MOLECULAR MECHANISMS OF GI PEPTIDE HORMONE PROCESSING

胃肠肽激素加工的分子机制

• 批准号: 2518351
• 负责人: CHRIS John DICKINSON
• 金额: $ 18.09万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518351
• 关键词: antisense nucleic acid; cell transformation; enzyme substrate; gastrins; gastrointestinal epithelium; gastrointestinal hormones; glycine; neoplastic cell culture for noncancer research; neuropeptide receptor; peptide hormone; peptide hormone biosynthesis; proteolysis; radioimmunoassay; receptor binding; site directed mutagenesis; transfection; zymogens

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.

胃肠肽类激素经历了广泛的翻译后 在达到其生理活性形式之前进行修饰。 它 重要的是要阐明这些处理机制，因为它们导致 不同生物活性肽的组织特异性产生。 为 胃泌素，这些修饰包括在成对的碱性磷酸酶的内切蛋白水解， 通过激素原转化酶和甘氨酸延伸的 羧基末端酰胺化胃泌素的加工中间体（G-Gly） (G-NH/2）通过酰胺化酶的作用。 在胃泌素的情况下， 我们有令人振奋的新证据表明G-Gly不仅仅是一种加工过程， 它是胃泌素生物合成的中间产物， 由不同于标准的受体介导的生物学作用 酰胺化胃泌素/CCK/B受体。 具体来说，G-Gly刺激胃 酸分泌，并与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加工 肠道功能的机制。 鉴于它们对胃泌素的重要性 生物合成和胃肠道生理学，我们将表征 胃泌素在体外加工的分子机制，在细胞 表达系统，在分离的胃窦G细胞，和胃肠道 肿瘤的 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 激素原内蛋白水解的决定因素，并证实在体内 通过共表达正义的激素原转化酶的底物特异性 和编码具有野生型或突变的胃泌素的酶的反义cDNA 内分泌细胞中的cDNA。 2)识别激素原转化酶 负责胃窦G细胞中前胃泌素的加工。 3)定义 胃泌素酰胺化酶对不同底物的相对选择性 G-Gly的分子形式，并检查这些生理意义 通过确定G-Gly与G-Gly结合的分子形式， 受体，以刺激肠道组织的生长。 4)由于 肽激素加工在肠道肿瘤中是完全未知的， 合成前胃泌素，我们将确定负责 前胃泌素在结肠癌和其他细胞系中的加工。 我们还将 表征介导胃泌素营养的G-NH/2和G-Gly受体 这些细胞的影响。 由于这些处理反应是共同的， 许多肽激素前体，这些研究的结果将 也有助于我们理解调节 其他组织中的激素原加工。