The primary goal of this project is to identify and characterize olfactory receptors for odorants. We plan to do this by developing assays for odorant receptor function in Xenopus oocytes. A pool of clones encoding a recently identified olfactory-specific 7-helix receptor family will be constructed and expressed in oocytes. The response to certain odorants thought to act by increasing inositol trisphosphate levels can then be directly assayed by electrophysiological measurements of C1- current. Most odorants are thought to increase cAMP levels, however, and no sensitive physiological assay for cAMP elevation in oocytes is currently available. We are thus attempting to develop such assays. The first assay method involves the construction of a Galpha protein that will interact with receptors that are normally coupled to adenylyl cyclase, but will instead cause activation of phospholipase C (leading to C1- channel opening). If this can be done, it will be of interest in its own right for G protein biology. The second and third methods will use expression of cAMP-regulated channels in oocytes to generate a current response to cAMP elevation. After a suitable assay is developed, we will use it to attempt to identify clones encoding receptors interacting with particular odorants. We will then express these receptors in cell lines to examine the mechanisms involved in olfactory signal transduction. We will also make receptor- specific antibodies to study in vivo receptor expression.
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