1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

9 Symposium Chemosensory Receptors Satellite“DEORPHANIZING” MAMMALIAN ODORANT RECEPTORSMatsunami H. 1 1 MGM, Duke University Medical Center, Durham, NCHow does mammalian olfactory system use hundreds or moreodorant receptors (ORs) to detect and discriminate a vast number ofvolatile odorants? To tackle this question, it is essential to understandhow structurally diverse chemicals activate different ORs. However, ithas been difficult to express mammalian ORs on the cell surface ofheterologous cells and assay their ligand-binding specificities, becauseOR proteins are typically retained in the endoplasmic reticulum and nottransported to the cell surface. We have identified RTP1 and RTP2 thatpromote functional cell-surface expression of ORs in heterologous cells.Structure-function analysis of RTP1 revealed important domainsfunctioning in trafficking of ORs. We have constructed a heterologousexpression system to identify new ORs that respond to various odorants.We have tested ~300 human and ~200 mouse ORs against a panel of~100 diverse odorant chemicals to determine the odorant-ORinteractions. This screening have resulted in identification of ~100human and mouse ORs that respond to a wide variety of odorantchemicals. Some ORs seem to respond small number of structurallysimilar chemicals while others seem to respond to many chemicals,suggesting variable tuning specificities of ORs. Supported by an NIHgrant DC0578211 Symposium Chemosensory Receptors SatelliteOLFACTORY DEFICITS IN MICE DEFICIENT FOR THETRANSIENT RECEPTOR POTENTIAL CHANNEL M5Restrepo D. 1 , Margolskee R.F. 2 , Lin W. 1 1 Cell and DevelopmentalBiology, University of Colorado Health Sciences Center, Aurora, CO;2 Neuroscience, Mount Sinai School of Medicine, New York, NYMice defective for the cyclic nucleotide-gated channel (CNGA2) have asevere olfactory deficit, but respond to putative pheromones implyingthe presence of other transduction pathways in addition to the canonicalcAMP pathway (Lin et al., 24:3703, 2004). We studied theresponsiveness of individual glomeruli in CNGA2 knockout mice bydetecting odor-induced Fos expression in periglomerular cells. While asubset of the glomeruli activated by putative pheromones were necklaceglomeruli where the second messenger cGMP is thought to mediatetransduction, the majority of active glomeruli in CNGA2 knockout micewere regular glomeruli targeted by olfactory sensory neurons (OSNs)that would normally have expressed CNGA2, and do not expresselements of the cGMP pathway. Interestingly, electroolfactogram(EOG) responses elicited by putative pheromones in CNGA2 knockoutmice are inhibited by the phospholipase C (PLC) inhibitor U73122implying an involvement of this pathway in olfactory transduction.Further, we find that the transient receptor potential channel M5, aneffector participating in the PLC pathway in taste cells is co-expressedwith CNGA2 in a subset of OSNs projecting to glomeruli that respondto putative pheromones and urine. While the olfactory deficits inTRPM5 knockout mice are relatively mild, we find that mice defectivefor both TRPM5 and CNGA2 have a dramatic phenotype includingseverely diminished size of the olfactory bulb and missing glomeruli indiscrete areas of the bulb. These data imply that, in a subset of OSNs,the PLC/TRPM5 and cAMP pathways are co-expressed and play a rolein olfactory transduction. Supported by NIH grants DC00566,DC04657, DC006070 (DR) and DC006828 (WL), DC03155 (RFM).10 Symposium Chemosensory Receptors SatelliteOLFACTION TARGETEDMombaerts P. 1 1 The Rockefeller University, New York, NYThe sense of smell is mediated by a repertoire of ~1000 odorantreceptor genes in mice. Each olfactory sensory neuron is thought toexpress just one of these genes. Its axon synapes with second-orderneurons within a glomerulus in the olfactory bulb. The axons of allneurons that express the same receptor gene converge to the sameglomeruli. The odorant receptor is a critical determinant of whichglomerulus is innervated. An olfactory sensory neuron thus faces twotasks during differentiation: to choose one odorant receptor gene forexpression, and to project its axon to a specific glomerulus.12 Symposium Chemosensory Receptors SatelliteMONITORING ODORANT DETECTION BY OLFACTORYRECEPTORS EXPRESSED IN YEAST AS A REPORTERSYSTEMMinic J. 1 , Grosclaude J. 2 , Persuy M. 1 , Aioun J. 1 , Connerton I. 3 , SalesseR. 1 , Pajot-Augy E. 1 1 Neurobiologie de l'Olfaction et de la PriseAlimentaire, Institut National de la Recherche Agronomique, Jouy-en-Josas Cedex, France; 2 Virologie et Immunologie Moléculaires, InstitutNational de la Recherche Agronomique, Jouy-en-Josas Cedex, France;3 Biosciences, University of Nottingham, Nottingham, United KingdomBreaking down the molecular mechanisms of odorant perception andcoding, and screening receptor-odorant couples primarily require thefunctional expression of olfactory receptors in a cellular system. Wehave developed techniques to optimize membrane expression ofolfactory receptors in engineered S. cerevisiae yeast. Receptorsfunctional activity is evaluated both in living cells, where receptorstimulation by its odorant ligand is monitored through thebioluminescence of a luciferase reporter, and in nanosomes membranefragments, where activation of the receptor upon odorant stimulationcan be assessed by monitoring surface plasmon resonance response. Wedemonstrate that olfactory receptors maintain their activity in membranefragments. A same bell-shaped concentration-dependence response isobtained, in terms of threshold concentration and optimal concentration,which gives evidence that this receptor functional response in the livingcell indeed arises from its own behavior upon odorant stimulation, withno artefactual contribution from the cellular transduction pathway.Olfactory receptors efficiently discriminate between odorant ligandsand unrelated odorants. This system can fruitfully serve to evaluate thecomparative coupling efficiency of olfactory receptors to various G alphaprotein subunits, without the interference of cellular contribution.Moreover, nanosomes can be used as sensing elements of bioelectronicsensors, at the basis of potentially powerful electronic noses with a newconcept of mimicking in vivo odorant specific detection anddiscrimination. This work was supported by the PICASSO program(HF2004-0055) funded by EGIDE and the SPOT-NOSED project (IST-38899) of the European Community.3