Báo cáo khoa học: Ligand-specific dose–response of heterologously expressed olfactory receptors potx

Ligand-specific dose–response of heterologously expressed olfactory receptors Gre ´ goire Levasseur 1 , Marie-Annick Persuy 1 , Denise Grebert 1 , Jean-Jacques Remy 2 , Roland Salesse 1 and Edith Pajot-Augy 1 1 INRA-Biotechnologies, Neurobiologie de l’Olfaction et de la Prise Alimentaire, Re ´ cepteurs et Communication Chimique, Jouy-en-Josas, France; 2 Unite ´ Neurogene ` se et Morphogene ` se au cours du De ´ veloppement et chez l’Adulte, UMR CNRS 6545, Institut de Biologie du De ´ veloppement de Marseille, Parc scientifique de Luminy, Marseille, France Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS 88, 2366–2369]. Maximal sti- mulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant con- centrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we deve- loped an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand- specific and dose-dependent. We thus demonstrate that a differential dose–response originates from the olfactory receptor itself, which is thus capable of efficient discrimin- ation between closely related agonists. Keywords:olfactoryreceptors;olfactorycoding;olfactory discrimination; odorants; intracellular calcium. Olfactory receptors (ORs) belong to the large family of G-protein coupled receptors (GPCRs) characterized by their seven transmembrane spanning domains. Investigation of olfactory receptors/odorant interactions is crucial to understand the molecular basis of olfactory coding. For this purpose, olfactory receptor genes have been heterologously expressed in various surrogate cells [1–4], in cell lines with a neuronal phenotype [5], or derived from the olfactory epithelium [5–7], or even directly in olfactory epithelium [8,9]. Individual olfactory sensory neurons have also been tested for their responsiveness to odorant stimulation [10– 15]. So far, due to the large number of potential ligands and the lack of a suitable screening system, only a few OR– odorant couples have been identified. The rat I7 receptor [16] was the first mammalian olfactory receptor for which a preferential ligand (octanal) was identified [8]. As such, it has been the subject of subsequent investigations [3,9], involving an impressive range of odorants and reporting a number of stimulating odorants. This raises the possibility that the receptor itself is capable of some olfactory discrimination, as suggested by the response of individual olfactory neurons to a few odorants at given concentrations [12,13], and that this is not only performed in higher olfactory centers (i.e. olfactory bulb). OR17-40 was the first characterized human olfactory receptor, for which helional represented the most effective odorant ligand [6]. An heterologous expression system in a mammalian host cell line was thus developed, using the full-length cDNA sequence instead of chimeric constructions. We report a functional expression of the rat I7 olfactory receptor in stably transfected COS cells, and of the human OR17-40 olfactory receptor in stably transfected ODORA cells tested by a spectrofluorimetric intracellular calcium assay. Both COS-I7 cells and ODORA OR17-40 cells exhibit a dose- dependent response to their ligands with optimal concen- trations in a subpico- to subnano-molar range. Moreover, COS-I7 cells responded differentially to odorants of the same family of aldehydes but with varying carbon chain length, in terms of concentration providing the optimal response. Thus, olfactory receptors themselves can not only efficiently discriminate broad families of odorants, but they are also able to differentiate close odorants of a given family. Correspondence to E. Pajot-Augy, INRA-Biotechnologies, Neuro- biologie de l’Olfaction et de la Prise Alimentaire, Re ´ cepteurs et Communication Chimique, 78352 Jouy-en-Josas Cedex, France. Fax: + 33 1 34 65 22 41, Tel.: + 33 1 34 65 25 63, E-mail: pajot@jouy.inra.fr Abbreviations: OR, olfactory receptor; GPCR, G-protein coupled receptor; COS, Cercopithecus aethiops SV40 transformed; PLC, phospholipase C; NaCl/P i , phosphate buffer saline; HEK, human embryonic kidney; FBS, fetal bovine serum. (Received 17 February 2003, revised 18 April 2003, accepted 15 May 2003) Eur. J. Biochem. 