Gherici Hassaine

Renaud Wagner

Juliette Kempf

Nadia Cherouati

Nouzha Hassaine

Cecile Prual

Nicolas André

Christoph Reinhart

Franc Pattus

Kenneth Lundstrom
email: kenneth.lundstrom@mepnet.org

Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells

Keywords: G protein-coupled receptors, Semliki Forest virus, Structural genomics, Structural biology, Recombinant expression, Large-scale production, Mammalian cell lines, Immunodetection, Binding assays GPCRs, G protein-coupled receptors, 3D, three dimensional, MBP, maltose binding protein, SFV, Semliki Forest virus, MePNet, Membrane protein network

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

G protein-coupled receptors (GPCRs) 1 are targets for 60-70% of drugs developed today [START_REF] Vanti | Patent status of the therapeutically important G protein-coupled receptors[END_REF]. Traditionally, the drug discovery process has relied on screening chemical libraries in search of compounds interacting favorably with the GPCR of interest [START_REF] Hodgson | Receptor screening and the search for new pharmaceuticals[END_REF]. Although this procedure has been relatively successful, there is still a huge demand on drugs with improved aYnity and selectivity. One solution has been to apply structure-based drug design and there are several examples of novel drugs developed based on the information of the three-dimensional (3D) structure of their target protein [START_REF] Blundell | High-throughput crystallography for lead discovery in drug design[END_REF]. However, the paradox with GPCRs is that of the therapeutically interesting targets, not a single high-resolution structure is available today. The only 3D structure on a GPCR has been solved for bovine rhodopsin [START_REF] Palczewski | Crystal structure of rhodopsin: a G protein-coupled receptor[END_REF] in which case the high receptor abundance in the cow retina made isolation from native tissue possible. In this context, one of the major bottlenecks for obtaining high-resolution structures for recombinant GPCRs has been the diYculties in producing quantitatively and qualitatively satisfactory material. Moreover, the need of detergents for receptor puriWcation has an additional negative impact on the stability of GPCRs. The crystallization in the presence of detergents as well as the receptor Xexibility and small hydrophilic loops, which reduce the potential crystal contacts, has further hampered the progress in structure determination of membrane proteins. The recombinant expression of GPCRs and other membrane proteins has been evaluated in all possible expression systems including bacterial, yeast insect, and mammalian vectors, and was recently reviewed by Sarramegna and co-workers [START_REF] Sarramegna | Heterologous expression of G protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and puriWcation[END_REF]. The results have been quite variable with generally relatively moderate yields in bacterial membranes. However, modiWcation of the coding sequence and fusion to appropriate partner proteins such as maltose binding protein (MBP) has resulted in signiWcantly improved expression yields of, for example, the rat neurotensin receptor [START_REF] Luca | The conformation of neurotensin bound to its G protein-coupled receptor[END_REF]. Alternatively, GPCRs can be directed to inclusion of bodies in bacteria, which results in high yields, but require a refolding process to restore functional activity of the receptor. Although refolding has generally proven to be ineYcient, success was achieved for the leukotriene BLT1 receptor [START_REF] Baneres | Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1[END_REF]. Yeast-based GPCR expression has been relatively successful especially when applying Pichia pastoris vectors, which has resulted in yields of 40 pmol/mg protein [START_REF] Weiss | Comparative biochemical and pharmacological characterization of the mouse 5-HT5A 5-hydroxytryptamine receptor and the human beta2adrenergic receptor produced in the methylotrophic yeast Pichia pastoris[END_REF]. One advantageous feature of yeast expression is that the extremely high biomasses are achieved in large-scale production although breaking the thick cell wall is a challenge. Moreover, insect cells have been frequently applied for GPCR expression using baculovirus vectors. For instance, the human neurokinin-1 receptor has been expressed in milligram quantities at 60 pmol/mg protein [START_REF] Mazina | Expression and solubilization of a recombinant human neurokinin-1 receptor in insect cells[END_REF]. Very recently, a study on 16 GPCRs expressed from baculovirus in three insect cell lines exhibited binding activities from 1 to 250 pmol/mg suggesting excellent applicability of insect cell-based expression to structural studies on GPCRs [START_REF] Akermoun | Characterization of 16 human G proteincoupled receptors expressed in baculovirus-infected insect cells[END_REF]. Expression of GPCRs in mammalian cells has been hampered by time-consuming and labor-intensive procedures with disappointingly low yields. However, rhodopsin expression from a tetracycline-inducible stable HEK293 cell line resulted in yields of 10 mg/L opsin [START_REF] Reeves | Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants[END_REF]. One system that has frequently been applied for GPCR expression in mammalian cells is based on Semliki Forest virus (SFV) vectors [START_REF] Lundstrom | Semliki Forest virus vectors for rapid and highlevel expression of integral membrane proteins[END_REF]. The attractive features of SFV, a member of the alphavirus family, are the rapid high-titer virus generation, the broad range of mammalian host cells and the high yields of recombinant GPCRs production. Several GPCRs have been expressed with a speciWc binding activity of 50-200 pmol/mg protein and the yields in large-scale production in mammalian cell suspension cultures have been up to 10 mg/L [START_REF] Lundstrom | Semliki Forest virus vectors: eYcient vehicles for in vitro and in vivo gene delivery[END_REF].

