The applicant argues that transmembrane signaling by hormones and neurotransmitters requires a cell surface receptor, a G protein, and an effector enzyme. Additional proteins are needed to modulate signaling. For example, protein kinases and arrestin contribute to desensitization of receptors. Much less is known about proteins that promote desensitization of the G proteins. The applicant's proposal seeks to test the hypothesis that G proteins undergo desensitization. Specifically, he will examine how two proteins, phosducin and Sst2, alter G protein function. These experiments will be carried out in the yeast strain S. cerevisiae because yeast express receptors and G proteins remarkably similar to those found in humans and a yeast homologue of phosducin has been recently identified in the applicant's laboratory. In addition, yeast is particularly well suited for in vivo genetic analysis. Preliminary experiments reveal that phosducin and Sst2 bind to G protein in vitro and are required for desensitization in vivo. To achieve his goals, the applicant proposes four Specific Aims.
Aim 1 will examine how phosducin affects G protein function. The hypothesis is that phosducin blocks G protein subunit association and further activation by the receptor in yeast. The functional consequences of phosducin binding to G proteins will be evaluated by measuring its effects on pheromone sensitivity in vivo (transcriptional activation, growth arrest) and G protein activity in vitro (subunit association, GTP hydrolysis) Aim 2 will examine how phosducin is regulated. The hypothesis is that phosphorylated phosducin does not interact with the G protein in vivo. To test this hypothesis, the phosphorylation site(s) will be mapped, and mutants that prevent modification will be evaluated for changes in pheromone signaling in vivo and G protein activation in vitro.
Aim 3 will examine how Sst2 affects G protein function. The hypothesis is that Sst2 promotes desensitization by stimulating G protein GTPase activity. To test this hypothesis, purified recombinant proteins will be used to measure G protein activity in the presence and absence of Sst2.
Aim 4 will examine how Sst2 is regulated. Sst2 undergoes a pheromone-stimulated change in electrophoretic mobility, reflecting some post-translational modification. To determine the nature and consequence of this change, the sites of modification will be mapped, and mutants that block modification will be tested for changes in pheromone signaling in vivo and G protein activity in vitro.
