THE MOLECULAR BIOLOGY OF THE MAMMALIAN GRP GENE

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The structure, function, and expression of mammalian prepro gastrin releasing peptide (GRP) genes was studied. Experiments were performed to a) characterize the structure and regulation of the rat prepro GRP gene, b) overexpress the human GRP prohormone protein to analyze post-translational processing and peptide hormone function, c) test the autocrine growth hypothesis by introducing a constitutively expressed GRP gene into cells which respond to exogeneous GRP hormone by increased cell growth. A. Rat prepro GRP gene. The structure and entire nucleotide sequence of the rat prepro GRP gene was determined, using genomic clones and cDNA clones isolated from a rat brain cDNA library. In contrast to the human gene, there is no alternative RNA processing observed in rat tissues expressing this gene. The coding regions for GRP and its associated peptide are evolutionarily conserved, consistent with a biologic function for both domains. The rat GRP gene is transcribed from a tissue specific promoter in the brain, creating an alternative route for regulating gene expression. B. The human prepro GRP gene was successfully overexpressed in a novel baculovirus protein expression system. Using this model system, sufficient protein can be produced to allow analysis of post-translational processing events needed to generate biologically active peptides, and purify those peptides to establish function. C. A constitutively expressed human GRP gene was introduced into mouse embryonic Swiss 3T3 fibroblasts using DNA transfection. These fibroblasts, which respond to added GRP by cell division, failed to fully process the GRP prohormone into biologically active peptides and consequently showed no growth changes. This experiment establishes that processing of GRP prohormone is restricted to specific cell types.