In this study, we have applied the SFV system for the overexpression of 101 GPCRs as part of the Membrane Protein Network (MePNet) program. In this consortium, this large number of GPCRs has been expressed in parallel in three expression systems based on Escherichia coli, P. pastoris, and SFV vectors [START_REF] Lundstrom | Structural genomics of GPCRs[END_REF]. Here, we concentrate on reporting on the results of the SFV expression by evaluating the expression levels for all targets by immunodetection. Selected targets were subjected to radioligand binding assays to determine the functional-ity of the expressed GPCRs. Furthermore, some GPCRs were subjected to expression optimization by addition of ligands, previously shown to enhance the binding activity [START_REF] Hovmann | Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells[END_REF]. Finally, a comparison was made on the expression of a few GPCRs from the conventional SFV vector and a mutant SFV vector [START_REF] Lundstrom | Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature-sensitivity for long-term enhancement of transgene expression[END_REF] with enhanced expression levels and prolonged host cell survival.

Experimental procedures

Materials

Restriction enzymes, SP6 RNA polymerase, RNAse inhibitor, and the CAP analogue m 7 G(5Ј)ppp(5Ј)G were purchased from New England Biolabs. Chymotrypsin and aprotinin were from Sigma.

Mammalian cell cultures

BHK-21 (baby hamster kidney), CHO-K1 (Chinese hamster ovary), and HEK293 (human embryonic kidney) cells were cultured in a 1:1 mixture of Dulbecco's modiWed F-12 medium and Iscove's modiWed Dulbecco's medium supplemented with 4 mM glutamate and 10% FCS using standard cell culture techniques. DU-145 cells (human prostate tumor cell line) were grown in Dulbecco's modiWed F-12 medium, 4 mM glutamate and 10% FCS and C8166 cells (human T lymphocyte cell line) in RPI medium, 4 mM glutamate and 10% FCS. All cell culture reagents were from Gibco BRL.

Engineering of SFV vectors, subcloning of GPCR cDNAs, and generation of recombinant SFV

The pSFV2genB and pSFV2genC vectors (Fig. 1) were engineered based on the previously described pSFV2gen vector [START_REF] Lundstrom | Semliki Forest virus vectors for large-scale production of recombinant proteins[END_REF]. The pSFV-PD vector has been previously described [START_REF] Lundstrom | Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature-sensitivity for long-term enhancement of transgene expression[END_REF]. All 101 GPCRs were originally cloned into the pCR4Blunt-TOPO vector (Invitrogen) and their sequences veriWed to those sequences available in publications and public databases before subcloning into the BamHI-SpeI region of the SFV expression vectors. Each clone was veriWed by restriction endonuclease digestions to conWrm the accuracy of obtained clones. Recombinant SFV particles were generated as described previously [START_REF] Lundstrom | Semliki Forest virus vectors for large-scale production of recombinant proteins[END_REF]. BrieXy, SFV vector constructs were linearized by NruI or SapI and conWrmed by restriction digestion analysis. In vitro transcribed RNA molecules from SFV expression and pSFV-helper2 vectors [START_REF] Berglund | Semliki Forest virus expression system: production of conditionally infectious recombinant particles[END_REF] were co-electroporated into BHK-21 cells and recombinant SFV particles harvested 24 h later. The conditionally infectious SFV particles were activated bychymotrypsin treatment and the reaction terminated by addition of aprotinin (trypsin inhibitor). determined by veriWcation of morphology changes on infected cells. Generally, titers in the range of 5 £ 10 8 to 1 £ 10 9 infectious particles per milliliter were obtained. Due to temperature sensitivity, virus stocks prepared from the pSFV-PD vector were cultured at 31 °C instead of the conventional temperature of 37 °C.

SFV-based GPCR expression in mammalian cell lines

Preliminary expression evaluation was done in adherently cultured BHK-21, CHO-K1, and HEK293 cells on 12-and 24-well plates. Cells at 60-70% conXuency were infected with various concentrations of recombinant SFV and cultured for 24, 36, 48, and 72 h before harvesting. Selected well-expressed targets were also tested for expression in DU-145 cells and in suspension cultures of BHK-21, CHO-K1, and C8166 cells. The human prostate tumor cell line DU-145 was chosen because of the high SFV-infection rate and high reporter gene expression previously observed [START_REF] Hardy | Recombinant Semliki Forest virus infects and kills human prostate cancer cell lines and prostatic duct epithelial cells ex vivo[END_REF]. As the human T lymphocyte cell line C8166 natively represents suspension growth, the expression of a few GPCRs was compared to those expressed in BHK and CHO cells adapted to suspension culture. Expression scale-up was performed in spinner or roller Xask cultures.

