1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

337 Poster Chemosensory Molecular Genetics andVNO/PheromoneFUNCTIONAL ANALYSIS OF THE MOUSE ODORANTRECEPTOR MOR42 SUBFAMILYAbaffy T. 1 , Matsunami H. 2 , Luetje C. 1 1 Pharmacology, University ofMiami, Miami, FL; 2 MGM, Duke University, Durham, NCPhylogenetic analysis groups mammalian odorant receptors (ORs)into two broad classes and numerous subfamilies, which may reflectfunctional organization. To investigate the receptive ranges of membersof OR subfamilies, we are using Xenopus oocytes as a heterologousexpression system. A variety of Class I and Class II mouse ORs(MORs) can be expressed in Xenopus oocytes and co-expression withGαolf and the cystic fibrosis transmembrane regulator allowsmeasurement of odorant responses using electrophysiological methods.All receptor constructs included the N-terminal 20 amino acid residuesof human rhodopsin and for 7 out of the 9 MORs tested, this wassufficient for functional expression. Co-expression of accessory proteins(RTP1, RTP2 and REEP1) allowed functional expression of theremaining 2 MORs. We screened the members of the MOR42subfamily against a panel of 31 aliphatic odorants varying in chemicalgroup and carbon chain length. MOR42-1 responded to dicarboxylicacids (C9-C12). MOR42-2 responded to monocarboxylic acids (C7-C10). MOR42-3 responded to dicarboxylic acids (C8-C10) andmonocarboxylic acids (C10-C12). Thus, the receptive range of eachreceptor was unique. However, overlap between the individualreceptive ranges shows that the members of this subfamily arecontributing to one contiguous subfamily receptive range, supportingthe idea that OR subfamilies constitute functional units. Support:MH66038 and DA08102 (CWL), DC05782 (HM)338 Poster Chemosensory Molecular Genetics andVNO/PheromoneEXPLORING THE MOLECULAR RECEPTIVE RANGES OFMAMMALIAN ODORANT RECEPTORSRepicky S.E. 1 , Matsunami H. 2 , Luetje C.W. 1 1 Molecular and CellularPharmacology, University of Miami, Miami, FL; 2 MGM, DukeUniversity, Durham, NCEach odorant receptor (OR) is thought to recognize a particular arrayof odorant molecules, termed the molecular receptive range (MRR) ofthe receptor. Several mouse ORs (MORs) have been shown respond tolinear aliphatic compounds. MOR 23-1, 31-4, 32-11, 40-1 and 40-4each respond to octanoic and nonanoic acid. In addition to thesecommon ligands, additional compounds activate one or more of thesereceptors suggesting that each MRR is unique. However, the extent towhich the MRRs of these receptors differ is unclear. We have expressedeach OR in Xenopus oocytes, along with Gαolf and the cystic fibrosistransmembrane regulator (CFTR), allowing measurement of ORfunction using two-electrode voltage clamp. MOR23-1 required anaccessory protein (RTP1) for functional expression, while the otherreceptors did not. We screened each MOR against a panel of 39 odorantcompounds that varied in chemical group and carbon chain length. EachMOR displayed a distinct MRR. For example, MOR40-4 had a narrowMRR, responding well only to 9-, 10- and 11-carbon monocarboxylicacids and the 11-carbon aldehyde. In contrast, MOR23-1 had a broadMRR, responding well to alcohols, aldehydes, monocarboxylic acids,and bromocarboxylic acids with carbon lengths ranging from 6 to 9.Thus, while the MRRs for these receptors show some overlap, the MRRfor each receptor is unique. Support: MH66038 (CWL) DA08102(CWL) and DC05782 (HM).339 Poster Chemosensory Molecular Genetics andVNO/PheromoneDECIPHERING THE MOLECULAR BASIS OF HUMANOLFACTORY THRESHOLD VARIATIONSMenashe I. 1 , Hasin Y. 1 , Doron L. 1 1 Molecular Genetics, WeizmannInstitute of Science, Rehovot, IsraelHumans are highly variable in their olfactory thresholds. Specificanosmia/hyposmia and specific hyperosmia, the two extremes of thisphenotypic spectrum are documented for dozens of odorants andsuggested to have a genetic basis. Olfactory receptor (OR) segregatingpseudogenes, having both functional and non-functional forms in thehuman population, are excellent candidates to underlie these phenotypes(Menashe et al., Nat. Genet. 2003). To examine this hypothesis, weassessed the olfactory thresholds for 6 diverse odorants (androstenone,isoamyl acetate, isovaleric acid, l-carvone, pentadecalactone and cineol)in 377 individuals, and genotyped the segregating single nucleotidepolymorphism in 30 ORs in this cohort. A strong association was seenbetween three OR genes on chromosome 14 and sensitivity to isovalericacid, suggesting a locus related to specific anosmia. We also examinedthe average individual sensitivity across all odorants. These were foundto span a wide range of >4 orders of magnitude, with differentindividuals having disparate values of average threshold. Such result,which stems from a concordance between odorant thresholds in eachindividual, implies a “general olfactory factor” that governs olfactorythresholds, in parallel to the odorant-specific determinants. Thus, wehave demonstrated that the extensive threshold variation among humanstowards particular odorants is a complex trait, contributed to both byolfactory-receptor-specific variations as well as by potential interindividualdifferences in downstream components in the olfactorysignaling pathway.340 Poster Chemosensory Molecular Genetics andVNO/PheromoneIDENTIFICATION OF AMINO ACIDS INVOLVED IN GPROTEIN ACTIVATION BY A MOUSE EUGENOLRECEPTOR, MOR-EGKato A. 1 , Katada S. 1 , Touhara K. 1 1 Department of IntegratedBiosciences, The University of Tokyo, Chiba, JapanThousands of odorants are recognized and discriminated by olfactoryreceptors (ORs) that belong to seven-transmembrane (TM) G proteincoupledreceptors (GPCRs). Once activated, GPCRs undergoconformational changes that trigger an intracellular signal transductioncascade by activating heterotrimeric G proteins. The aim of this studyis to elucidate molecular mechanisms underlying G protein activationby an OR. We have previously shown that a eugenol receptor, mOR-EG, is functionally expressed in HEK293 cells such that odorantresponsiveness is determined by intracellular Ca 2+ increase via cotransfectedGα15 or by an increase in cAMP level via endogenous Gαs.We introduced site-directed mutations within highly-conserved residuesin the third intracellular loop and the C-terminal tail of mOR-EG. Eachmutant was characterized by assays of Ca 2+ and cAMP levels inHEK293 cells. Some mutations resulted in dramatic decreases incAMP production, whereas no effect was observed on Ca 2+ responses.The results suggest that these amino acid residues appear to be involvedin coupling to Gαs but not to Gα15. Next, we introduced mutationswithin the cytosolic half of TM6 to investigate a role of TM6 inreceptor dynamics upon odorant activation. One of mutations exhibiteda dramatic increase in cAMP responsiveness, suggesting that TM6 isinvolved in regulation of G protein-coupling efficiency. The presentstudy is the first to provide insight into the interaction of an OR with Gproteins at the molecular level. [supported by PROBRAIN, Japan]85