GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

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The acid stimulatory action of gastrin is dependent on its biological activation via post-transnational processing and the binding of the active peptide to its receptor on gastric parietal cells as well as other cells in the gastric mucosa. The focus of this project has been t o elucidate the molecular basis for these crucially important steps at the juncture of hormone-target cell interaction. During the previous funding period, we have characterize the enzymes and substrates involved in gastrin processing and defined the critical role of glycine-extended processing intermediates (G-gly) as the substrates for carboxyl-terminal amidation, a reaction that confers acid secretagogue activity to gastrin. Although G-gly had been thought to be biologically inactive, we have discovered that G-gly has a potent mutagenic effect on AR4-2J cells and induces H+,K+-ATPase gene expression in gastric parietal cells via high affinity receptors that can be distinguished form gastrin receptors. We propose to extend these studies by isolating a cDNA clone encoding the G-gly receptor and characterizing this receptor's properties, including differential binding of gastrin and G-gly, induction of cell proliferation, induction of tyrosine kinase activity, and induction of H+,K+-ATPase gene expression. The cloning effort will involve three approaches: homology screening, screening for G-gly binding in a COS-cell expression system, and a novel approach by screening for induction of H+,K+-ATPase transcription. In other experiments conducted during the previous funding period, we cloned the human gastrin/CCKb receptor (hG/CCKbR) gene and determined that it gives risk to two different receptors as a result of alternative RNA splicing. We propose to explore the functional significance of the two hG/CCK receptors by first expressing them in heterologous cell lines and characterizing them as to their gastrin/CCK peptide selectivity, linkage to both the G-proteins, Gq and Gi, ability to induce H+,K+-ATPase gene expression, and affinity for the selective antagonist PD134308. Although both gastrin and carbachol induce turnover of membrane inositol phospholipids in parietal cells, carbachol is a far more potent stimulant of aminopyrine uptake than gastrin. Three properties of the hG/CCKBR that we characterized during previous funding period may account for the differences: its rapid desensitization, its inability to activate Ca++channels independent of those associated with depletion of intracellular Ca++pools, and its linkage to Gi. We will examine the molecular basis for these properties of the human M3 muscarinic and CCKA receptor inserted. The functional importance of these properties will be explored by examining the effect of the chimeric receptors on parietal cell activity. Through the proposed studies, we hope to shed light on the molecular basis for the physiological actions of gastrin and, further, to provide insight into the potential utility of gastrin receptor agonist and antagonists as therapeutic agents in treatment of human diseases.