Immunodetection by Western blotting

Western blotting was performed as follows. Cell samples from various virus concentrations and harvest times were lysed as previously described and subjected to 10% SDS-PAGE [START_REF] Lundstrom | Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature-sensitivity for long-term enhancement of transgene expression[END_REF]. A 30 min electrotransfer to Hyband ECL nitrocellulose membrane was followed by treatment with 5% milk in TBST (TBS with 0.1% Tween 20) at +4 °C, the primary antibodies (anti-FLAG and/or anti-His) (Sigma) and the secondary anti-mouse antibody (Sigma). The visualization of speciWc bands was carried out using the ECL chemilumeniscence kit from Amersham.

Radioligand binding assays

The membrane fraction was isolated from SFVinfected mammalian cells as previously described [START_REF] Hovius | Characterization of a mouse serotonin 5-HT 3 receptor puriWed from mammalian cells[END_REF]. BrieXy, cell pellets were resuspended in 10 mM HEPES, 1 mM EDTA (pH 7.4) and homogenized with an Ultra-Turrax homogenizer for 20 s. Membranes were centrifuged at 55,000g and resuspended in 50 mM Tris, 150 mM NaCl, and 20% glycerol. Radioligand binding assays were performed as previously described [START_REF] Hovmann | Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells[END_REF][START_REF] Lundstrom | High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system[END_REF][START_REF] Lundstrom | Proposed schizophrenia-related gene polymorphism: expression of the Ser9Gly mutant human dopamine D3 receptor with the Semliki Forest virus system[END_REF][START_REF] Harikumar | Modulation of antagonist binding to serotonin1A receptors from bovine hippocampus by metal ions[END_REF][START_REF] Domenech | Characterization of human serotonin 1D and 1B receptors using [ 3 H]-GR-125743, a novel radiolabelled serotonin 5HT1D/1B receptor antagonist[END_REF][START_REF] Audinot | Inverse agonist properties of antipsychotic agents at cloned, human (h) serotonin (5-HT)(1B) and h5-HT(1D) receptors[END_REF][START_REF] Adham | The rat 5-hydroxytryptamine1B receptor is the species homologue of the human 5-hydroxytryptamine1D beta receptor[END_REF][START_REF] Millan | Agonist and antagonist actions of yohimbine as compared to Xuparoxan at alpha(2)-adrenergic receptors (AR)s, serotonin (5-HT)(1A), 5-HT(1B), 5-HT(1D) and dopamine D(2) and D(3) receptors, SigniWcance for the modulation of frontocortical monoaminergic transmission and depressive states[END_REF][START_REF] Marjamaki | Use of recombinant human alpha 2-adrenoceptors to characterize subtype selectively of antagonist binding[END_REF][START_REF] Klotz | Comparative pharmacology of human adenosine receptor subtypes-characterization of stably transfected receptors in CHO cells[END_REF][START_REF] Weiss | PuriWcation and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli[END_REF][START_REF] Peralta | Distinct primary structures, ligand-binding properties, and tissue-speciWc expression of four human muscarinic acetylcholine receptors[END_REF][START_REF] Bastian | IdentiWcation of a key region of kinin B(1) receptor for high aYnity binding of peptide antagonists[END_REF][START_REF] Hill | Cloning, expression, and comparison of the binding characteristics of the known human dopamine receptors[END_REF][START_REF] <www | euroscreen[END_REF][START_REF] Campbell | Reduced inXammation in genetically hypertensive rat airways is associated with reduced tachykinin NK(1) receptor numbers[END_REF][START_REF] Grisshammer | Expression of rat NK-2 (neurokinin A) receptor in E. coli[END_REF][START_REF] Oury-Donat | Functional characterization of the nonpeptide neurokinin3 (NK3) receptor antagonist, SR142801 on the human NK3 receptor expressed in Chinese hamster ovary cells[END_REF][START_REF] Cabrele | Y-receptor aYnity modulation by the design of pancreatic polypeptide/neuropeptide Y chimera led to Y(5)-receptor ligands with picomolar aYnity[END_REF][START_REF] Befort | Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition[END_REF][START_REF] Tahara | Pharmacological characterization of YM087, a potent, nonpeptide human vasopressin V1A and V2 receptor antagonist[END_REF]. The binding experiments were carried out as single point measurements in triplicates at radioligand concentrations close to the 10-fold theoretical K d value.

