Gastrointestinal peptide hormones undergo extensive post-translational modification before they achieve their physiologically active forms. It is important to elucidate these processing mechanisms since they result in the tissue-specific generation of distinct bioactive peptides. For gastrin, these modifications include endoproteolysis at paired basic residues by a prohormone convertase and conversion of a glycine-extended processing intermediate (G-Gly) to carboxyl-terminally amidated gastrin (G-NH/2) via the action of the amidating enzyme. In the case of gastrin, we have exciting new evidence that G-Gly is not merely a processing intermediate but a distinct end-product of gastrin biosynthesis with a biological actions mediated by a receptor distinct from the standard amidated gastrin/CCK/B receptors. Specifically, G-Gly stimulates gastric acid secretion and works cooperatively with G-NH/2 as a trophic agent. Thus, for the first time we can link post-translational processing mechanisms to gut function. Given their importance to gastrin biosynthesis and gastrointestinal physiology, we will characterize the molecular mechanisms of gastrin processing in vitro, in a cellular expression system, in isolated antral G-cells, and gastrointestinal tumors. Toward the goal of elucidating these mechanisms, our studies will focus on the following specific objectives: 1) Define the molecular determinants of prohormone endoproteolysis and confirm the in vivo substrate specificity of the prohormone convertases by coexpressing sense and antisense cDNAs encoding the enzymes with wild-type or mutated gastrin cDNAs in endocrine cells. 2) Identify the prohormone convertases responsible for the processing of progastrin in antral G-cells. 3) Define the relative selectivity of the gastrin amidating enzyme for the different molecular forms of G-Gly and examine the physiologic implications of these findings by determining which molecular forms of G-Gly bind to the G-Gly receptor to stimulate growth of gut tissues. 4) Since the mechanisms of peptide hormone processing are completely unknown in gut tumors that synthesize progastrin we will identify the enzymes responsible for the progastrin processing in colon cancer and other cell lines. We will also characterize the G-NH/2 and G-Gly receptors that mediate gastrin's trophic effects in these cells. Since these processing reactions are common to many peptide hormone precursors, the results of these investigations will also aid in our understanding of the molecular mechanisms that regulate prohormone processing in other tissues.
