Myr-Ric-8A enhances G(alpha15)-mediated Ca2+ response of vertebrate olfactory receptors.
The determination of ligand specificities of odorant receptors will contribute to the understanding of how odorants are discriminated by the olfactory system. To date, the ways in which some olfactory receptors (ORs) pair with their cognate ligands has been studied using a Ca(2+) imaging technique. This approach has been used to investigate orphan G protein-coupled receptors expressed in heterologous cells; however, most attempts to functionally express ORs on the cell surface of heterologous cells have failed. Recently, receptor-transporting protein 1 and Ric-8B have been identified as proteins involved in targeting receptors to the cell membrane and amplifying receptor signals, and thus, they are able to facilitate cellular responses via ORs in a heterologous cell system. Here, we describe a technique in which we employed a myristoylation sequence-conjugated mutant of Ric-8A (Myr-Ric-8A) as a signal amplifier and show Myr-Ric-8A greatly enhances G(alpha15)-mediated Ca(2+) responses of ORs in HEK293 cells. Coexpression of Myr-Ric-8A enabled us to deorphanize a mouse OR and to determine its molecular receptive range. Our results suggest that Myr-Ric-8A should be helpful in functional characterization of ORs in heterologous cells using Ca(2+) imaging.
Yoshikawa
Keiichi
K
Department of Integrated Biosciences, Rm201, The University of Tokyo, Kashiwa, Chiba, Japan.
Touhara
Kazushige
K
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000378:metabolism*; D018035:Receptors, Odorant / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D018035:Receptors, Odorant; C485884:Ric8a protein, mouse; C559967:Ric8b protein, mouse; D000242:Cyclic AMP; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium