title: Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants.
abstract: Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.
authors.affiliation: Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Shirokova
authors.forename: Kristin
authors.initials: K
authors.lastname: Schmiedeberg
authors.forename: Peter
authors.initials: P
authors.lastname: Bedner
authors.forename: Heiner
authors.initials: H
authors.lastname: Niessen
authors.forename: Klaus
authors.initials: K
authors.lastname: Willecke
authors.forename: Jan-Dirk
authors.initials: JD
authors.lastname: Raguse
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Meyerhof
authors.forename: Dietmar
authors.initials: D
authors.lastname: Krautwurst
mesh_terms: D000080462:Acyclic Monoterpenes; D000447:Aldehydes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D039821:Monoterpenes / Q000494:pharmacology; D018035:Receptors, Odorant / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000080462:Acyclic Monoterpenes; D000447:Aldehydes; D008024:Ligands; D039821:Monoterpenes; D018035:Receptors, Odorant; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D007545:Isoproterenol; C108217:citronellal; D002118:Calcium