Defining a role for Ggamma13 in olfaction and biochemical studies of olfactory receptor activation and AC3 modulation

This thesis focuses on initial signaling events in the cilia of olfactory sensory neurons, examining mechanisms of ligand activation to the termination of the cellular response. The goal was to gain insight into the structure-function relationships of various components involved in olfactory signal transduction, particularly olfactory receptors, G, proteins, and adenylyl cyclase type III (AC3).; A role for the G, protein subunit, G_γ13 in olfactory signaling was defined. G_γ13 mRNA distribution is restricted to olfactory neurons, and the G_γ13 protein is markedly expressed in olfactory cilia. The association of G_γ13 with G_β1 , a protein also highly enriched in olfactory cilia, suggests G _γ13 mediates mammalian olfactory signaling in vivo.; An assay was developed to test whether olfactory receptors are phosphorylated as a means of terminating olfactory signaling and to biochemically screen olfactory receptors for functional activity. In a whole-cell phosphorylation assay, chimeric and full-length olfactory receptors did not show ligand-dependent phosphorylation, suggesting that receptor phosphorylation may not be utilized for odorant desensitization.; Odorant-evoked adenylyl cyclase activity was examined in olfactory cilia of a genetically-altered mouse expressing the I7 olfactory receptor in all olfactory sensory neurons. The goal was to quantify the binding affinity of I7 for a known ligand, heptanal. No significant heptanal-stimulated activation was seen, indicating the amount of I7 protein made in each cell is significantly lower than in a cell in which the I7 receptor is endogenously produced.; Previous reports that RGS2 associates with AC3 to inhibit olfactory signaling led to the investigation of the effect of RGS2 on cyclase activity, with the goal of further examining RGS2-AC3 physical interaction. In a cyclase assay, RGS2 caused slight increases in forskolin-stimulated activity levels in cells transfected with AC3. RGS2 had no effect in cells co-transfected with AC3 and a mutant G_αs which constitutively activates adenylyl cyclase. These data suggest that no RGS2-AC3 interaction takes place.