and transferred to a nitrocellulose membrane prior to the detection ). This absence of signal sequence processing was reported 289 for the mouse 5HT 5A receptor also expressed in P. pastoris [START_REF] Weiß | Comparative biochemical and 563 pharmacological characterization of the mouse 5HT 5A 5-hydroxytryptamine receptor and the human b 2 -adrenergic receptor produced in the methylotrophic yeast Pichia pastoris[END_REF]. ). Such SDS-resistant dimers/higher order oligomers have 303 been reported for an ever-increasing number of GPCRs [START_REF] Javitch | The ants go marching two by two: oligomeric structure of Gprotein-coupled receptors[END_REF]. the pre-hydrolysis of the beads which we included prior to streptavidin coupling most probably leads to a lower density of SAV on the beads and thus reduces the steric hindrance problems that could be associated with a higher SAV density.

After solubilization, receptors were captured on SAV-coated beads in a batch mode. Surprisingly, very small quantities of the receptors bound to the beads within the first hours, although the streptavidin-biotin interaction is the strongest non-covalent biological interaction (K a = 10 15 M À1 ) between a protein and its ligand known to date. The bond formation between SAV and biotin is normally rapid and essentially non-reversible, suggesting that most probably, in our case, steric hindrance due to the interaction of the biotinylated C-terminal part of the tagged GPCR with neighboring parts of the protein prevented the rapid capture of biotin by SAV. However, the quantity of bound protein increased with time, and an overnight contact at 4 °C proved to be optimum. Longer incubation times, even at higher temperatures (20-37 °C), did not increase the yield (data not shown). The optimal amount of home-made SAV-agarose resin necessary for the purification, starting from 20 mg of membrane proteins, was calculated to be 1.2 ml of resin. Increasing the resin quantity did not improve the yield of purified protein but actually slightly increased the amount of contaminants.

After capture of CNR2 on SAV, the beads were incubated in presence of an endoglycosidase (PNGase F). They were further extensively washed to remove non-specific contaminants. Several washing buffers were tested, including high and low pH buffers, high detergent and high salt buffers. The best results in terms of final receptor purity consisted in sequential washes with buffers containing (i) 1% CHAPS for removal of contaminants interacting through hydrophobic bonds, (ii) 1.5 M NaCl for contaminants bound through hydrophilic interactions and in some cases and (iii) the chaotropic agent NaSCN (1.0 M) for very strongly bound contaminants. Then, the beads were equilibrated in TEV buffer containing 2 mM DTT which is an essential component to maintain the functionality of the TEV protease over long periods of time. The TEV protease cleaves off the receptors from the SAV-coated beads to which the receptors are bound by their C-terminal biotinylated tag, and also releases the N-terminal tags from the receptors. After addition of the protease, a time-course was performed to estimate the optimal incubation time: Fig. 3 shows that the cleaved receptor can be detected after 1 h of incubation (lane 2). Its quantity increased from 1 to 3 h (lanes 1-4). However, the maximum amount of receptor was obtained either after 3 h (lane 4) or after overnight incubation (lane 5). Two bands can be seen, the main one at 36 kDa and a more hazy band at $40 kDa. Both were identified as the cannabinoid receptor by mass spectrometry (see below). The 40 kDa band most likely represents a post-translationally modified version of the receptor.

Several purified GPCRs

The one step purification method described above was tested out on several other GPCRs belonging to the rhodopsin-related class A subfamily. Fig. 4 shows the results obtained with five of these receptors, the human neurokinin-3 receptor (NK3R_HUMAN, theoretical molecular weight 52 kDa), the human purinergic receptor type-1 (P2RY1_HUMAN, 42 kDa), the human j-opioid receptor (OPRK_HUMAN, 43 kDa), the human histamine H 1 receptor (HRH1_HUMAN, 56 kDa), the human a 2B -adrenergic receptor (ADA2B_HUMAN, 50 kDa). All of these receptors were obtained in a relatively pure state (Fig. 4). NK3R (lane 1) and ADA2B (lane 5) migrate at the expected molecular weights, but P2RY1 (lane 2), OPRK (lane 3) and HRH1 (lane 4) migrate slightly faster than among GPCRs [START_REF] Gan | Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI À cells[END_REF]. They may bind more SDS than other proteins of similar size, due to a higher level of hydrophobicity and thus migrate faster during SDS-PAGE.

In order to confirm the identities of the purified receptors, the SDS-PAGE bands of purified OPRK, CNR2 and ADA2B, respectively, were excised and submitted to in-gel digestion followed by MAL-DI-TOF mass fingerprinting. As shown in Table 1, a total of 4, 8 and 11 peptides were identified for OPRK, CNR2 and ADA2B, respectively. Their masses were in concordance with the calculated molecular masses of theoretical tryptic peptides derived either from the cytoplasmic loops or from the N-and C-terminal domains. These analyses provided an unambiguous confirmation of the identities of the purified receptors. In the case of the CNR2 receptor, that gives two bands on SDS-PAGE (Fig. 3), both bands gave a similar pattern, thus confirming that the upper band likely represents a post-translationally modified version of the receptor.

Ligand binding capacity of purified CNR2 and ADA2B receptors

Binding assays were realized for two purified, SAV-agarose bound receptors, CNR2_HUMAN and ADA2B_HUMAN, using specific 3 H-labelled ligands (CP 55940 and rauwolscine, respectively). Two purification conditions were tested, the first including 0.1% DDM in all buffers, the second with 0.1% DDM and 0.02% cholesteryl hemisuccinate (CHS), a well-known GPCR stabilizer. For CNR2, the binding level was 9 nmol/mg in the first condition and 10.5 nmol/mg in the second, corresponding, respectively, to 36% and 42% of active receptors in the preparation. For the ADA2B receptor, the values were much lower: 0 and 0.2 nmol/mg in the two conditions tested, respectively, corresponding to only 0% and 1% of active receptors.