270, 2905–2912 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03672.x Materials and methods Constructs The I7 full-length sequence was amplified by PCR from genomic rat DNA with cloned Pfu DNA polymerase (Stratagene) and inserted in a pGEM-T vector (Promega) for subcloning and control sequencing (Genome Express). I7 was inserted in the pCMV-Tag3 expression vector (Stratagene), in frame with the translationalstarting sequence (10-base Kozak consensus sequence) and the 10 amino acids long tag from the human c-myc gene of this vector, using sites PstIandKpnI of the MCS. Similarly, OR17-40 full-length sequence was cloned into a pGEM-T vector, then inserted in the pCMV-Tag3 expression vector using sites BamHI and XhoI of the MCS. Therefore, the c-myc epitope is located at the 5¢-terminus of olfactory receptor sequences. Cell lines and transfection COS-7 cells (Cercopithecus aethiops kidney cells transformed by an origin-defective mutant of SV-40) were grown in Dul- becco’s Modified Eagle’s Medium containing 10% decom- plemented foetal bovine serum in a 5% (v/v) CO 2 atmosphere at 37 °C, and transfected at a 50% confluence using ExGen 500 from Euromedex in six-well dishes. Geneticin (G418) was used at a final concentration of 0.5 mgÆmL )1 to select stable clones. Culture media, G418 and trypsin-EDTA were from Gibco BRL, fetal bovine serum(FBS)fromPerbio. ODORA cells [17] consist of a conditionally immortalized cell line derived from the olfactory sensory neuron lineage, obtained from rat olfactory epithelium. They were grown at 33 °C in the same medium as COS cells. Transfection, and selection of stable clones, were performed similarly. Human embryonic kidney (HEK) 293 cells were grown and transfected in the same conditions as COS cells. For further experiments, transfected cells were used 24 h after transfection. RT-PCR on extracted RNA RNAs from established stable clones were prepared from 10 7 cells following the modified procedure of Chomczynski [18] proposed by Puissant and Houdebine [19]. RT-PCR was performed on DNase-treated RNA samples (RQ1 DNase, RNase-free from Promega). First-strand synthesis was achieved with Gibco BRL SuperScript kit using oligo(dT) 12)18 as the primer. Specific primers were designed with I7 or OR17- 40 sequences and used to specifically amplify the target cDNA by PCR on the first strand: 5-ATggAgCAgAAACTC ATCTCTgAA-3¢ and 5¢-TTCTgCAgCTAACCAATTTTg CTgCCTTTgTT-3¢ for I7, 5-CgggATCCATgCAgCCA gAATCTggggCC-3¢ and 5¢-CCgCTCgAgTCAAgCCAg TgACCgCCTCCC-3¢ for OR17-40. Each PCR consisted of 40 cycles: 94 °C/60 °C/72 °Cwith 1 min steps, with a final elongation of 10 min at 72 °C. PCR products were sequenced (Genome Express). Immunofluorescence microscopy Cells were cultured on glass slides, coated with either FBS or 0.01% poly L -Lysine. They were washed with NaCl/P i (Na 2 HPO 4 8m M ,KH 2 PO 4 1.5 m M , NaCl 150 m M ,KCl 3m M )for4· 5 min. Fixation was performed with 2.5% paraformaldehyde in NaCl/P i for 20 min at room tempera- ture. Cells werewashed again with NaCl/P i for 4 · 5min.No permeabilization was performed. Preincubation was carried out for 1 h at room temperature in NaCl/P i +2%BSA (Sigma). A mouse monoclonal anti-(c-myc Ig) (Roche) was used in combination with a FITC-coupled secondary anti- body (Jackson Immunoresearch Laboratories). The primary antibody was diluted at 1/800 from the 1 mgÆmL )1 stock solution and incubated for 18 h at 4 °CinNaCl/P i incuba- tion buffer. Cellswere washed four times in NaCl/P i +0.2% BSA, then incubated for 1 h at room temperature in the dark witha 1/800 dilutionof FITC-coupled goatanti-(mouse IgG). Cells werewashed four times in NaCl/P i +0.2%BSA. After a final NaCl/P i rinsing, slides were mounted with Vectashield (Vector), and kept at 4 °C in the dark. They were examined under a fluorescent microscope (Leica DMRB) equipped with the appropriate filter for fluorescein, or on a Carl Zeiss LSM 310 confocal laser scanning microscope at 488 nm excitation using helium-neon ion laser, and optimal depth resolution. It was checked with another membrane receptor with the same c-myc tag at its N-terminus, expressed in the same type of cells, that this procedure indeed induces only a membrane-located fluorescence (data not shown, prolactin receptor expression vector by courtesy of I. Gourdou-Jacovella, NOPA, INRA Jouy-en-Josas). Odorants Octanal, heptanal, nonanal, octanol and octanoic acid were from Sigma-Aldrich. Helional, lyral and lilial were free gifts from Roche. Stock solutions (10 )1 M ) were prepared each day in dimethylsulfoxide, and 10 )4 M dilutions in water were made extemporaneously, directly from the 10 )1 M stock solution. EtOH was used instead of dimethylsulfoxide for lyral and lilial, with further extemporaneous dilutions starting from 10 )3 M dilutions in water. Further dilutions were prepared extemporaneously by successive 1 : 10 dilu- tions in water. Diacetyl (Sigma-Aldrich) solutions were prepared directly in water. Spectrofluorimetric intracellular calcium assay Stable cells were seeded at about 200 cellsÆmm )2 on glass coverslips of adequate size coated previously with either FBS or 0.01% poly( L -lysine), grown until a uniform layer of subconfluent cells was obtained, and placed in a 1% FBS medium 