Expression optimization by ligand addition

The ligand Atropin (Sigma) was added at 100, 300, and 500 M concentrations to cell culture medium at the time of infection with recombinant SFV particles expressing the human muscarinic M1 (ACM1_HUMAN) and the pig muscarinic M2 (ACM2_PIG) receptors, respectively. Radioligand binding was performed as described above to determine the eVect of ligand addition on the speciWc binding activity. Expression optimization was also evaluated by comparison of the expression of the mouse muscarinic M1 receptor from the pSFV2genC vector to the less cytopathogenic pSFV-PD vector [START_REF] Lundstrom | Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature-sensitivity for long-term enhancement of transgene expression[END_REF].

Results

Novel SFV expression vectors

Although previously developed SFV vectors had proven successful for the expression of a large number of GPCRs [START_REF] Lundstrom | Semliki Forest virus vectors for rapid and highlevel expression of integral membrane proteins[END_REF], and additional vectors had been engineered to contain tags for identiWcation and puriWcation [START_REF] Lundstrom | Semliki Forest virus vectors: eYcient vehicles for in vitro and in vivo gene delivery[END_REF] two novel SFV vectors were generated within the MePNet program (Fig. 1). These vectors contain at the N-terminus an optimized Kozak translation sequence [START_REF] Kozak | Point mutations deWne a sequence Xanking the AUG initiator codon that modulates translation by eukaryotic ribosomes[END_REF], a signal sequence from the inXuenza hemagglutinin (HA) gene to potentially enhance the receptor transport to the plasma membrane and a FLAG-epitope for immunodetection. Downstream of the gene of interest (GPCR), a deca-histidine (His-10) tag was engineered for immunodetection and metal aYnity chromatography puriWcation. The pSFV2genC vector diVers from pSFV2genB by an additional biotinylation sequence from Propriobacterium shermanii downstream of the His-10 tag. The biotin tag was introduced into the vector because it would allow streptavidin aYnity puriWcation and the presence of a biotin tag has been demonstrated to provide improved stability of GPCRs expressed in yeast cells [START_REF] Weiss | Comparative biochemical and pharmacological characterization of the mouse 5-HT5A 5-hydroxytryptamine receptor and the human beta2adrenergic receptor produced in the methylotrophic yeast Pichia pastoris[END_REF]. Previously, the expression of the human neurokinin-1 receptor with a C-terminal biotin tag from SFV vectors in CHO cells showed that the expression yields were as high as for the wild-type receptor [START_REF] Lundstrom | Functional activity of a biotinylated human neurokinin-1 receptor fusion expressed in the Semliki Forest virus system[END_REF]. Moreover, the receptor binding characteristics and the coupling to G proteins were unaVected. Both pSFV2genB and pSFV2genC contain Tev protease cleavage sites Xanking the GPCR insert, which makes it possible to cleave oV all tags of the puri-Wed receptor prior to crystallization.

Expression evaluation by immunodetection

Initial expression evaluation was performed by immunodetection in western blots (Fig. 2). Of the 101 GPCR targets, 47 were evaluated for the pSFV2genB vector and 99 for the pSFV2genC vector (Table 1). The expression levels were divided into three categories: low (+), medium (++), and high (+++). Combining the results from both pSFV2genB and pSFV2genC vectors showed that in only six cases no signiWcant signal of the expected molecular weight was detected for the speciWc GPCR (¡). Even so, despite being negative in western blots, one of these GPCRs, the rat serotonin 5-HT7 receptor (5H7_RAT) showed speciWc binding activity (see below). As the expression levels were similar for the two SFV vectors, in interest of the progress of the MeP-Net program, a large number of GPCRs were only tested in one of the vectors, mainly pSFV2genC. For this reason, of those six GPCRs with no signal from pSFV2genC only one was tested and also found negative in pSFV2genB. Among the positive signals obtained, 14 showed low (+), 10 medium (++), and 11 high (+++) from the pSFV2genB vector. The corresponding numbers for pSFV2genC were 28 (+), 23 (++), and 42 (+++). Although it is rather diYcult to give exact numbers of expression levels based on immunodetection, we estimate that the category of medium expression is in the range of 0.5-1 mg and high expression 1-10 mg/L culture. The targets compatible to structural biology were therefore deWned as those belonging to categories of medium (++) and high (+++) expression levels. For pSFV2genB 60.0% and for pSFV2genC 69.9% fell into these categories, suggesting that the biotin tag did not have a negative impact on the expression levels. Taken together, the total number of structural compatible GPCRs was 70.

