MOLECULAR MECHANISMS OF GI PEPTIDE HORMONE PROCESSING

胃肠道多肽激素经历广泛的翻译后 在它们达到生理活性形式之前进行修饰。它 对于阐明这些处理机制很重要，因为它们会导致 特定于组织的不同生物活性多肽的生成。为 胃泌素，这些修饰包括配对基础上的内蛋白分解 前激素转化酶的残基和甘氨酸延伸的转化 加工中间体(G-甘氨酸)至羧基末端酰胺化胃泌素 (G-NH/2)通过酰胺化酶的作用。在胃泌素的情况下， 我们有令人兴奋的新证据表明G-Gly不仅仅是一个过程 胃泌素生物合成的中间产物，但不同的最终产物 由不同于标准的受体介导的生物作用 酰胺化胃泌素/CCK/B受体。具体地说，G-Gly刺激胃部 酸分泌，与G-NH/2作为营养剂协同工作。 因此，我们第一次可以将翻译后处理 肠道功能的机制。鉴于它们对胃泌素的重要性 生物合成和胃肠生理学，我们将描述 细胞内胃泌素体外加工的分子机制 胃窦G细胞和胃肠G细胞的表达系统 肿瘤。为了阐明这些机制，我们的研究将 重点关注以下具体目标：1)定义分子 激素原内切酶的决定因素及其在体内的确认 共表达正义的前激素转换酶的底物特异性 以及编码带有野生型或突变胃泌素的酶的反义cDNA 内分泌细胞中的cDNA。2)鉴定前激素转换酶 负责胃窦G细胞孕激素的处理。3)定义 胃泌素酰胺化酶对不同物种的相对选择性 G-Gly的分子形式及其生理学意义 通过确定G-Gly的哪些分子形式与G-Gly结合的研究结果 刺激肠道组织生长的受体。4)由于 在肠道肿瘤中，肽激素的处理是完全未知的 合成孕激素我们将确定负责 结肠癌和其他细胞系中孕激素的加工。我们还将 介导胃泌素营养的G-NH/2和G-Gly受体的特征 对这些细胞的影响。由于这些加工反应是常见的 许多肽类激素前体，这些研究的结果将 也有助于我们理解调控的分子机制 激素原在其他组织中的加工。

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.