24 h before experiments. Cells to be transfected were seeded at about 100 cellsÆmm )2 on glass coverslips previously coated with either FBS or 0.01% poly( L -lysine), grown for 24 h, transfected as described above, and used 24 h later. Prior to the assay, cells were washed in a Hank’s Hepes buffer, pH 7.4 (137 m M NaCl, 5.4 m M KCl, 0.441 m M KH 2 PO 4 ,0.16m M NaH 2 PO 4 ,0.885m M MgCl 2 , 5.55 m M glucose, 1.25 m M CaCl 2 ,25m M Hepes; buffer A). They were then loaded with 1 l M fluorescent marker fura-2- acetoxy-methyl [20] (Molecular Probes) for 30 min in the dark at room temperature, and washed three times in buffer A. Fura-2-acetoxy-methyl is an EGTA-derived cal- cium chelator that enters the cells and is transformed in Fura-2 by nonspecific esterases. Coverslips were introduced 2906 G. Levasseur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 in an adapted cuvette, with excitation and emission beams at 45 ° relative to the surface. Experiments were performed on a Hitachi F-2500 spec- trofluorimeter using a double wavelength excitation (k1 ¼ 340 nm, k2 ¼ 380 nm, excitation slits at 10 nm). Emission intensities F(k1) (calcium-chelating Fura-2) and F(k2) (nonchelating Fura-2) were monitored at 510 nm for 10 min (emission slit at 10 nm). Each measurement was calibrated by final injection of 25 l M digitonin (Sigma) to obtain the maximum of calcium-chelating Fura-2 [providing F max (k1) and F min (k2)], followed by an injection of EGTA 4m M Tris 30 m M , pH 8, to reach the minimum non- chelating Fura-2 [providing F min (k1) and F max (k2)]. The intracellular calcium concentration is provided by the spectrofluorimeter using: [Ca 2+ ] i (nM) ¼ K · (R ) R min )/ (R max ) R)where,R min ¼ [F min (k1) ) Z1]/[F min (k2) ) Z2] and R max ¼ [F max (k1) ) Z1)/[F max (k2) – Z2] and R ¼ [F(k1) ) Z1)/F(k2) – Z2] and K ¼ K d · F 0 /F s ,where K d is Fura-2 dissociation constant (224 n M ), F 0 is the 510 nm emission signal (380 nm excitation) in the absence of calcium, and F s is the 510 nm emission signal (380 nm excitation) with a saturating concentration of calcium. Z1 and Z2 are the intrinsic fluorescence intensities of the sample excited at k1andk2. Odorant stimulation was performed by injection of a 30-lL volume of a given dilution of the odorant in the spectrofluorimeter cuvette containing 3 mL buffer A, indu- cing a further odorant dilution of 1/100. A magnetic stirrer ensures efficient homogenization of odorant in the medium in less than 2 s. As there is no buffer aspiration and thus no rinsing of the odorant, a new coverslip must be used for each odorant stimulation and for each concentration. Measure- ments were performed several times at each concentration and for each odorant. Experiments with solvents at the same dilution used in odorant samples were also performed under the same conditions with new coverslips. Data plots mention first quartile, median (second quartile) and third quartile of all significant data. In the case of a single odorant, all data points are also plotted as a scatter chart. Results Clonal transfected cell lines The presence of I7 mRNA in COS-I7 cells and of OR17-40 mRNA in ODORA OR17-40 cells was tested by RT-PCR in the stable clones, after a DNase treatment to eliminate a potential genomic DNA contamination. The expected bands (980 bp for I7 and 950 bp for OR17-40) were detected on agarose–ethidium bromide gels for COS-I7 cells and ODORA OR17-40 cells, respectively (Fig. 1, lanes 2 and 4). Negative controls were obtained by RT-PCR performed on mRNAs from untransfected cells (Fig. 1, lanes 1 and 3), and by PCR on nonretro-transcripted mRNAs (not shown). Sequencing the PCR product confirmed that the RT-PCR products indeed had the expected OR sequences. Fluorescence microscopy In order to visualize the recombinant expression of I7 and OR17-40 olfactory receptors, we performed immuno- fluorescence microscopy on nonpermeabilized COS-I7 and ODORA OR17-40 stable clonal cell lines, using an anti- (c-myc Ig) and a fluorescein isothiocyanate (FITC)-coupled secondary antibody. Stable COS-I7 cells never exhibited any detectable labeling, nor did stable ODORA OR17-40 cells. Detection was thus attempted on various types of cells transiently transfected with I7 or OR17-40 expression vectors. Observations were performed with a confocal microscope. COS cells transiently transfected with I7 showed no specific labeling either. In the case of OR17-40, positive labeling was observed in a number of transiently transfected cell lines (Fig. 2). About 1% of OR17-40 transiently trans- fected COS cells showed a positive labeling (Fig. 2A). A cortical localization is particularly visible in HEK293 OR17- 40 cells, in which about 10% of the cells exhibited this pattern (Fig. 2B). As for