The objective of this research is to learn the precise molecular details of both the biosynthesis of peptide hormones and the mechanism of action of peptide hormones. Secreted bioactive peptides (peptide hormones, growth factors, neuroregulatory pep- tides) are indispensable agents of the intercellular interactions that control both embryonic development and the coordination and integration of life functions in the adult. Numerous human diseases are associated with the under- (or over-) production of regulatory peptides, or with the inability to respond properly to such signals. Even the unicellular eukaryotic microorganism Saccharomyces cerevisiae (baker's yeast) secretes and responds to peptide hormones (""""""""mating pheromones"""""""")- Because incisive genetic and biochemical studies can be performed in yeast cells, particularly those that utilize recombinant DNA, study of peptide hormone biogenesis and signal transduction in yeast may provide useful insights for understanding the molecular basis of diseases that affect the human neuroendocrine system. Four specific areas of investigation are proposed: (1) The molecular basis of cell type-specific gene expression will be studied by examining the transcriptional regulation of the MF a1 gene (which encodes a precursor, prepro-a-factor, of one of the mating pheromones)- Genetic and biochemical experiments are proposed for further defining both the cis-acting sites and the trans-acting factors responsible for the control of this gene, and for exploring the role of a particular """"""""zinc finger""""""""- containing transcription factor (STE5 gene product). (2) The enzymic basis of pre= cursor maturation at sites comprised of pairs of basic residues will be explored by determination of the three-dimensional structure of a yeast precursor cleaving enzyme (KEX2 gene product) with this specificity and by using probes based on this yeast gene to isolate its mammalian homolog. (3) How a plasma membrane receptor acts as a ligand-triggered molecular switch and how its function is regulated during desensitization will be elucidated using the cloned alpha-factor receptor gene (STE2), its coupled G a subunit (GPAl gene product), and a regulatory factor (SST2 gene product) of as yet unknown function. (4) Hormonal induction of gene transcription will be studied by genetic and biochemical experiments designed to identify and characterize the protein factors that interact with an upstream pheromone-response element (PRE).
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