Yields of purification

The yields obtained for several receptors are displayed in Table 2.

They were mainly determined by two methods. The first consisted of protein measurements made after the removal of the TEV protein and the N-terminal free tags over a gel filtration column (Superose Fig. 3. Time course of CNR2 release by the TEV protease. CNR2 was solubilized and bound to streptavidin-agarose beads; the beads were washed and samples were taken at different time points after addition of the TEV protease and denatured with SDS sample buffer. Lane 1, time 0 after addition of the TEV protease; lane 2, 1 h after addition; lane 3, 2 h; lane 4, 3 h; lane 5, 14 h (overnight). The method allows easy exchange of buffers, salts and detergents, as well as addition of stabilizing additives early on in the purification process, or only at the last step involving elution by TEV. In our hands, no strong inhibition of the TEV protease could be observed, whatever the additives (specific ligands and/or CHS) or detergents used. In a previous study, the protease was shown to be inhibited by several detergents, however, the detergent concentrations used were much higher, at least by an order of magnitude, than the concentrations used in our study [START_REF] Mohanty | Inhibition of tobacco etch virus protease activity by detergents[END_REF].

For most GPCRs used in this study, substantial amounts of tagfree proteins have been purified in quantities and at purity levels sufficient to initiate structural studies. Among the receptors tested, one could not be purified and a few were obtained only in low amounts. Most of these refractory receptors were nevertheless expressed and biotinylated to levels comparable to receptors purified with good yields, indicating that the problem most probably lies in the organization of the C-terminal part of the proteins, where the biotin moiety could be hindered. A possible remedy would be to add a linker between the GPCR and the biotinylation domain, thus rendering the biotin able to interact with SAV.

Besides structural studies, a possible application of the method would be to use streptavidin-agarose bound receptors to go ''fishing" for receptor-interacting proteins in soluble/membrane extracts of various types of cells or tissues, in the presence or absence of added ligands. We recently obtained preliminary results

showing that b-arrestin-1, a protein involved in GPCRs internalization, can be retrieved with SAV-agarose bound CNR2 receptor from an E. coli extract containing arrestin expressed in low amounts (results not shown). The method described here would allow (i) a direct biochemical confirmation of interactions detected by other methods such as immunoprecipitation, (ii) the identification of novel interacting partners and (iii) the possibility to release the GPCR-interacting partner complexes with the TEV protease and to realize a biochemical study of such complexes. The yields are expressed in mg of purified GPCR, obtained from 1 L of induced culture.

  198purified on an IMAC column (His Trap, GE healthcare) followed by 199 a desalting step and a cation exchange chromatography at pH 6.1 200 on a Mono S column (GE healthcare). After elution with a 201 0-1.0 M NaCl gradient, protease containing fractions, which eluted 202 at 750 mM NaCl, were aliquoted and stored at À80 °C. 203 Ligand binding experiments 204 Binding assays were performed as previously described [9], 205 using 10 nM tritiated ligands CP55940 and rauwolscine for 206 CNR2_HUMAN and ADA2B_HUMAN, respectively. The binding 207 buffer used for the CNR2 receptor contained 50 mM Tris-HCl 208 pH 7.4, 2.5 mM EDTA, 5 mM MgCl 2 and 0.5 mg/ml bovine serum separated protein bands were excised from the gel 212 and the proteins were digested according to the Shevchenko proto-213 col [11]. The gel plugs were washed three times with 100 ll of 214 25 mM ammonium hydrogen carbonate (NH 4 HCO 3 ) and 100 ll of 215 acetonitrile. The cysteine residues were reduced by 50 ll of 216 10 mM dithiothreitol at 56 °C and alkylated by 50 ll of 55 mM 217 iodoacetamide. After dehydration with acetonitrile, the proteins 218 were cleaved with 15 ll of a 12.5 ng/ll solution of proteomics 219 grade trypsin (Sigma) in 25 mM NH 4 HCO 3 . The digestion was per-220 formed overnight at room temperature. The resulting peptides 221 were extracted with a 65% acetonitrile solution containing 5% acid 222 formic and 0.05% SDS for 1 h. MALDI-TOF mass measurements were 223 carried out on an AutoFlex II TOF/TOF (Bruker Daltonik GmbH, Bre-224 men, Germany) operating in positive reflectron mode. The samples 225 were prepared by standard dried droplet preparation on stainless 226 steel MALDI targets using a-cyano-4-hydroxycinnamic acid as ma-227 trix. The samples were washed twice with 5% formic acid. External 228 calibration of MALDI mass spectra was carried out using singly 229 charged monoisotopic peaks of a mixture of bradykinin 1-7 230 (m/z = 757.400), human angiotensin II (m/z = 1046.542), human 231 angiotensin I (m/z = 1296.685), substance P (m/z = 1347.735), bom-232 besin (m/z = 1619.822), ACTH 1-17 (m/z = 2093.087) and ACTH 233 18-39 (m/z = 2465.199). Monoisotopic peptide masses were auto-234 matically annotated using Flexanalysis 3.0.

Fig. 1 .

 1 Fig. 1. Schematic representation of the method. A solubilized receptor is captured on immobilized streptavidin. The TEV protease then cleaves the N-and C-terminal tags, thus releasing the receptor and the N-terminal tag. The C-terminal tag remains bound to streptavidin.

  U N C O R R E C T E D P R O O F 273 of biotinylated proteins with alkaline phosphatase-linked strepta-274 vidin. For yeast transformed with an empty vector, several endog-275 enous biotinylated proteins were detected in the soluble fractions, 276 but none could be detected in the membrane fractions (results not 277 shown). Fig. 2A (lanes 1-4) shows that all four receptors tested, 278 respectively, ADA2B_HUMAN, CNR2_HUMAN, OPRK_HUMAN and 279 NK3R_HUMAN are biotinylated in P. pastoris. The sizes of the 280 detected proteins are in good agreement with those expected for 281 ADA2B (observed Mr: 65 kDa) and CNR2 (55 kDa), taking into 282 account the size contribution of the N-and C-terminal tags. For 283 OPRK (85 kDa) and NK3R (100 kDa), the observed sizes are higher 284 than expected. This is likely to be due to a lack of processing of the 285 N-terminal a-factor signal sequence by internal Kex2 endopepti-286 dase of P. pastoris. The same probably applies to the additional 287 $85 kDa doublet observed for the ADA2B receptor (Fig. 2, lanes 1 288 and 5

290

  When the membranes were treated with exogenously added 291 biotin-protein ligase (BirA), only a slight increase in the level of 292 biotinylation could be observed (Fig.2A, compare lanes 1-4 and 293 5-8), indicating that, in vivo, the biotinylation of the proteins 294 was almost complete. Fig. 2B shows the same receptors detected 295 with an anti-flag M2 monoclonal antibody; the bands observed 296 are essentially the same as those detected with streptavidin-phos-297 phatase. Taken together, these results indicate that both the N-and 298 C-terminal parts of the proteins are intact since they are detected 299 by the anti-flag antibody and streptavidin-phosphatase, respec-300 tively. Dimers and higher order oligomers are also detected, espe-301 cially for CNR2 and OPRK (Fig. 2A, lanes 2, 3, 6 and 7; Fig. 2B, lanes 302 2 and 3

304

  These results along with the efficient in vivo biotinylation of 305 these receptors prompted us to perform further purification tests.306With the aim of developing a generic method (see Fig.1), the 307 human peripheral cannabinoid receptor (CNR2_HUMAN) was used 308 as a prototypical GPCR. 309 CNR2_HUMAN capture onto streptavidin-coated beads and its release 310 by TEV protease 311 Membrane proteins (1 mg/ml) containing CNR2_HUMAN were 312 solubilized with 1% DDM (n-dodecyl b-D-maltopyranoside) and 313 then mixed with SAV-agarose beads. Commercial beads were 314 tested in preliminary experiments, but the results were not as good 315 as with our in-house made SAV beads prepared as described in Experimental section Q1 . The difference is most likely due to the different levels of substitution of the agarose beads with SAV:

Fig. 2 .

 2 Fig. 2. Detection of four GPCRs expressed in Pichia pastoris. Membrane proteins were subjected to SDS-PAGE, and transferred to nitrocellulose. The receptors were detected with (A) Alkaline phosphatase-linked streptavidin or (B) anti-Flag M2 monoclonal antibody. Lanes 1 and 5: ADA2B_HUMAN; lanes 2 and 6: CNR2_HU-MAN; lanes 3 and 7: OPRK_HUMAN; lanes 4 and 8: NK3R_HUMAN. Membranes from lanes 5 to 8 were subjected to in vitro biotinylation with the BirA proteinbiotin ligase prior to SDS-PAGE. Arrows point to receptor bands at the expected size. Asterisks show receptors for which the signal sequence was not processed, leading to molecular masses higher than expected.

Fig. 4 .

 4 Fig. 4. Several purified GPCRs. Proteins purified with streptavidin beads were separated by SDS-PAGE and stained with coomassie blue. Lane 1, NK3R_HUMAN; lane 2, P2Y1_HUMAN; lane 3, OPRK_HUMAN; lane 4, HRH1_HUMAN; lane 5, ADA2B_HUMAN; lane 6, TEV protease. Arrowheads indicate purified GPCRs and asterisks indicate probable dimers.

Table 1

 1 Tryptic fragments observed by MS actually conserving the N-terminal tags in position on the streptavidin protein and exploiting TEV elution to liberate the protein of interest. The strength of the streptavidin-biotin interaction also allows, when necessary, the use of harsh washing conditions such as very high or low pH, or high concentrations of denaturants such as urea or guanidine.

	Receptor	Measured masses	Sequence

454

lot of development and optimization

[START_REF] Klammt | Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation[END_REF][START_REF] Klammt | Reprint of ''Cell-free production of G protein-coupled receptors for functional and structural studies[END_REF][START_REF] Ishihara | Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors[END_REF]

.

455

Purification methods for membrane proteins that use the 456 avidin-biotin interaction have been developed in a few cases 457

[START_REF] Krepkiy | Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2[END_REF][START_REF] Jidenko | Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain[END_REF]

. Usually, monomeric avidin bound to agarose has been 458 used, but some problems, mainly concerning elution of the 459 proteins, have been frequently reported

[START_REF] Jidenko | Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain[END_REF]

. The streptavidin-bio-460 tin is not generally used because of the very high strength of this 461 interaction, making it virtually impossible to elute the protein of 462 interest in mild conditions. We turned this drawback to our advantage by

Table 2

 2 Purification yields for 10 GPCRs

	Receptor	Yield (mg)
	CNR2_HUMAN	0.9
	NK1R_HUMAN	0.05
	NK3R_HUMAN	0.4
	ADA2B_HUMAN	0.2
	ADA1B_MESAU	0.1
	HRH1_HUMAN	0.5
	D2DR_HUMAN	0.05
	P2Y1_HUMAN	0.5
	OPRD_HUMAN	0.7
	OPRK_HUMAN	0.8
	ADA2C_RAT	<0.02
	PAR1_HUMAN	<0.02

Please cite this article in press as: T. Magnin et al., A novel, generic and effective method for the rapid purification of G ..., Protein Expr. Purif. (2008), doi:10.1016/j.pep.2008.09.007

Acknowledgments

We are grateful to Rim Benmamaar for improving the English of the article. This study was funded by the European Membrane Protein consortium (eMeP).