OR17-40 transiently transfected ODORA cells, only1& of thecells yieldeda discretepunctuate labeling at the level of the plasmic membrane (Fig. 2C). Intracellular calcium assay Characteristics of the calcium response to odorant stimulation. Figure 3 shows representative curves obtained during spectrofluorimetric intracellular calcium assays on COS-I7 cells from a stable clone, each with a single odorant stimulation performed, respectively, at heptanal 10 )13 M , octanal 10 )10 M and nonanal 10 )12 M (final concentrations), and on a ODORA OR17-40 stable clone stimulated with helional 10 )12 M (final concentration). The response of the cells consists of a transient peak of intracellular calcium concentration, with a maximum reached about 10 s after injection, and prompt return to the baseline. The late broad increase results from the calibration procedure. Specificity and dose-dependence of the calcium response: I7 response from COS-I7 cells. Specificity of olfactory receptors responses to odorant stimulation was investigated Fig. 1. Detection of rat I7 mRNA in RT-PCR products from cell strains. Lane 1, native COS 7 cells; lane 2, COS-I7 clone obtained through stable transfection (see conditions in the text). A 980 bp band corresponding to the expected sequence size of the PCR product is detected. Lane 3, native ODORA cells; lane 4, ODORA OR17-40 clone obtained through stable transfection. A 950 bp band corres- ponding to the expected sequence size of the PCR product is detected. Size marker is DRIgest III from Amersham. Ó FEBS 2003 Ligand-specific dose–response of olfactory receptors (Eur. J. Biochem. 270) 2907 on both rat I7 and human OR17-40 receptors. I7 was expressed in COS cells that represent a widely used, multipurpose cell factory. A number of odorants were tested on COS-I7 cells from the same clone. A series of aliphatic aldehydes (heptanal, octanal, nonanal), aromatic aldehydes (lyral, lilial) and odorants with same carbon chain length but a different chemical function from octanal (octanol and octanoic acid), and diacetyl were used. We obtained a very specific response of I7 receptor with the three aliphatic aldehydes, but not with other odorants (Fig. 4). Solvents at the same dilution used in odorant samples did not induce any stimulation of the cells. Negative controls were obtained with native COS cells, for which no calcium response was ever obtained with any odorant stimulation (aldehydes, lyral, lilial or diacetyl). ATP disodium salt (10 )4 M ) was used as positive control. Fig. 2. Confocal fluorescence microscopy on OR17-40 transiently transfected cells. Immunolabeling was performed with a mouse monoclonal anti- (c-myc Ig), and a FITC-coupled secondary antibody. A, COS cells; B, HEK293 cells; C, ODORA cells. 2908 G. Levasseur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The different COS-I7 clones gave comparable responses to odorant stimulations. In a given clone, plotting [Ca 2+ ] i increase in response to various concentrations of heptanal yields a narrow bell-shaped curve, with a maximum for a 10 )13 M concentration of heptanal (Fig. 4), and no response for higher concentrations. Specificity and dose-dependence of the calcium response: OR17-40 response from ODORA OR17-40 cells. In an effort to reproduce more closely natural conditions, OR17- 40 was expressed in ODORA cells that are more representative of the native tissue expressing olfactory receptors. Stable ODORA OR17-40 cells were submitted to helional or other odorant stimulation. Only helional induced a response from the cells. No response was obtained with solvents used at the same dilution as in odorant samples. Native ODORA cells never exhibited any response to any odorant stimulation tested. Again, the dose–response profile to helional stimulation is a narrow bell-shaped curve, with a maximum for 10 )11 M helional, almost no response for 10 )10 M and above, few responses for 10 )12 M helional, and no response for lower concentrations (Fig. 5). In addition, stimulation experiments were attempted on ODORA, COS and HEK cells transiently transfected with OR17-40 expression vector, as immunodetection was able to reveal various levels of receptor expression at the membrane in those cells. Although the same global response pattern was observed as in ODORA OR17-40 stable cells (not shown), both response level and reproducibility were too low to allow accurate data processing in any of those transiently transfected cells. Differential dose–response to a family of linear aldehydes for I7. In the case of I7, the response of the receptor to a family of linear aldehydes was investigated. For each aldehyde, a bell-shaped dose–response curve was obtained. The maximal signal amplitude is of the same order of magnitude for each