HORMONE-REGULATED GTP-BINDING PROTEINS

一类鸟嘌呤核苷酸的相互作用机制 结合蛋白（G蛋白）作为中间体， 细胞表面的激素受体及其细胞内效应物 系统将进行调查。 至少有四个成员 大多数具有多种亚型，用于协调 至少六种不同的细胞内调节活性， 途径。 这些蛋白质介导重要的激素或类激素 对所有哺乳动物细胞都有影响。 他们都有一个类似的 亚基组成并共享一个共同亚基。 本申请 建议研究这种共同的亚基如何可逆地 与蛋白质的其他亚基结合，并提供了一种机制， 它们相互影响，从而协调 细胞内调节途径。 这有五个组成部分 项目 （具体目标1）研究完整的 来自培养物中生长的S49小鼠淋巴瘤细胞的膜。 这些研究 将表明亚基解离的行为中的作用， 未用去污剂从膜上溶解的蛋白质 并随后从其他蛋白质中纯化出来， 它们的功能。 （具体目的2）G亚基解离的研究 从牛脑或人红细胞或类似物中纯化的蛋白质 重组蛋白，在洗涤剂溶液中;这将决定什么 调节不同蛋白质之间的相互作用， 亚单位 （具体目标3）研究 这些纯化的G蛋白的可逆亚基相互作用， 洗涤剂溶液，通过检查GTP结合动力学测定 水解得到纯化蛋白。 （具体目标4） 这些可逆的亚基相互作用是如何影响 在S49细胞的完整膜中， cAMP-腺苷酸环化酶系统。 （具体目标5） 蛋白质上的亚基结合位点，主要由 分离化学修饰的蛋白质片段， 阻断亚基相互作用。 这些研究 将提供重要 这些蛋白质的结构信息以及它们的亚基 互动.

The mechanisms for the interactions of a family of guanine nucleotide binding proteins (G proteins) which act as intermediaries between hormone receptors on the cell surface and their intracellular effector systems will be investigated. There are at least four distinct members of this family, most with multiple isoforms, that serve to coordinate the activities of at least six different intracellular regulatory pathways. These proteins mediate important hormone or hormone-like effects in essentially all mammalian cells. They all have a similar subunit composition and share one common subunit. This application proposes to investigate how this common subunit reversibly associates with the other subunits of the proteins and provides a mechanism by which they affect each others function, thereby coordinating intracellular regulatory pathways. There are five components of this project. (Specific Aim 1) The study of subunit interactions in intact membranes from s49 mouse lymphoma cells grown in culture. These studies will indicate the role of subunit dissociation in the behavior of proteins which have not been solubilized from membranes with detergents and subsequently purified away from other proteins which may modulate their function. (Specific Aim 2) The study of subunit dissociation of G proteins purified from bovine brain or human erythrocytes, or analogous recombinant proteins, in detergent solution; which will determine what regulates the interactions of the different proteins with their shared subunit. (Specific Aim 3) The study of the functional consequences of these reversible subunit interactions of purified G proteins in detergent solution, determined by examining the kinetics of GTP binding and hydrolysis to the purified proteins. (Specific Aim 4) The study of how these reversible subunit interactions affect a specific intracellular regulatory system in intact membranes from s49 cells, the CAMP-adenylyl cyclase system. (Specific Aim 5) The identification of the subunit binding sites on the proteins, determined primarily by isolating chemically-modified fragments of the proteins responsible for blocking subunit interactions. These studies will provide important structural information about these proteins and how their subunits interact.