Safwan Alfattani

Wael Jaafar

Yassine Hmamouche

Halim Yanikomeroglu

Abbas Yongaçoglu

Dũng Ðào

Peiying Zhu

Aerial Platforms with Reconfigurable Smart Surfaces for 5G and Beyond

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

As the world steps into the deployment of 5G, researchers continue addressing the challenges of ubiquitous connectivity. The role of aerial platforms for wireless networks is an increasingly important one for meeting the communication requirements of 5G/B5G. Indeed, the Third Generation Partnership Project (3GPP) has considered aerial platforms to be a new radio access for 5G (TR 38.811, TR 22.829, and TS 22.125), and several projects have been initiated to provide ubiquitous Internet services, such as Google Loon and Thales Stratobus. Due to their 3D-mobility, flexibility, and adaptable altitude, aerial platforms can efficiently support the connectivity of terrestrial and aerial users by enhancing their capacity and coverage, or even providing backhaul links. Based on their operation altitudes, we distinguish two main types of aerial platforms, Unmanned Aerial Vehicles (UAVs) and High Altitude Platform Stations (HAPS). UAVs operate at low altitudes of few hundred meters and act as flexible and agile relays or base stations (UxNB) (TR 22.829). Their use is generally time-limited, ranging from a few minutes to a few hours due to limited onboard energy. Alternatively, tethered UAVs can fly for much longer owing to the continuous supply of power from the tether. By contrast, HAPS operate at higher altitudes of 8 to 50 km above ground (TR 38.811), with current HAPS projects focusing on the 20 km altitude. They allow wider coverage areas and longer flight times compared to UAVs (e.g., Google Loon's flight record is 223 days). To further reduce costs and make aerial platforms more attractive for 5G/B5G networks, researchers are constantly searching for new horizons of improvements, including fuselage materials, battery, fuel, and solar-cell technologies, as well as enhancing trade-offs in energy efficiency (EE) and communication performance.

From the communication perspective, passive reconfigurable smart surface (RSS) 1 has emerged as a potential substitute for active communication components [START_REF] Liaskos | A New Wireless Communication Paradigm through Software-Controlled Metasurfaces[END_REF]. RSS is a thin and flexible metasurface equipped with programmable passive circuits to "reflect" received signals in a controlled way. An RSS can then positively exploit the propagation environment to enhance the quality of a communication link between a transmitter and a receiver, known as RSS-assisted communication, or to transmit signals more effectively, identified as RSS-initiated communication. Efforts have been made to evaluate and explore the benefits of RSS. For instance, NTT DOCOMO collaborated with Metawave and demonstrated an increase in data rate of approximately ten times using RSS at 28 GHz. Other notable initiatives include the VisoSurf consortium for RSS prototyping, and Greenerwave, which is already marketing RSS that operate at frequencies up to 100 GHz.

Considering the opportunities presented by RSS technology, we discuss in this paper a novel paradigm where RSS is deployed in aerial platforms, and propose a control architecture workflow for communication, mobility, and sensing. We then describe potential use cases where RSS-equipped HAPS and UAVs aim to achieve communication objectives, such as coverage extension and low-cost network densification. Finally, related challenges are presented. To the best of our knowledge, this is the first work that presents an in-depth discussion of the use of RSS-equipped aerial platforms, their control architecture, as well as their potential applications and challenges.

OVERVIEW OF RSS IN COMMUNICATIONS

Background

Researchers have traditionally focused on improvements in hardware and transmission techniques to enhance link quality between a transmitter and a receiver. This research stream has sought ways of mitigating the impact of environmental objects on the quality of wireless signals. However, a different approach has emerged whereby the environment between transmitters and receivers can be managed to initiate or support wireless communications. For initiated communications, various ambient energy sources, (RF, solar, etc.), can power ultralow-power electronics to enable sensing functions and backscatter communications through different environmental objects (see [START_REF] Kimionis | Zero-Power Sensors for Smart Objects: Novel Zero-Power Additively Manufactured Wireless Sensor Modules for IoT Applications[END_REF] and references therein). For assisted communications, the idea of controlling surfaces and objects in wireless propagation environments was proposed earlier, around two decades ago. The objective was to mitigate undesired interference and improve the desired signal through the use of frequency selective surfaces (FSS). These FSS resemble bandpass or bandstop filters, and they can be integrated into indoor walls. Also, they can be equipped with sensors and diode switches for flexibly controlled response.

While FSS's key function has been to filter signals and alleviate interference, another vision of utilizing the propagation environment is to combine the scatters of the desired signals and direct them to the targeted users. In this context, passive reflectors (a.k.a., mirrors) consisting of metallic sheets or high conductivity materials have been designed for indoor and outdoor environments, aiming to enhance the received power. For outdoor environments, authors in [START_REF] Peng | An Effective Coverage Scheme With Passive-Reflectors for Urban Millimeter-Wave Communication[END_REF] suggested using passive reflectors on top of buildings to focus reflected signals from ground base station (gBSs) toward users. Compared to conventional coverage enhancement approaches, such as relaying and gBSs densification, passive reflectors made of lowcost elements offer flexible deployment, easy maintenance, and improved EE. Nevertheless, passive reflectors are constrained by Snell's law of reflection, and once designed, they have a fixed response. Hence, they are inherently unable to accommodate the dynamics of wireless environments. Alternatively, to capture the wireless environment dynamicity, "active" surface technologies were proposed in [START_REF] Hu | Beyond Massive MIMO: The Potential of Data Transmission with Large Intelligent Surfaces[END_REF], where the walls consist of a massive number of electromagnetically radiating and sensing elements working as large smart antennas, and regarded as an extension to the massive multiple-input multiple-output (MIMO) concept. While passive reflectors utilize material properties to direct electromagnetic waves, active surfaces require integrating electronics, antennas and communication circuits in walls to work as full transceivers. Although they might have better performances, the circuit complexity, material costs, and energy consumption are higher than for passive surfaces.

