GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

胃泌素的促酸作用依赖于其生物学特性。 通过翻译后处理激活和绑定 胃壁细胞表面活性多肽对其受体的作用。的目标是 这项建议侧重于阐明这两种物质的生化基础。 荷尔蒙-靶细胞交界处的关键步骤 互动。在以前的研究中，我们已经探索了生物合成 Gastin等人提出的翻译后加工模型 前身。胃泌素的生物激活需要形成一种 甘氨酸延伸孕激素的羧基末端酰胺部分 加工中间体。来探索这方面的结构要求 和我们建议表达的其他胃泌素处理反应，在 各种内分泌来源的细胞系，胃泌素cDNA克隆 其他人之前也曾孤立过。由于它们合成了其他 肽激素，这些细胞系已知能够传导 有些处理反应，但可能不会有其他反应。我们将研究 具有位点特异性突变的胃泌素基因克隆的表达 三个加工位点，Arg57-Arg58残基被切割形成 胃泌素-34，被裂解形成胃泌素17的Lys74-Lys75残基， 和羧基末端的Gly93-Arg94-Arg95复合体，它发出信号 酰胺化过程。因为几乎所有的多肽都是通过 二碱裂解反应，一半以上发生羧基末端 这些研究的结果可能具有广泛的影响。 与多种激素调节的胃肠道有关 功能。在其他研究中，我们探索了 壁细胞胃泌素受体的研究 对酰胺化胃泌素的需求，b)其结构为单亚基 74kD蛋白，以及c)其与细胞肌醇磷脂的连接 周转和蛋白激酶C易位。我们建议将这些措施延长 纯化胃泌素受体，测定其部分受体的研究 氨基酸序列，并使用构建在 在此序列的基础上分离编码该受体的cDNA克隆 构建了一个壁细胞cDNA文库。图书馆将进行筛选，此外， 产生一种针对纯化的胃泌素受体的抗体。 我们将采用的另一种方法是胃泌素的表达克隆 非洲爪哇卵母细胞中的受体。分离胃泌素后 受体cdna，我们将在非内分泌和内分泌中表达它。 细胞系。将应用定点突变技术 选择性地结合受体的跨膜和胞浆内结构域 分别探讨配体结合的结构要求 以及与信号转导机制的联系。通过这些研究，我们 希望深入了解多肽激素受体的结构，并 它们与健康和疾病中的生理功能有关。

The acid stimulatory action of gastrin is dependent on its biological activation via post-translational processing and the binding of the active peptide to its receptor on gastric parietal cells. The aims of this proposal focus on elucidation of the biochemical basis for these two crucially important steps at the juncture of hormone-target cell interaction. In previous studies we have explored the biosynthesis of gastrin and developed a model for post-translational processing its precursor. Biological activation of gastrin requires the formation of a carboxyl-terminal amide moiety from a glycine-extended progastrin processing intermediate. To explore the structural requirements for this and other gastrin processing reactions we propose to express, in a variety of endocrine-derived cell lines, gastrin cDNA clones that we and others have previously isolated. By virtue of their synthesis of other peptide hormones, these cell lines are known to be capable of conducting some processing reactions but possible not others. We will examine the expression of gastrin cDNA clones that have site-specific mutations at three processing sites, the Arg57-Arg58 residues that are cleaved to form gastrin-34, the Lys74-Lys75 residues that are cleaved to form gastrin 17, and the carboxyl-terminal Gly93-Arg94-Arg95 complex that signals the amidation process. Because virtually all peptides undergo processing via dibasic cleavage reactions and more than half undergo carboxyl-terminal amidation, the results of these studies may have broad implications relevant to a large variety of hormonally regulated gastrointestinal functions. In other studies we have explored the biochemistry of the parietal cell gastrin receptor by demonstrating a) its selective requirement for amidated gastrins, b) its structure as a single subunit 74kD protein, and c) its linkage to cellular inositol phospholipid turnover and protein kinase C translocation. We propose to extend these studies by purifying the gastrin receptor, determining a portion of its amino acid sequence, and using an oligonucleotide constructed on the basis of this sequence to isolate a cDNA clone encoding the receptor from a parietal cell cDNA library. The library will be screened, in addition, with an antibody generated against the purified gastrin receptor. Another approach that we will pursue is expression cloning of the gastrin receptor in oocytes of Xenopus laevis. After isolating the gastrin receptor cDNA, we will express it in both non-endocrine and endocrine cell lines. The techniques of site-directed mutagenesis will be applied selectively to the receptor's transmembrane and intracytoplasmic domains to explore the structural requirements for, respectively, ligand binding and linkage to signal transduction mechanisms. Through these studies we hope to gain insight into the structure of peptide hormone receptors and their linkage to physiological functions in health and disease.