aldehyde studied. However, the concentration–response curves are shifted along the concentration axis as a function of the odorant carbon chain length. COS-I7 clones cells exhibit a response to heptanal in a low concentration range (10 )14 to 10 )12 M ), to nonanal in an intermediate concentration range (10 )13 to 10 )10 M ), and to octanal for higher odorant concentrations over a broader range (10 )12 to 10 )7 M ). Fig. 3. Spectrofluorimetric intracellular calcium assays. The curves show representative responses of cells to single odorant stimulation. COS-I7 cells were stimulated with aldehydic odorants, and ODORA OR17-40 cells with helional. The final concentration indicated for the respective odorants is reached by injection of a dilution of the odorant at t 0 indicated by the arrow. A typical curve resulting from pure dimethylsulfoxide (or ethanol) injection is also shown. Fig. 4. Differential dose-response of rat I7 receptor expressed in a COS-I7 clone. Dose-response curves were plotted by measuring the intracellular calcium concentration increase in response to stimulation by aldehydic odorants over a large concentration range (final con- centrations). Heptanal, j; octanal, • ;nonanal,m. First quartile, median (second quartile), and third quartile of all significant data are plotted. The symbols for the medians are enlarged and curves are drawn from these points for each odorant. Fig. 5. Dose-response of human OR17-40 receptor expressed in an ODORA-OR17-40 clone stimulated by helional over a large concen- tration range (final concentrations). All significant data points are plotted. First quartile, median (second quartile), and third quartile are shown, and the curve based on the median is traced. Ó FEBS 2003 Ligand-specific dose–response of olfactory receptors (Eur. J. Biochem. 270) 2909 Discussion Expression of olfactory receptors in surrogate cells is necessary to study molecular interaction with odorants and signalling pathways used for olfactory coding. A number of attempts have already been reported in hetero- logous systems, as well as in cells presenting neuronal phenotypes (primary neurons cultures or immortalized olfactory cell lines), to express olfactory receptors properly inserted into the plasma membrane [1,4,5,7,17,21]. How- ever, it is still a matter of debate whether nonengineered, native receptors, can indeed be functionally expressed. As we expect to use the designed system to study not only ligand/receptor interaction, but also the functional charac- terization and desensitization of stimulated signalling path- ways, we assumed that any modification of the expressed protein could interfere with its functional interactions. Therefore, unlike in previous studies, we expressed the olfactory receptor without any molecular manipulation of the coding sequence of the receptor, such as addition of an import sequence to enhance protein translocation to the membrane [2,6,22–24], or engineering chimeric constructs with only part of the coding sequences of olfactory receptors [3,24]. Only the c-myc tag was added at the 5¢-terminus of I7 sequence. Olfactory receptor specific and dose-dependent response The cells expressing recombinant I7 exclusively exhibit a response to odorants of the aldehyde family (namely heptanal, octanal and nonanal) consistent with the results of previous in vivo [8] or in vitro studies [3,9]. However, in those studies, octanal was reported as being the main ligand for rat I7 receptor. Further analysis shows that these results and ours are in fact complementary. Zhao and Araneda’s experiments on adenovirus-infected olfactory epithelium were conducted with varying carbon chain length aldehydes using a single odorant concentration of 10 )3 M . At this concentration, octanal was reported to show the largest response – with a response amplitude (electro-olfactogram) of 1.7 relative to the control, whereas aldehydes with shorter or longer chains exhibited lower responses (1.5 for nonanal, 1.45 for decanal, 1.35 for heptanal). These results compare to our own results at the highest odorant concentrations used (10 )10 or 10 )9 M ), which induced the largest calcium response with octanal, a less intense response with nonanal, and no response at all with heptanal. Nevertheless, shifting to lower and more physiological concentrations highlighted a different ranking of the odorants, heptanal singled out as the preferential odorant at a 10 )13 M concentration, more efficient than nonanal, and octanal no longer inducing any response at this concentration. These observations allow us to conclude that heptanal can in fact be defined as the preferential odorant ligand for rat I7, inducing a response at the lowest concentration. Moreover, the concentration range of odorants