Inspired by recent advances in the physics of metasurfaces and meta-materials, passive RSS technology has been presented as an interesting add-on to various applications in wireless communications [START_REF] Liaskos | A New Wireless Communication Paradigm through Software-Controlled Metasurfaces[END_REF]. Metasurfaces are distinct manmade 2D meta-materials, consisting of thin layers of metallic/dielectric inclusions. Metasurface units can be arranged in arrays and designed to have peculiar electromagnetic (EM) features with customizable interactions with the EM waves. Indeed, these units can realize abrupt phase shifts and anomalous reflections and refractions, where the phase of the wavefronts can be altered, a process known as phase discontinuity [START_REF] Yu | Light Propagation with Phase Discontinuities: Generalized Laws of Reflection and Refraction[END_REF]. Moreover, the reflected and refracted EM waves can have an arbitrary direction beyond Snell's law, subject to "the generalized laws of reflection and refraction" [START_REF] Yu | Light Propagation with Phase Discontinuities: Generalized Laws of Reflection and Refraction[END_REF]. To bypass the fixed response of metasurfaces, digital meta-materials were proposed in [START_REF] Cui | Coding Metamaterials, Digital Metamaterials and Programmable Metamaterials[END_REF], where the manipulations of the EM waves were reconfigurable by digitally controlling the metasurface units, i.e., switching between different meta-material responses using biased diodes. Subsequently, an advanced layout is proposed in [START_REF] Liaskos | A New Wireless Communication Paradigm through Software-Controlled Metasurfaces[END_REF], wherein a programmable metasurface layer was integrated with an Internet-of-Things gateway and a control layer to form a thin film, called "HyperSurface tile", coating surfaces and environmental objects. Hence, the interaction of the surfaces with the EM waves can be programmed and controlled remotely via software.

Types of Communications with RSS

Given the capabilities demonstrated in physics for controlling and reconfiguring EM waves with meta-materials, wireless communication researchers have started investigating the use of RSS in different ways, particularly for RSS-assisted communications and RSS-initiated communications.

RSS-Assisted Communications

Aiming for efficient delivery of transmitted signals, most research has focused on using RSS as a relay to enhance the received signal at end-users. Different objectives have been considered, such as maximizing data rates or spectral efficiency, extending the coverage area, mitigating interference, and minimizing consumed energy. For instance, the authors of [START_REF] Wu | Intelligent Reflecting Surface Enhanced Wireless Network via Joint Active and Passive Beamforming[END_REF] showed that by optimizing the RSS phase shifts, quadratic increase in the signal-to-noise ratio (SNR) is realized, and better spectral efficiency than relays can be achieved. To practically substantiate the advantages of RSS-assisted communications, an experimental testbed was implemented in [START_REF] Tan | Enabling Indoor Mobile Millimeter-wave Networks Based on Smart Reflect-arrays[END_REF] demonstrating that RSS can solve the coverage-hole problem caused by obstacles in millimeterwave (mmWave) networks. Additionally, motivated by the EE potential of RSS, researchers have focused on supporting energy-constrained communications. For instance, authors in [START_REF] Huang | Energy Efficient Multi-user MISO Communication Using Low Resolution Large Intelligent Surfaces[END_REF] demonstrated that, even with low-resolution phase shifters in RSS-assisted communications, EE can be improved by 40% compared to conventional relaying systems.

RSS-Initiated Communications

In contrast to previous works, RSS is being tested as an energy-efficient and low-complex transmitter. The idea is to connect the RSS with a nearby RF source and, through phase shifting, data can be encoded and transmitted. While the reliability of RSS-initiated communications were analyzed in [START_REF] Basar | Wireless Communications Through Reconfigurable Intelligent Surfaces[END_REF], authors of [START_REF] Dai | Wireless Communications through a Simplified Architecture Based on Time-Domain Digital Coding Metasurface[END_REF] validated through experiments the design of a high order modulation RSS-based transmitter, where a digital video file was modulated and transmitted through the RSS without requiring filters, mixers, or power amplifiers.

AERIAL PLATFORMS WITH RSS: INTEGRATION AND CONTROL ARCHITECTURE

The current research works on RSS are focused on its use in terrestrial environments, e.g., RSS equipping buildings facades. However, motivated by the numerous aforementioned RSS capabilities, and the advanced features of aerial platforms, we envision that the usage of RSS in aerial platforms will offer great flexibility and support for wireless networks. Integrating RSS on aerial platforms can be done in several ways depending on the platform's shape. For instance, the RSS can coat the outer surface of a balloon or an aircraft, or it can be installed as a separate horizontal surface at the bottom of a UAV, as shown in Fig. 1. The reconfiguration of such surfaces can be realized through lumped elements embedded in the reflector units or via the distributed control of some material properties. Examples of lumped elements include PIN diodes, varactor diodes, and radio frequency micro-electro-mechanical systems. By tuning the lumped elements, the resonant frequency of the reflectors can be changed, hence achieving the desired wave manipulation and phase shift. To manage high frequency signals, e.g., mmWave and free-space optical (FSO) communication, tunable materials such as liquid crystal and graphene are used to design the metasurface reflector units.

