THE MECHANISM OF ACTION OF THYROTROPIN-RELEASING HORMONE

促甲状腺激素释放激素的作用机制

• 批准号: 3483461
• 负责人: THOMAS F. J. MARTIN
• 金额: $ 19.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3483461
• 关键词: G protein; affinity chromatography; calcium flux; hormone receptor; hormone regulation /control mechanism; laboratory rabbit; laboratory rat; lipid metabolism; liposomes; membrane reconstitution /synthesis; phorbols; phosphatidylinositols; phospholipase C; phospholipids; protein kinase C; protein purification; receptor coupling; second messengers; thyrotropin releasing hormone; tissue /cell culture

Hormones that bind to cell surface receptors trigger a cascade of biochemical events which result in second messenger generation and cellular activation. The membrane components involved in the receptor-regulated adenylate cyclase system, receptors, G proteins and cyclase, have been purified and functionally reconstituted in vitro. However, the actions of over 25 hormones, transmitters and growth factors are known to involve a separate transduction mechanism of receptor-regulated phosphoinositide hydrolysis and the generation of lipid-derived second messengers. Membrane components involved in this transmembrane signalling system, G proteins and phospholipase C (PL C), remain to be identified and characterized. Thyrotropin-releasing hormone (TRH) is a well-studied representative of this class of Ca2+-mobilizing hormones. Our previous studies demonstrated the GTP-dependent stimulation of PL C activity by TRH in GH3 cell plasma membranes. TRH stimulation was shown to be mediated through a novel pertussis toxin-, cholera toxin-insensitive G protein (Gp). We have developed methods to solubilize and separate GH3 cell membrane Gp and PL C. In addition, we have reconstituted functional Gp-PL C coupling in artificial liposomes using the separated components. With these techniques, it is now possible to purify and characterize the components involved in the transduction system utilized by TRH. Our specific aims for this project period are: 1. To purify and characterize the Gp that mediates receptor activation of PL C; and 2. To purify and characterize the membrane PL C that is regulated by the receptor and Gp. In additional studies, the regulation of Gp and PL C by the TRH receptor and by protein kinase C will be studied utilizing reconstituted systems. Hence, the additional specific aim will be: 3. To reconstitute TRH receptor-Gp-PL C coupling in artificial liposomes and to elucidate the mechanism for reduced Gp-PL C coupling in phorbol ester-treated GH3 cells. The complete characterization of components involved in TRH-initiated signal transduction will significantly contribute to a broader understanding of the mechanism by which Ca2+-mobilizing hormones trigger cellular responses mediated through cell surface receptors.

与细胞表面受体结合的激素触发级联 生化事件，导致第二信使生成和细胞 激活。 受体调节的膜成分 腺苷酸环化酶系统，受体，G蛋白和环化酶已经是 在体外纯化并在功能上重构。 但是， 已知超过25种激素，发射器和生长因子涉及 受体调节的磷酸肌醇的单独转导机制 水解和脂质衍生的第二信使的产生。 膜 该跨膜信号系统，G蛋白和 磷脂酶C（PL C）仍有待鉴定和表征。 促甲状腺蛋白释放激素（TRH）是一个良好的代表 这类Ca2+摩托激素。 我们以前的研究表明 TRH在GH3细胞等离子体中依赖GTP的刺激PL C活性 膜。 TRH刺激被证明是通过小说介导的 百日咳毒素，霍乱不敏感的G蛋白（GP）。 我们有 开发了溶解和分离GH3细胞膜GP和PL C的方法。 此外，我们已经重组了功能GP-PL C耦合 人造脂质体使用分离的成分。 与这些 技术，现在可以净化和表征组件 参与TRH使用的转导系统。 我们的具体目标 这个项目期是： 1。纯化和表征介导受体激活的GP PL C;和 2。纯化和表征由膜PL C调节 受体和GP。 在其他研究中，TRH受体对GP和PL C的调节 并将使用重构系统研究蛋白激酶C。 因此，另一个具体目标是： 3。重建人造脂质体中的TRH受体-GP-PL C耦合 并阐明phorbol中GP-PL C耦合降低的机制 酯处理的GH3细胞。 组件的完整表征 参与TRH发射的信号转导将显着贡献 更广泛地了解Ca2+润滑的机制 激素触发通过细胞表面介导的细胞反应 受体。