giving rise to signal detection is in the submicro- to subpico-molar concentration range that seems to be close to reported physiological detection limits for some odorants in humans (10 )7 to 10 )11 M [6,25]) or in dogs (10 )14 to 10 )17 M [25,26]). All previous studies have been performed using much higher odorant concentrations – thus, far above the physiological range – and a much narrower concentration range than in the present work: 1 l M to 100 m M range [8], 1–30 l M range [3], 640 l M [23]. We have also performed heterologous expression of I7 in a yeast system, where a specific dose-dependent response was obtained exclusively in response to heptanal stimulation, in the 10 )8 to 10 )5 M range [27], which corroborates the present results in terms of preferential ligand, even though the odorant concentration range needed for stimulating the receptor response in the yeast system is much higher than in COS cells. This modulation could arise from modifications in the lipidic environment differing among cellular types [28]. In the case of OR17-40 human olfactory receptor, only helional, among all other odorants tested, elicited a response from the cells expressing the receptor. This is true for stable ODORA cells, but also for transiently transfected ODORA cells or COS cells. This specificity had already been reported, but only for an odorant concentration of 50 l M [22], whereas in our experimental set-up, an optimum was obtained for 10 )11 M helional, a dose far below those usually tested in other systems. Bell-shaped dose-dependent response The bell-shaped odor dose–response curves obtained here for both human OR17-40 and rat I7 olfactory receptors expressed in various cell types clearly differs from ÔclassicalÕ pharmacological GPCR dose–response curves exhibiting a plateau at high ligand concentration. However, some previous studies already seemed to yield a similar dose– response curve, though shifted to higher concentrations: in Krautwurst’s study [3], involving expression in HEK293 cells of a chimeric receptor including the N-terminus of rhodopsin and full-length I7 sequence and G a15,16 ,octanal induced a response at 10 l M but also a smaller response at 1 l M and 30 l M . Other measurements reported in the literature fall short of answering the question of the shape of the dose–response curves [12] as the concentration ranges for the odorants that were explored lie within the submil- limolar to millimolar range, thus far from physiological concentrations and far from the concentrations used in this study. For the highest concentrations, experiments are prevented by the toxic effect of both the solvent and the odorant chemical itself, and by solubility problems. Kajiya et al. [29] also reported cAMP elevation and [Ca 2+ ] i increase when using recombinant olfactory receptors expressed in HEK293; dose–response curves seemed to downturn at the highest ligand concentration (1 m M for mOR-EG, 3 m M for mOR-EV). Similarly, increasing odorant concentrations elicit increasingly larger responses from isolated olfactory neurons [11,14], while even higher concentrations seem to yield relatively smaller responses [14]. Moreover, the results obtained by Ronnett et al.,on populations of primary olfactory neuronal cultures exposed to odorant stimulation, had previously exhibited concen- tration-related patterns in terms of intracellular cAMP levels and adenylate cyclase activity, where maximal stimu- lation occurred for intermediate concentrations, whereas adenylate cyclase activity declined for both low and high odorant concentrations [30]. In the present study, we followed the response of populations of cells expressing a single olfactory receptor, either I7 or OR17-40. The results 2910 G. Levasseur et al. (Eur. J. Biochem. 270) Ó FEBS 2003 obtained tend to support the interpretation that the bell- shaped dose–response curve indeed arises from an intrinsic response of the olfactory receptor itself. Desensitization mechanisms may be evoked to account for the shape of the dose–response curves Desensitization of the receptors depends on their phos- phorylation, as well as downstream mechanisms with contribution of GRKs and beta-arrestins [31], and on their internalization [32]. We infer that some inhibition of the receptor or saturation of its transduction pathway might occur at high concentration, as it had been evoked for isolated olfactory neurons [14]. In the present experimental set-up with no rinsing after odorant application, this could involve a blocked, ÔsaturatedÕ conformation of the receptor, with bound ligand but no activation of the transduction pathway. In other experimental set-ups with extensive washing following the stimulation, highly concentrated odorants may nonetheless elicit