GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

胃泌素的酸刺激作用取决于其生物学特性。 通过跨国后加工和结合的激活 活性肽与胃壁细胞以及其他细胞上的受体结合 胃粘膜中的细胞。 该项目的重点是 阐明这些至关重要的步骤的分子基础， 细胞与靶细胞相互作用的结合点。 在过去的融资中 期间，我们已经表征了参与的酶和底物， 胃泌素的加工和定义的关键作用甘氨酸延长 加工中间体（G-gly）作为羧基末端的底物 酰胺化，一种赋予胃泌素酸性促分泌活性的反应。 虽然G-gly被认为是生物学上不活跃的，但我们已经发现了它。 发现G-gly对AR 4 - 2 J细胞具有有效的诱变作用， 诱导胃壁细胞H ~+，K ~+-ATP酶基因表达 可与胃泌素受体区分开的亲和受体。 我们 我建议通过分离编码以下蛋白的cDNA克隆来扩展这些研究： G-gly受体及其特性，包括 胃泌素和G-gly的差异结合，细胞诱导 增殖、酪氨酸激酶活性的诱导和 H+，K+-ATP酶基因表达。 克隆工作将涉及三个 方法：同源性筛选，在COS细胞中筛选G-gly结合 表达系统，并通过筛选诱导的新方法， H+，K+-ATP酶转录。 在其他实验中， 在上一个资助期，我们克隆了人类胃泌素/CCK b受体 (hG/CCKbR）基因，并确定它给两个不同的风险， 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，首先 在异源细胞系中表达它们并表征它们， 它们的胃泌素/CCK肽选择性，与两种G蛋白的连接， Gq和Gi，诱导H+，K+-ATP酶基因表达的能力，和亲和力 选择性拮抗剂PD 134308。 虽然胃泌素和 卡巴胆碱诱导壁细胞膜肌醇磷脂转换 细胞，卡巴胆碱是一种更有效的刺激氨基比林吸收 而不是胃泌素。 我们表征的hG/CCKBR的三个性质 在上一个供资期间可能会造成差异：其快速 脱敏，它不能激活Ca++通道独立于 与细胞内Ca++池耗竭相关的那些，及其 与Gi的联系我们将研究这些性质的分子基础 插入的人M3毒蕈碱和CCKA受体。 功能 这些属性的重要性将探讨通过检查的影响， 嵌合受体对壁细胞活性的影响 通过 提出的研究，我们希望阐明的分子基础上， 胃泌素的生理作用，并进一步提供洞察 胃泌素受体激动剂和拮抗剂作为 用于治疗人类疾病的治疗剂。

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.