In order for the RSS-equipped aerial platforms to perform communication functions properly, the channel information needs to be acquired and the RSS should be accurately configured. The control can be managed at two levels: Ground control station and aerial platform, as illustrated in Fig. 1.

Ground Control Station

This controller consists mainly of a processing unit that analyzes the sensed data from the aerial platform, and the access control and users localization conditions, acquired through information exchange with gBSs and/or gateways. Given a set of global policies (e.g., flying regulations, power, etc.) and objectives (e.g., coverage, SNR, EE, etc.), the processing unit ensures the joint management of the aerial platform's flying and communication functions. Moreover, it estimates the channel state information (CSI) and angle of arrival (AoA) between users and the aerial platform to determine the best RSS configuration setup.

Onboard Aerial Platform Control

The onboard aerial platform's controller consists of two units, namely the flight control unit, and the RSS control unit. The former receives motion commands from the ground station, and ensures the platform's stabilization. By contrast, the RSS control unit receives the optimized RSS configuration, translates it into a switch control ON/OFF activation map, and applies it to the metasurface layer for directing incident signals to targeted directions.

AERIAL PLATFORMS WITH RSS: POTENTIAL USE CASES

To demonstrate the potential benefits of integrating RSS in aerial platforms, we detail here several novel integration use cases.

HAPS with RSS to Support Remote Areas

Connecting sparse users in remote areas via terrestrial networks is deemed to be expensive and unprofitable to operators due to the high costs of typical gBSs and wired backhauling infrastructure. Satellites have been proposed to serve remote areas; however, due to high deployment costs, large communication delays, and excessive path-loss, satellites are considered as a last resort solution. Alternatively, HAPS have been introduced as a cost-effective solution for connecting remote users [START_REF] Alzenad | FSO-Based Vertical Backhaul/Fronthaul Framework for 5G+ Wireless Networks[END_REF]. HAPS are quasi-stationary platforms located in the stratosphere at a fixed point relative to the earth. Since the wind's speed in this region is weak, low power is required to stabilize the platform. Moreover, the coverage area of a HAPS is greater than that of a gBS. For instance, the International Telecommunication Union (ITU) suggested that the HAPS' coverage radius be up to 500 km (ITU-R F.1500); however, current HAPS projects have less than 100 km coverage radius to achieve high area throughputs.

HAPS are mostly powered by solar/fuel energy, and their flight duration may vary from several hours to a few years. The energy consumed by a HAPS is mainly for propulsion, stabilization, and communication operations. Typically, a HAPS is used in communications either as a BS (HAPS-BS) or as a relay station (HAPS-RS). Although a HAPS-BS has more capabilities than a HAPS-RS, it involves a higher cost, a heavier payload, and an increased energy consumption. When solar cells are used, the harvested energy may not be sufficient for operations. For this reason, other power sources are added to complement the solar energy. Nevertheless, the EE of HAPS is still an open problem, which directly impacts the flight duration.

In designing a HAPS deployment, low energy consumption, light payload, and reliable communication need to be achieved. To this end, motivated by the benefits of RSS, we propose using a HAPS equipped with RSS (HAPS-RSS) for wireless traffic backhauling from remote area BSs. The envisioned scenario is depicted in Fig. 2, where BSs handle the traffic of a few clustered users and transmit the traffic to the HAPS. Through the HAPS' RSS, the received signals are smartly "reflected" toward a gateway station, which is connected to the core network. If a gateway station is not within the HAPS coverage range, the RSS can be configured to "reflect" the signals toward another HAPS and thereby reach the gateway station in a dual or multi-hop fashion.

Using HAPS-RSS has several advantages. First, it reduces the used power for communication functions, since directing signals can be realized in a nearly-passive way, without RF source or power amplifiers, and only minimum amount of power is required for the RSS control unit. Second, since RSS is made of thin and lightweight materials, the HAPS payload is lighter even if a large number of reflectors is deployed. Third, given the reduced communication components and payload, the required stabilization energy is minimized. The simplification in communication circuits and minimization of consumed energy yield to extended flight duration and reduced HAPS deployment costs.

The performance of RSS-assisted communications strongly depends on the number of reflectors. The dimensions of a metasurface reflector unit (a.k.a., meta-atom) are proportional to the wavelength [START_REF] Liaskos | A New Wireless Communication Paradigm through Software-Controlled Metasurfaces[END_REF]. Typically, a HAPS is a giant aircraft with a length between 30 and 200 m, which has the potential to accommodate a large number of reflectors, especially in high frequencies. The large number of reflectors would have a significant impact on the directivity gain, hence better SNR performance than HAPS-RS can be achieved [START_REF] Wu | Intelligent Reflecting Surface Enhanced Wireless Network via Joint Active and Passive Beamforming[END_REF]. In terms of energy consumption, the switches on the metasurface layer Low (Low-cost RSS, extended flight duration) consume very low power. A recent experiment showed that each meta-atom consumes 0.33 mW when its switch diode is set to ON [START_REF] Tang | Wireless Communications with Reconfigurable Intelligent Surface: Path Loss Modeling and Experimental Measurement[END_REF]. Thus, for a HAPS with 10,000 metaatoms, the maximal power consumed by the metasurface layer when all switches are activated would be about 3.3 W, which constitutes a very promising result. Hence, HAPS-RSS is expected to achieve a better EE than HAPS-RS [START_REF] Huang | Energy Efficient Multi-user MISO Communication Using Low Resolution Large Intelligent Surfaces[END_REF]. Finally, it is worth noting that RSS supports full-duplex communication [START_REF] Wu | Intelligent Reflecting Surface Enhanced Wireless Network via Joint Active and Passive Beamforming[END_REF]. Indeed, unlike relays experiencing high noise and residual loop-back self-interference, which are issued from active RF components, RSS bypasses these constraints by only exploiting the intrinsic properties of the reflecting material. Thus, HAPS-RSS would be more spectrally efficient than HAPS-RS. Table I summarizes the main differences between HAPS-RS and HAPS-RSS communications.

UAVs with RSS to Support Terrestrial Networks

RSS can be combined with UAVs and their attendant benefits, namely, agility, flexibility, and rapid deployment, to assist terrestrial cellular networks in a cost-effective manner. Typically, RSS can be carefully mounted on a swarm of UAVs (UxNB-RSS) to create an intermediate reflection layer between gBSs and isolated users, as illustrated in Fig. 3. In this way, the UxNB-RSS allows a smooth mechanical movement of the RSS layer, while the RSS enables digitallytuned reflections of incident signals to improve service for isolated users. Hence, besides its low manufacturing cost, UxNB-RSS can be rapidly deployed to increase operators' revenue per user by filling coverage holes and meeting users' high-speed broadband needs.

Coverage holes or blind spots can occur for various reasons, such as disruption of some gBSs due to maintenance, failures, or natural disasters. Also, future communication networks are exploring the use of higher frequencies with wider spectrum bandwidths, such as mmWave and FSO, to tackle the increasing throughput demands and the spectrum scarcity problem. A major challenge hindering their widespread use is their vulnerability to blockages in the propagation environment, which requires perfect line-of-sight (LoS) links but may create coverage holes in regions with impenetrable blockages. In such a context, UxNB-RSS, which has a panoramic view of the environment, can provide full-angle 360 • reflection and thus can reduce the number of reflections compared to terrestrial RSS [START_REF] Lu | Enabling Panoramic Full-angle Reflection via Aerial Intelligent Reflecting Surface[END_REF]. This approach can also be adopted to deliver new signals to users served by a heavily loaded gBS, and thus help in balancing the traffic of congested cells.

In the absence of channel knowledge, UxNB-RSS can be used for spatial diversity to combat channel impairments (e.g., fading and path-loss). A typical user can actually receive several copies of the desired signal over different transmission paths generated by reflections through the swarm of UxNB-RSS, which enhances the reliability of the reception. Alternatively, fading can be seen as a source to increase the degrees of freedom. A high-rate data stream can be divided into many lower-rate streams and transmitted over different transmission paths using the swarm of UxNB-RSS. Thus, the user can retrieve independent streams of information with sufficiently different spatial signatures, resulting in improved data throughput. It is also worth noting that RSS can absorb a certain amount of the impinging signals and leverage them to recharge the UxNB-RSS.

Tethered Balloons with RSS to Support Terrestrial/Aerial Users

UxNB has been viewed as a candidate solution for several wireless challenges, such as coverage and capacity extension. Although they have promising advantages, such as flexible placement and LoS communication links, there are also several limitations that militate against using UxNB. First, the limited onboard energy makes UxNB exclusively suitable for shortterm deployments. Second, backhauling from a UxNB is typically through a wireless link, which has limited reliability and capacity compared to wired backhauling links. To bypass these limitations, a tethered UxNB has been recently introduced. The tether provides the UxNB with both data and power. Thus, longer flight times and reliable backhaul links are achieved. However, a tethered UxNB consumes a considerable amount of energy for flying, hovering and transmitting signals, which yield to increased deployment and operational costs. A more cost-effective alternative that we propose here is using a tethered balloon equipped with RSS (TBAL-RSS), as depicted in Fig. 4.

Inspired by recent works and prototypes that use RSS as transmitters [START_REF] Basar | Wireless Communications Through Reconfigurable Intelligent Surfaces[END_REF], [START_REF] Dai | Wireless Communications through a Simplified Architecture Based on Time-Domain Digital Coding Metasurface[END_REF], we point out that TBAL-RSS can be used as a low-cost wireless access point to connect or enhance the capacity of urban users. By illuminating the outer surface of the TBAL-RSS with an RF feeder, the information provided by the tether data link can be encoded and transmitted. Thus, the RSS controller adjusts the phases of the reflected signals to generate different beam directions that can serve multiple users. Compared to a tethered UxNB, TBAL-RSS has several advantages. Unlike a small tethered UxNB, a TBAL is large and can accommodate a higher number of reflectors. Also, compared to typical gBSs, network densification with TBAL-RSS in urban environments is more feasible and cost-effective. Since TBAL-RSS provides near-field communications with strong LoS links to users, it is expected to provide better spectral efficiency than MIMO gBSs, considered as a farfield communication system with many NLoS links, high interference, and pilot contamination. Furthermore, unlike down-tilted antennas in gBSs, the TBAL-RSS can sustain reliable connectivity to UAV users (UAV-UEs) such as delivery drones. Thus, both terrestrial and aerial users can be served simultaneously.

Moreover, a TBAL does not continuously drain power for lifting and hovering, as it uses lifting gases. Although the altitude of a TBAL is limited by the tether's length, some flexibility in its position is available by controlling the tether's direction to offer better coverage in certain spots. Besides, a backhaul fiber optic link along the tether can be installed to guarantee the TBAL's connectivity to the core network. A processing unit is also placed at the TBAL's launching point to control its movement, determine the targeted users, and send the optimum RSS configuration.

CASE STUDY

To emphasize the potential gains when using RSS-equipped aerial platforms, we present a case study where the communication between a terrestrial transmitter and a terrestrial receiver is assisted by an RSS-equipped aerial platform, namely, a UAV, a HAPS, or a low earth orbit (LEO) satellite. Each platform has a typical coverage footprint and deployment altitude, where typically higher altitude platforms have wider footprints, i.e., larger coverage radius ( ). For instance, a UAV may have = 2 km, while a HAPS has ∈ [50, 100] km. For the sake of simplicity, we assume that the aerial platform is located at the middle distance between the transmitter and receiver, which are located at opposite edges of the coverage footprint. Moreover, platforms have different sizes that delimit the practical RSS mounting areas on them. Given the RSS area and operating frequency, the maximum number of reflectors on the RSS can be bounded.

