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NOVEL N-PROTEIN COUPLES TRH RECEPTOR TO PHOSPHOLIPASE C

新型 N 蛋白将 TRH 受体与磷脂酶 C 偶联

基本信息

批准号：
3295304
负责人：
THOMAS E COTE
金额：
$ 7.93万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-01 至 1992-06-30
项目状态：
已结题

项目摘要

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.

该研究项目的长期目标是提供存在新型 GTP 结合的实验证据似乎充当膜传感器耦合的蛋白质某些细胞表面磷脂酶受体 C. 磷脂酶 C 的激活导致形成第二信使三磷酸肌醇 (IP3)，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，例如神经递质或激素释放或平滑肌收缩取决于细胞类型。这是提出假定的 GTP 结合蛋白连接细胞- 磷脂酶 C 的表面受体在细胞对激动剂的反应。本次活动的具体目标提案应确定以下内容： (1) 要求鸟嘌呤核苷酸对 TRH 受体刺激 IP3 将确定 7315c 细胞裂解物中的形成；能力 GTP、GTP gamma S、GDP 和 GDPbetaS 的增强或抑制将研究 TRH 对 IP3 的刺激； (2)TRH的能力将测试刺激 GTP 酶； (3)鸟嘌呤的能力将测试影响激动剂结合的核苷酸； (4) 作为第一朝着分离 TRH 受体及其 GTP 结合迈出了一步蛋白质，溶解活性和 GTP- 的最佳条件将确定敏感的TRH受体； (5) 尝试分离 TRH 受体（与其密切相关） GTP 结合蛋白）在小麦胚芽凝集素柱上，然后 TRH 激动剂亲和柱色谱法，(6) 如果 TRH 受体及其GTP结合蛋白被成功分离，将尝试用磷脂重构它们囊泡；受体与其 GTP 结合的相互作用 TRH 刺激的能力将证明蛋白质该制剂中的 GTP 酶活性，以及 (7) 一些细菌将测试毒素催化 ADP 的能力与 TRH 相关的 GTP 结合蛋白的核糖基化受体。