some response from the receptors. At the other end of the concentration range, the present experiments clearly established the threshold odor- ant concentration, above which the receptor is able to trigger a cellular response. Olfactory receptor discrimination ability The I7 olfactory receptor exhibits a differential dose- dependent response to odorants of a same chemical family. It was already known that an olfactory receptor could recognize a number of odorants, that a given odorant could be detected by a number of different receptors, and that different odorants could be recognized by different combi- nations of olfactory receptors (Ôcombinatorial receptor codingÕ [12,33]). This observation can also be related to the behavior of individual ORNs, which have a different reaction profile according to the odorant tested, and a different concentration threshold for each odorant [11,14]. Here again, as in the case of the shape of the dose–response curves, we demonstrate that a single receptor exhibits an elaborate discrimination ability, responding differentially to closely related odorants, with a different coding of the olfactory information in terms of odorant concentration. This may arise from a modulation of the odorant–receptor interactions depending on odorant chain length, within the putative ligand-binding site determined by transmembrane domains IV–VII, which will be further investigated by bio-informatic docking studies. Visual estimation of the olfactory receptors expression level Comparison between immunofluorescence results and spec- trofluorimetric calcium measurements performed in the various cell lines indicate that no direct correlation exists between immunodetection and functional response levels. Indeed, transiently transfected HEK cells yield no better intracellular calcium results than stable ODORA OR17-40 cells. Expression of olfactory receptors in only limited amounts could lead to adequate membrane trafficking and functional expression, whereas overexpression could be detrimental to functionality, leading to intracellular receptor aggregation or to unphysiological receptor coupling as already observed in the case of other GPCRs [34]. A few experiments performed on stable COS-I7 cells with heptanal 10–13 M using calcium imaging in B. Dufy’s laboratory in Bordeaux (CNRS UMR 5543, France) showed that less than 10% of the cells are in fact responsive to the stimulation by this odorant. Similar observations were made from other cell types (e.g. CHO) stably transfected with the same expression vector (results not shown). This confirms that the absence of immunodetection of the receptor at the cell surface is not synonymous to an absence of functional response. The observations also suggest that not all cells of a stable cell line under continuous Geneticin selection pressure may at a given time yield a functional response. Several phenomena could be respon- sible for this behavior: the physiological state of the cell may influence receptor expression efficiency, the adequacy of the effector pathway may limit the responsiveness, and consti- tutive activity of olfactory receptors could induce receptor internalization and recycling without odorant stimulation [35]. Thus, future experiments involving our nonengineered receptors could largely benefit from implementing calcium imaging experiments as an alternative technique to calcium spectrofluorimetry. Taken together, our results indicate that olfactory recep- tors themselves exhibit a complex pharmacology. Consid- ering the high number of olfactory receptor genes and the large spectrum of odorants detected by a given receptor, this adds another level of complexity to the olfactory receptor world. The combination of these properties could account for the exquisite adaptation of the olfactory perception to the amazing complexity of the odor space. Acknowledgements We are grateful to Miche ` le Lieberherr for supporting G. L. concerning the spectrofluorimetric intracellular calcium measurements, and for fruitful discussion. The authors wish to warmly thank Bernard Dufy, Pierre Vacher and Thomas Ducret (CNRS UMR 5543, Bordeaux 2 University) for their generous offer to perform calcium imaging experiments using their equipment, their skills, and their time. We acknowledge the generous gift of helional, lyral and lilial samples by Roche (Dubendorf, Switzerland) through the courtesy of Boris Schilling. This research was supported in part by Institut National de la Recherche Agronomique. G. 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J. Biochem. 270) 2909 Discussion Expression of olfactory receptors in surrogate. Ligand-specific dose–response of heterologously expressed olfactory receptors Gre ´ goire Levasseur 1 , Marie-Annick