The olfactory system has the task of detecting, recognizing, and classifying the very large number of odorant chemicals that an animal encounters in its environment. Olfactory signal transduction in mammals is likely to proceed primarily via binding of odorants to a very large family of olfactory 7-helix receptors (encoded by - up to 1000 genes) whose activation facilitates GTP binding to Gomicronlfalpha a Gs-like Galpha protein expressed at high levels in olfactory neurons. GTP-Gomicronlfalpha in turn activates the olfactory adenylyl cyclase, producing an elevation of intracellular cAMP levels. cAMP opens the olfactory cyclic nucleotide-gated (cng) cation channel, depolarizing the neuron. Ca2+ also flows into the neuron through this channel, which is highly permeable to Ca 2+, The major gap in our understanding of olfactory signaling is that the odorant specificity of the olfactory receptors is unknown. In fact, there is as yet no conclusive evidence that any of these receptors recognize a specific odorant. Without understanding receptor specificity, one cannot determine the nature of the information flow during the first stages of olfactory processing. The primary goal of this project is to identify odorant ligands for mammalian olfactory receptors. To do this, we have developed a number of assay systems in heterologous cells, and have characterized receptor expression in mammalian cells and Xenopus oocytes. We will test mammalian receptors for function in several systems. First, we have developed a permanent human cell line expressing the olfactory cng channel and Gomicronlfalpha. This will be transfected with receptor plasmids and odorant responses assayed by Ca2+ imaging using fura-2. Secondly, we have obtained and characterized human olfactory neuroblastoma lines, one of which may be capable of responding to odorants. We will transfect these lines with receptors as well. Third, we will collaborate with D. Anderson's group at Caltech (project 8) to analyze receptor expression and odorant responses in neurons derived from immortalized olfactory neuronal precursors. Fourth, we will generate recombinant adenoviruses that can express olfactory receptors in primary neuronal cultures and in other cells that cannot be transfected efficiently. Finally, we will collaborate with Cori Bargmann's group at UCSF to develop functional assays for a 7-helix receptor from C. elegans. ODR-10, which mediates specific behavioral responses to the odorant diacetyl.
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