Expression comparison in diVerent cell lines

Although the majority of the GPCRs were expressed in BHK-21 cells, a cell line frequently used for SFV production and also pharmacological characterization of GPCRs, a number of receptors were also evaluated in CHO-K1 and HEK293 cells (Table 2 expressed both in CHO-K1 and HEK293 cells, but not in BHK-21 cells. Interestingly, LSHR_RAT and GLR_HUMAN were preferentially expressed in DU-145 cells. The CKR7_HUMAN was expressed at high levels not only in C8166 cells, but also in suspension cultures of BHK-21 cells. These Wndings suggested that the host cell background is very important for achieving high expression levels as previously shown for opioid receptors [START_REF] Lundstrom | Semliki Forest virus vectors applied to receptor expression in cell lines and primary neurons[END_REF].

Radioligand binding on selected targets

Selected GPCRs expressed from SFV vectors were subjected to radioligand binding assays. In total, 41 GPCRs were analyzed for speciWc binding, which demonstrated a huge variation in binding values (Table 3). The binding activity levels were divided into three categories, low (< 1 pmol/mg), medium (1-10 pmol/mg), and high levels (> 10 pmol/mg). Only 2 GPCRs showed no speciWc binding, 10 showed low, 16 medium, and 13 high binding activities. The highest binding activity of 287 pmol/mg was observed for the human adenosine A2A receptor (AA2A_HUMAN).

Comparison of immunodetection and speciWc binding

The expression levels evaluated by immunodetection were compared to the binding data obtained (Table 2). Interestingly, the rat serotonin 5-HT7 receptor (5H7_RAT), which showed no speciWc signal in western blotting, resulted in a binding activity of 2.18 pmol/mg. Among the 10 targets in the category of low (+) immunodetection, 2 showed no binding activity, 3 low, 4 medium, and 1 high speciWc binding. The six GPCRs in the category of medium (++) expression were evenly distributed (2 each) in the binding activity groups. Of the group of 24 high (+++) expressing targets 2 GPCRs showed low, 11 medium, and 11 high binding activity.

Expression optimization

In attempts to further improve the expression levels of GPCRs in mammalian cell lines, the supplementation of ligands was investigated. Previously, it was demonstrated that the addition of ligand to the cell culture medium, signiWcantly increased the binding activity of the histamine H2 receptor in SFV-infected COS-7 cells [START_REF] Hovmann | Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells[END_REF]. Here, we tested the addition of increasing concentrations of a speciWc ligand, Atropin, to CHO suspension cell cultures infected with SFV expressing two musca- rinic receptors. In this context, the binding activity of the human muscarinic M1 receptor (ACM1_HUMAN) could be enhanced from 0.75 to 5 pmol/mg (Fig. 3). Similarly, ligand binding improved approximately Wve-fold to 22 pmol/mg (data not shown) for the pig muscarinic M2 receptor (ACM2_PIG). In contrast, ligand addition did not aVect the binding activity of the mouse muscarinic M1 receptor (ACM1_MOUSE) (data not shown).

Application of mutant SFV vectors

Several mutant SFV vectors have been engineered with the aim to reduce their inherent cytotoxicity on host cells [START_REF] Lundstrom | Semliki Forest virus vectors: eYcient vehicles for in vitro and in vivo gene delivery[END_REF][START_REF] Perri | Replicon vectors derived from Sindbis virus and Semliki Forest virus that establish persistent replication in host cells[END_REF]. In addition to the lower cytotoxicity, the SFV-PD vector also showed elevated levels of reporter protein expression [START_REF] Lundstrom | Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature-sensitivity for long-term enhancement of transgene expression[END_REF]. Here, we could demonstrate that the binding activity of the mouse muscarinic M1 was enhanced four-to Wvefold when expressed from the SFV-PD vector in comparison to the pSFV2genC vector (Fig. 4). Interestingly, in contrast to the lack of any eVect on binding levels in the pSFV2genC vector after ligand addition, the SFV-PD generated signiWcantly higher binding activity (4.5 pmol/mg).