Following the scattering paradigm-based link budget analysis conducted in [START_REF] Alfattani | Link Budget Analysis for Reconfigurable Smart Surfaces in Aerial Platforms[END_REF], where perfect RSS phase shifting and no reflection losses are assumed, we compare in Fig. 5 the received powers when the communication is assisted by RSS in aerial platforms or in terrestrial environments. The characteristics of each platform are shown in the legend boxes. We set the transmit power and the frequency to 40 dBm and 30 GHz, respectively. Also, we assumed unit antenna gains at both transmitter and receiver, and a path-loss exponent equal to 4 for the terrestrial environment. We notice that at a fixed number of reflectors, the UAV achieves the best performance. This is expected due to strong LoS links compared to the terrestrial RSS (where blockages degrade the communication links), and the other aerial platforms (where longer distances are traveled by the transmitted signal). However, since the UAVs' RSS area are limited and some platforms can host a larger number of RSS reflectors (red squares), HAPS become better than UAVs. Ultimately, the HAPS is preferred due to its achieved performance and sustainability.

RELATED RESEARCH CHALLENGES

As the research of RSS in wireless communications is still in its infancy, several challenges and open problems remain to be addressed. In the following, we discuss some key issues.

Accurate RSS and Aerial Channel Models

Most of the existing research works consider RSS with perfect manipulation of the EM wave and ideal phase shifts. However, there is a paramount need for practical RSS models that consider the configuration capabilities and the reliability with different communication frequencies and diverse numbers and sizes of RSS units.

Furthermore, although several air-to-ground channel models have been developed, integrating RSS in aerial platforms is a new direction that requires the investigation of new aerial models that consider RSS capabilities with aerial platforms properties. Some important factors that could affect path-loss models need to be analyzed, such as reflection loss, correlation between RSS units, and atmospheric attenuation. Moreover, the mobility of aerial platforms may cause fluctuations and beam misalignments which have to be considered, especially for HAPS and high frequency communications.

RSS Deployment on Aerial Platforms' Surfaces

Aerial platforms have different surface shapes and types. They might be flat, like the bottom of a UAV, or curved, like the outer surface of an aircraft or balloon. How such an RSS deployment may affect metasurface configuration and performance has not been investigated yet.

Channel Estimation and RSS Control Update

One of the fundamental assumptions in most current RSS research is the availability of CSI, which is essential for performing perfect reflection. However, gaining such knowledge within the channel's coherence time in typical dynamic wireless environments with mobile platforms is still an open problem. One solution to this is by incorporating low-cost sensors in metasurfaces [START_REF] Kimionis | Zero-Power Sensors for Smart Objects: Novel Zero-Power Additively Manufactured Wireless Sensor Modules for IoT Applications[END_REF], which frequently sense the environment and send measurements to the The latter computes and sends back the updated optimal RSS configuration. Alternatively, the reciprocity of the channel can be exploited to estimate the CSI for pre-determined phase shifting configurations, or machine learning techniques, such as supervised learning and reinforcement learning, can be leveraged to find the optimal RSS configuration. However, such techniques require large amounts of data and/or long training times. Hence, the delay impact due to distance and CSI estimation process merits further investigation.

CONCLUSION

In this article, we discussed the potential integration of the recent RSS technology into aerial platforms as a novel 5G/B5G First, we provided an overview of RSS technology, its operations, and types of communication. Then, we proposed a control architecture workflow for the integration of RSS in aerial platforms. Potential use cases were discussed where different types of aerial platforms, namely HAPS, UAVs and TBALs, were exploited. Integrating RSS in aerial platforms provides several advantages, including energy efficiency, lighter payload, and lower system complexity, which results in extended flight durations and more cost-effective deployment to support wireless networks. However, several issues need to be tackled for the smooth integration of this novel paradigm.

••Figure 1 .

 1 Figure 1. Control architecture of RSS-equipped aerial platforms.

Figure 2 .

 2 Figure 2. Backhauling support of isolated users in remote rural areas using HAPS-RSS.

Figure 3 .

 3 Figure 3. Communication support of terrestrial networks with blockages using UxNB-RSS.

Figure 4 .

 4 Figure 4. Communications support of terrestrial and aerial users using TBAL-RSS.

Figure 5 .

 5 Figure 5. Received power vs. number of reflectors (different platforms).

Table I COMPARISON

 I BETWEEN HAPS-RS AND HAPS-RSS FOR

		COMMUNICATION
	Aspect	HAPS-RS	HAPS-RSS
	Energy Consumption	Medium (Process, amplify, and forward signals)	Very low (RSS control)
	Payload	Heavy (Complex circuits: converters, mixers, amplifiers, and antennas)	Light (RSS designed as a thin sheet film coating HAPS outer surface)
	No. of Commun. Units	Limited by frequency and energy constraints, for circuits. and by allocated space	Limited by HAPS size and frequency band.
	Performance (directivity gain)	Good performance, low EE	High EE, performance depends on RSS size and reconfiguration capability
	Spectral efficiency	Good in full-duplex, but limited by noise and self-interference.	High, always full-duplex and no noise or interference.
		High (Several active
	Cost	components, short flight
		duration)

The term "Smart" is selected in accordance with previous 3GPP standards for similar technologies (e.g., TR

22.867, TR 25.842, TR 22.890, TR 36.848).