Pheromone response in the yeast Saccharomyces cerevisiae provides a microbial model for studying general features of hormone action and cell division control. When alpha-factor pheromone binds to specific receptors on the surface of yeast a cells, it causes the cells to arrest division in the G1 phase of the cycle. The occupied receptors are continually internalized and resynthesized. After prolonged exposure to alpha factor, the cells """"""""adapt"""""""" to the pheromone and reenter the mitotic cycle. Genetic studies suggest that the receptor belongs to the same structural class as rhodopsin and the adrenergic receptor and that signal transduction is mediated by a heterotrimeric GTP-binding regulatory protein (G protein). Detailed understanding of this very basic process in yeast should provide information for a more general understanding of receptor action and cell division control. A combined genetic and biochemical approach will be applied to this problem. The PI will define ligand/receptor contacts by seeking receptor mutants that block alpha-factor binding, by seeking alpha-factor mutants that compensate for these binding site defects and by examining the ability of the mutant receptors to distinguish specific ligand variants. There results will be useful for defining spatial relationships at the binding site and for defining the packing arrangement of the seven transmembrane helices. He will use in vitro assays to monitor the structure of their mutant receptors that affect the allosteric transition. He will provide a genetic dissection of the early alpha-factor-mediated endocythic events that occur at the plasm membrane. To this end, he will develop assays that are sensitive to these early events and isolate receptor mutants with specific defects. He will identify the residues in the G proteins subunits that contact the receptor by isolating receptor mutants that are defective for these contacts. He will determine the composition of the protein aggregated that contain the complex, and will determine the effect of alpha-factor exposure on these aggregates. He will evaluate the physiological significance of a """"""""hyper-constrained"""""""" form of the receptor by testing the ability of mutants to block the acquisition of this conformational state.
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