Discussion

We have evaluated the expression of 101 GPCRs applying SFV-based expression in mammalian cell lines. In Western blots, 95 of the 101 targets gave a positive signal. The expression levels were divided into three categories-low, medium, and high-and the results indicated that 70 GPCRs were expressed at structural biology compatible levels. Two SFV vectors harboring various tags and other accessory sequences (Fig. 1) were applied for the study. The pSFV2genB and pSFV2genC vectors were otherwise identical except for the C-terminal biotin tag present in the latter. As no major diVerences in expression levels were observed between the two vectors after analysis of half of the targets, it was decided to concentrate on the pSFV2genC vector for the evaluation of the rest of the GPCRs. Of the six GPCRs that did not show positive signals in western blots, the human dopamine D2 (D2DR_HUMAN), the rat serotonin 5-HT7 (5H7_RAT), and the human metabotropic glutamate R2 (MGR2_HUMAN) receptors have previously been successfully expressed from SFV vectors [START_REF] Lundstrom | Semliki Forest virus vectors for rapid and highlevel expression of integral membrane proteins[END_REF][START_REF] Schweitzer | Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors[END_REF]. Also the endothelin 1B receptor has been well expressed before [START_REF] Cramer | Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q[END_REF]. In a similar way, the human NK1R (NK1R_HUMAN) [START_REF] Lundstrom | Semliki Forest virus vectors: eYcient vehicles for in vitro and in vivo gene delivery[END_REF], the human adrenergic 2B (A2AB_HUMAN) [START_REF] Sen | Functional expression and direct visualization of the 2B -adrenergic receptor and 2B -AR green Xuorescent fusion protein in mammalian cells using Semliki Forest virus vectors[END_REF], and the rat NK1R (NK1R_RAT) have previously showed 5-to 20-fold higher binding activity in comparison to values obtained in this study [START_REF] Lundstrom | Semliki forest virus-based expression for versatile use in receptor research[END_REF]. In contrast, the AA2A_HUMAN receptor showed extremely high binding activity (287 pmol/mg) in comparison to the earlier observed 40 pmol/mg [START_REF] Lundstrom | Alphaviruses as tools in neurobiology and gene therapy[END_REF]. This discrepancy may be at least partly explained by the diVerences in SFV vector constructs. Particularly, the presence of the signal sequence from inXuenza HA at the large extracellular domain of the mGluR2 may have presented a negative eVect on the expression pattern. Additionally, variation in virus batches and cell culture conditions may have aVected the results. Most importantly, in most of the previously published studies at least some optimization of the expression conditions had taken place. In contrast, as described for the 101 GPCRs evaluated in this study, only limited optimization through ligand addition and vector selection was applied. The main purpose of this study was to evaluate the expression levels of a large number of GPCRs in parallel. The introduction of a signal sequence upstream of a GPCR sequence has previously demonstrated improved transport to the plasma membrane and enhanced binding activity in baculovirusinfected insect cells [START_REF] Guan | Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide[END_REF]. Here, we have not included transport and localization studies on GPCRs expressed 337 338 from the pSFV2genB and C vectors in comparison to standard SFV vectors. However, binding assays suggested no signiWcant superiority of either type of vector and only future localization studies will reveal whether any improvement in receptor delivery to the plasma membrane can be achieved. The successful expression of many GPCRs of which several showed high speciWc-binding activity forms a good basis for further studies. It will now be essential to optimize the expression conditions and binding activity for selected GPCRs in attempts to carry out further puriWcation and structural characterization with the ultimate goal of obtaining high-resolution structures of several GPCRs.

Fig. 1 .

 1 Fig. 1. Schematic presentation of various SFV vector constructs. nsP1-4, SFV non-structural genes; K, Kozak sequence; ss, signal sequence from inXuenza hemagglutinin (HA) gene; FLAG, FLAG-epitope tag; Tev, Tev protease cleavage site; His, 10 histidine-tag; Biotin, biotin tag from Propriobacterium shermanii; **, point mutations in nsP2 (S256P and R650D).

Fig. 2 .

 2 Fig. 2. Immunodetection of SFV-based expression of selected GPCRs. Expression was compared for GPCRs expressed from either the pSFV2genB (B) or pSFV2genC (C) vector as indicated. Lane 1, 5H1AHUMAN/B; lane 2, 5H1A_HUMAN/C; lane 3, 5H1B_HUMAN/B; lane 4, 5H1B_HU-MAN/C; lane 5, 5H2A_HUMAN/C; lane 6, 5H7-HUMAN/C; lane 7, AA1R_HUMAN/C; lane 8, AG2R_HUMAN/C; lane 9, BRB1_HUMAN/C; lane 10, CB2R_HUMAN/C; lane 11, CCR3_HUMAN/C; lane 12, CKR6_HUMAN/C; lane 13, EDG3_HUMAN/C; lane 14, ETR1_HUMAN/C; lane 15, HH2R_HUMAN/B; lane 16, HH2R_HUMAN/C; lane 17, STE3_YEAST/B; lane 18, STE3_YEAST/C. An anti-FLAG antibody (1:500) was used for detection.

Fig. 3 .Fig. 4 .

 34 Fig.3. Optimization of SFV-based expression of muscarinic receptors by ligand addition. Increasing concentrations (0-500 M) of Atropin was added to the medium of cells infected with the pSFV2genC vector carrying the human M1 (ACM1_HUMAN). Binding assays were carried out applying 24 nM 3 H-QNB (Perkin Elmer) as ligand.

