Peptide hormones are involved in numerous biological processes including growth, reproduction, basal metabolism, memory and pain perception. We propose to investigate the interaction of peptides with cells using the Saccharomyces cerevisiae alpha-factor, a tridecapeptide mating pheromone, as a prototypical peptide hormone. Studies will be focussed on the interaction of alpha-factor with its membrane bound receptor and on the structure of the alpha-factor receptor. Specifically, the topography of the receptor in the S. cerevisiae cell membrane, the binding domain of the alpha-factor within the receptor, and the biologically relevant conformation of the pheromone will all be examined. These investigations will require the synthesis of pheromone will all be examined. These investigations will require the synthesis of receptor peptides for production of antireceptor antibodies and the synthesis of alpha-factor analogs containing biotin, 125I, or a photoactivatable moiety for tagging the receptor binding domains. Functional regions in the receptor will be defined using molecular genetic procedures to generate mutations in the receptor that will related to a specific phenotype. A combination of random mutagenesis and site-specific mutagenesis will be used to ultimately determine the receptor residues involved in alpha-factor binding and signal transduction. 13C- and 1H-NMR will be used to discern the conformation of a biologically active cyclic analog of alpha-factor in solution and in the presence of phospholipid vesicles. Studies will be initiated to see if it is feasible to determine the conformation of alpha-factor bound to its receptor. If successful, these studies should help define the mechanism of action of alpha-factor and should provide insights into the molecular interactions between mammalian peptide hormones and their receptor.
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