NOVEL N-PROTEIN COUPLES TRH RECEPTOR TO PHOSPHOLIPASE C

该研究项目的长期目标是提供 实验证据表明存在一种新的GTP结合 一种蛋白质，似乎作为一个膜传感器耦合 磷脂酶C的某些细胞表面受体。 磷脂酶C的激活导致磷脂酶C的形成。 第二信使三磷酸肌醇（IP 3），反过来， 刺激细胞内钙释放。 一个 细胞内钙离子的升高， 细胞反应，如神经递质或激素释放， 平滑肌收缩取决于细胞类型。 是 提出了假定的连接细胞的GTP结合蛋白- 磷脂酶C的表面受体在 细胞对激动剂的反应 具体目标是 建议确定以下内容：（1）要求 用于IP 3的TRH受体刺激的鸟嘌呤核苷酸 将建立7315 c细胞裂解物中的形成; GTP、GTP γ S、GDP和GDP β S的结合，以增强或抑制 研究TRH对IP 3的刺激作用;（2）TRH对IP 3的刺激作用 将测试刺激GT3的能力;（3）鸟嘌呤的能力 将测试影响激动剂结合的核苷酸;（4）作为第一个 分离TRH受体及其GTP结合的步骤 蛋白质，溶解活性物质和GTP的最佳条件， 将确定敏感的TRH受体;（5）将尝试 分离TRH受体（与其 GTP结合蛋白），然后在小麦胚芽凝集素柱上， 在TRH-激动剂亲和柱上进行层析，（6）如果TRH 成功地分离了受体及其GTP结合蛋白， 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 蛋白质将通过TRH刺激 该制剂中的GT3活性，和（7）许多细菌 毒素将被测试其催化ADP的能力， TRH相关GTP结合蛋白的核糖基化 受体的

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.