Table 1

 1 Expression evaluation by immunodetection of 101 GPCRs from pSFV2genB and pSFV2genC

	M#	SFVB	SFVC	M#	SFVB	SFVC

M#, MePNet number; nd, not done; SFVB, pSFV2genB; SFVC, pSFV2genC.

of Pages 9; DTD=5.0.1 ARTICLE IN PRESS 7 July 2005 Disk Used Marry Anitha (CE) / Vijayakumar (TE)

Table 2

 2 Comparison of SFV-based expression in various mammalian cell lines (A) The number of GPCRs showing positive (ID+) and negative (ID¡) signals evaluated by immunodetection and the total number of GPCRs. (B) The number of GPCRs originally negative in various cell lines (ID¡s), which showed positive signals in other cell lines.

		ID+	ID±	Total
	(A)
	BHK-21	71	21	92
	CHO-K1	18	32	50
	HEK293	11	16	27
	ID±	ID+/BHK-21	ID+/CHO-K1	ID+/HEK293
	(B)
	BHK-21	-	9	6
	CHO-K1	7	-	3
	HEK293	4	2	-

Table 3

 3 Comparison of SFV-based expression evaluation by immunodetection and binding activity M#, MePNet number; SFV ID, SFV-based expression evaluated by immunodetection; SFV BA, SFV-based expression evaluated by radioligand. binding assays.

	MePNet# SEV ID	SFV BA

Acknowledgments

We are grateful to Gilles Moreau for technical assistance in subcloning and expression activities. The Wnancial support received from the industrial supporters of the MePNet program.


Juliette Chabassier
email: juliette.chabassier@inria.fr

Augustin Ernoult

Olivier Geber

Augustin Humeau

Alexis Thibault

Robin Tournemenne

Tobias Van Baarsel

THE VIRTUAL WORKSHOP OPENWIND : A PYTHON TOOLBOX ASSISTING WIND INSTRUMENT MAKERS

Our project develops the software OpenWInD for wind instrument making. A first feature is the prediction of the acoustical response of the instrument from the knowledge of its shape (bore and holes). This can be done in the harmonic (impedance computation) and temporal (sound computation) domains. It can account for various physical situations (non constant temperature, coupling with an embouchure, ...). Discretization is done in space with 1D spectral finite elements and in time with energy consistent finite differences. The second feature is the reconstruction of the shape of an instrument that fulfils a certain objective. This can be used for bore reconstruction, and instrument design. The latter is based on a strong interaction with makers and musicians, aiming at defining interesting design parameters and objective criteria, from their point of view. After a quantitative transcription of these criteria, under the form of a cost function and a design parameter space, we implement various gradient-based optimization techniques. More precisely, we exploit the fact that the sound waves inside the instruments are solution to acoustic equations in pipes, which gives us access to the Full Waveform Inversion technique (FWI) where the gradient is characterized as the solution to another wave equation. The computational framework is flexible (in terms of models, formulations, coupling terms, objective functions...) and offers the possibility to modify the criterion by the user. The goal is to proceed iteratively between the instrument makers and the numerical optimisation tool (OpenWInD) in order to achieve, finally, criteria that are representative for the makers. In the presentation, we will demonstrate and discuss some comparisons between measurements and simulation on real instruments.

INTRODUCTION

OpenWInD software aims at solving state-of-the-art models in musical acoustics for wind instruments with accurate, robust and efficient techniques from scientific computing science and numerical analysis. It allows the direct computation of the acoustical frequential response (input impedance) of a given instrument (knowing its bore and side holes geometrical parameters), or of the sound of this instrument when coupled to different types of excitators (reed, lips). It also relies on inversion techniques used in the field of geophysics (Full Waveform Inversion, [START_REF] Virieux | An overview of fullwaveform inversion in exploration geophysics[END_REF]) to perform reconstructions from measurements and investigate some sensitivity characteristics of the instruments.

DESCRIPTION

Finite Elements Method (FEM) is used in OpenWInD to perform the spatial discretisation of the 1D pipe equations (Telegraphist' equations with viscothermal effects, also called Zwikker-Kosten equations, or their approximations [START_REF] Zwikker | Sound absorbing materials[END_REF][START_REF] Tijdeman | On the propagation of sound waves in cylindrical tubes[END_REF][START_REF] Thibault | Viscothermal models for wind musical instruments[END_REF]). A quantitative comparison between Transfer Matrices (TMM) and 1D FEM methods for the computation of a trumpet input impedance [START_REF] Tournemenne | A comparison a one-dimensional finite element method and the transfer matrix method for the computation of wind music instrument impedance[END_REF] yields the result displayed in Fig. 1 : for arbitrary bores and in the presence of viscothermal effects, the FEM provides more accurate results (relative 2 error smaller than 1 %) for a lower computational cost, which makes it a good candidate for musical acoustics purposes. It constitutes a variational approximation of the pipe equations, suitable for energy consistent time-stepping strategies allowing the coupling with various elements such as nonlinear excitators, pipe junctions or radiation conditions (see [START_REF] Thibault | time-domain simulation of a dissipative reed instrument[END_REF]). The FEM generalizes the method used in [START_REF] Bilbao | Direct simulation of reed wind instruments[END_REF] where a low order approximation is performed. Moreover, it provides an efficient framework for the computation of the gradient of the acoustic field with respect to design parameters, as the bore radius or the side holes parameters (see [START_REF] Ernoult | Bore reconstruction of woodwind instruments using the full waveform inversion[END_REF]).

