The structure, function, and expression of mammalian prepro gastrin releasing peptide (GRP) gene 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) obtain cDNA clones for other bombesin family prohormone genes. 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. The brain specific promoter is used only in selected nuclei in the rat brain when analyzed at the cellular level by in situ hybridization. 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. We have designed and synthesized consensus oligonucleotide probes specific for the receptor-binding biologically active carboxyl heptapeptide of bombesin family peptides. This oligonucleotide probe is currently being used to obtain and characterize other bombesin family genes expressed in neural tissue.
