GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

胃泌素的促酸作用依赖于其生物学特性。 通过跨国后处理激活和绑定 胃壁细胞及其他细胞上其受体的活性多肽 胃粘膜中的细胞。这个项目的重点一直是 阐明这些至关重要的步骤的分子基础 激素与靶细胞相互作用的结合点。在之前的资助期间 期间，我们已经表征了参与其中的酶和底物 胃泌素的加工和定义甘氨酸延伸的关键作用 加工中间体(G-Gly)作为羧基末端的底物 酰胺化反应，使胃泌素具有促酸性分泌活性。 尽管G-Gly被认为在生物学上是不活跃的，但我们已经 发现G-Gly对AR4-2J细胞有较强的诱变作用 高密度脂蛋白诱导胃壁细胞H+，K+-ATPase基因表达 可与胃泌素受体区分的亲和受体。我们 建议通过分离编码该基因的cDNA克隆来扩展这些研究 G-Gly受体，并鉴定该受体的特性，包括 胃泌素与G-甘氨酸的差异结合及细胞诱导 增殖，诱导酪氨酸激酶活性，并诱导 H+，K+-ATPase基因表达。克隆工作将涉及三个 方法：同源筛选、COS细胞中G-Gly结合的筛选 表达系统，以及一种新的筛选诱导方法 H+，K+-ATPase转录。在进行的其他实验中， 在之前的资助阶段，我们克隆了人胃泌素/CCKb受体 (HG/CCKbR)基因，并确定它给两个不同的 受体作为选择性RNA剪接的结果。我们建议探索 两种HG/CCK受体的功能意义 在异源细胞系中表达它们并鉴定它们的特性 它们的胃泌素/CCK多肽的选择性，与G-蛋白的连接， GQ和GI，诱导H+，K+-ATPase基因表达的能力和亲和力 用于选择性拮抗剂PD134308。虽然胃泌素和胃泌素 卡巴胆碱诱导顶膜肌醇磷脂的周转 细胞，卡巴胆碱是一种更有效的氨基比林摄取刺激剂 而不是胃泌素。我们表征的HG/CCKBR的三个性质 前一个资金期的差异可能解释：它的快速 脱敏，它不能独立地激活钙离子通道 与细胞内钙离子库耗竭有关的因子及其 与GI的联系。我们将研究这些特性的分子基础。 插入了人M3 M受体和CCKA受体。功能界别 这些属性的重要性将通过检查影响来探索 嵌合受体对壁细胞活性的影响。通过 提出的研究，我们希望阐明分子基础上的 胃泌素的生理作用，并进一步提供对 胃泌素受体激动剂和拮抗剂作为 人类疾病治疗中的治疗剂。

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.