NOVEL N-PROTEIN COUPLES TRH RECEPTOR TO PHOSPHOLIPASE C

这项研究项目的长期目标是提供 存在一种新的GTP结合的实验证据 一种看起来像是膜转导偶联的蛋白质 磷脂酶C的某些细胞表面受体。 磷脂酶C的激活导致 第二信使三磷酸肌醇(IP3)，进而， 刺激细胞内储存的钙的释放。一个 细胞内钙的升高然后触发适当的 细胞反应，如神经递质或激素释放或 根据细胞类型的不同，平滑肌收缩。它是 推测连接细胞的GTP结合蛋白- 磷脂酶C的表面受体在 细胞对激动剂的反应。这样做的具体目的是 提案要确定以下几点：(1)要求 鸟嘌呤核苷酸对TRH受体刺激IP3的作用 将建立7315c细胞裂解产物的形成； 对GTP、GTP伽马S、国内生产总值和GDPbetaS的增强或抑制 将研究TRH对IP3的刺激；(2)TRH对IP3的刺激能力 刺激GTP酶的能力将被检测；(3)鸟嘌呤的能力 将测试影响激动剂结合的核苷酸；(4)作为第一次 分离TRH受体及其GTP结合的研究进展 蛋白质，溶解活性和GTP的最佳条件- 将确定敏感的TRH受体；(5)将尝试 用来分离TRH受体(与其密切相关 GTP结合蛋白)在小麦胚凝集素柱上 TRH-激动剂亲和层析柱，(6)如果TRH 成功地分离了受体及其GTP结合蛋白，并 将尝试在磷脂中重建它们 囊泡；受体与其GTP结合的相互作用 蛋白质将被TRH刺激的能力所证明 此制剂中的GTP酶活性，以及(7)一些细菌 毒素将被测试它们催化ADP的能力 与TRH相关的GTP结合蛋白的核糖化 受体。

The long-term objective of this research project is to provide experimental evidence for the existence of a novel GTP-binding protein that appears to act as a membrane transducer coupling certain cell-surface receptors to the enzyme phospholipase C. Activation of phospholipase C results in the formation of the second messenger inositol trisphosphate (IP3) which, in turn, stimulates the release of calcium from intracellular store. An elevation of intracellular calcium then triggers the appropriate cellular response such as neurotransmitter or hormone release or smooth muscle contraction depending on the cell type. It is proposed that the putative GTP binding protein linking the cell- surface receptor to phospholipase C plays a crucial initial step in the response of a cell to an agonist. The specific aims of this proposal are to determine the following: (1) the requirement of guanine nucleotides for TRH receptor stimulation of IP3 formation in lysates of 7315c cells will be established; the ability of GTP, GTP gamma S, GDP and GDPbetaS to enhance or inhibit TRH stimulation of IP3 will be investigated; (2) the ability of TRH to stimulate GTPase will be tested; (3) the ability of guanine nucleotides to affect agonist binding will be tested; (4) as a first step toward isolating the TRH receptor and its GTP binding protein, optimal conditions for solubilizing an active and GTP- sensitive TRH receptor will be determined; (5) an attempt will be made to isolate the TRH receptor (in close association with its GTP binding protein) on a wheat germ lectin column followed by chromatography on a TRH-agonist affinity column, (6) if the TRH receptor and its GTP binding protein are successfully isolated, an attempt will be made to reconstitute them in phospholipid vesicles; the interaction of the receptor with its GTP binding protein will be evidenced by the ability of TRH to stimulate GTPase activity in this preparation, and (7) a number of bacterial toxins will be tested for their ability to catalyze the ADP ribosylation of the GTP-binding protein associated with the TRH receptor.