Fig. 2 shows the modular organisation of OpenWInD. Several features are available in the public version of OpenWInD [START_REF] Chabassier | Open wind instrument design -a python toolbox assisting instrument makers[END_REF], released under GPLv3 licence, as input impedance computation of arbitrary shaped instruments with side holes (see [START_REF] Tournemenne | A comparison a one-dimensional finite element method and the transfer matrix method for the computation of wind music instrument impedance[END_REF]), sound computation with reeds and lips, sensitivity maps (see Fig. 4) and bore reconstruction from measurements.

ASSISTING INSTRUMENT MAKING

In the context of our collaboration with Humeau Factory, a measurement impedance sensor has been built following the principles described in Gibiat with five micro- since only T-junctions are available at this day. Indeed, the third and fourth holes are made of two small holes for improving the altered notes intonation, while the ninth hole is composed of two opposite holes on the bell. Finally, the eighth and ninth holes are equipped with keys which is not accounted for in the current version of OpenWInD. For all these reasons, some discrepancies can be observed between measurement and simulation. Refined models and better geometric reconstruction should yield a better accordance.

Fig. 4 displays a sensitivity map of a Humeau baroque oboe (logarithm of the modulus of the gradient of the input impedance with respect to the hole position, in a frequency range corresponding to ± 10 cent around the played frequency). This map indicates how much a hole displacement will affect the different notes of the oboe. Other maps can be generated for other hole parameters as its length or its radius. They can prove useful in the development of new instruments in order to assess quantitatively which holes parameters are preponderant in the tuning of a given note, or conversely, which notes will be affected by the modification of one hole. This feature is based on the evaluation of the gradient in the framework of the Full Waveform Inversion (see [START_REF] Ernoult | Bore reconstruction of woodwind instruments using the full waveform inversion[END_REF]).

Sound computation can be done from an arbitrary shaped instrument, coupled with a valve (reed, see [START_REF] Thibault | time-domain simulation of a dissipative reed instrument[END_REF] on a french bassoon, or lips, see [11] on trumpets of the national collection Besson of Cité de la Musique -Philharmonie de Paris). The tuning of the valve parameters influences greatly the instrument sound, leading to various sounds ranging from "beginner" sound to "artificial mouth" sound, and musical sound. Even if it is not yet representative of what a musician would sound like on this instrument, this feature could prove useful for instrument makers by virtually assessing the influence of a geometric change on several targets as the ease of playing, the intonation, and even the relative tone quality.

PROSPECTS

One purpose of the OpenWInD project is to implement robust, accurate and efficient numerical methods to model wind musical instruments with state-of-the-art models. The public version of the software is available under GPLv3 licence [START_REF] Chabassier | Open wind instrument design -a python toolbox assisting instrument makers[END_REF] along with examples for using the different features. One other purpose is to provide instrument makers with tools to virtually assess some changes in the characteristics of the instruments under geometric alterations. The currently implemented models are making some important approximations which still prevent quantitative comparisons, but show very promising for qualitative assessment. Possible future improvements will include finer models for propagation, toneholes, radiation of toneholes with keys, or automatic embouchure control ; all these should bring numerical results closer to the fine degree of precision required in instrument making.

1 Z

 1 -Z conv F EM Z conv F EM TMM FEM N=35, r=3 FEM N=35, r=4 adaptative FEM (b) CPU time w.r.t. impedance relative 2 error.

Figure 1 .

 1 Figure 1. Comparison between TMM and FEM method for the computation of a trumpet input impedance with viscothermal effects. Number of elements N , order of elements r.

Figure 2 .

 2 Figure 2. Schematic view of the different features of OpenWInD

Figure 3 .

 3 Figure 3. Measurement of a Humeau baroque bassoon with our impedance sensor.

Figure 4 .

 4 Figure 4. Sensitivity map of a Humeau baroque oboe, sensitivity of the input impedance w.r.t. the holes position. Open holes of each fingering are circled in red.

Figure 5 .

 5 Figure 5. modulus (top) and phase (bottom) of a Humeau baroque oboe with the first hole closed (B1 note). Simulation : blue. Measurement : orange. Played frequency : vertical line.