REGULATORY DOMAINS OF G-PROTEIN-COUPLED RECEPTORS

G蛋白是一种异三聚体鸟嘌呤核苷酸结合蛋白， 调节多条细胞通路以响应荷尔蒙。这些 蛋白质形成一个具有相似激活模式的多基因家族。 与它们的整体结构同源性有关。荷尔蒙受体 激活的G蛋白在结构上也是同源的，似乎可以调节 G蛋白的活性通过类似的机制发挥作用。发现了 癌基因，如ras，也是一种GTP结合蛋白，与 正常的G蛋白和MAS，实际上是血管紧张素受体，表明 G蛋白及其受体密切参与细胞 生长控制以及对cAMP水平、离子通道和 PI周转，本身就是多条细胞通路的调节器。视紫红质， 哺乳动物视杆细胞的光感受器调节 CGMP磷酸二酯酶通过GT，杆状细胞G蛋白。这盏灯- 激活过程直接类似于激素介导的激活 其他G蛋白偶联受体。本提案描述了 定点有序突变修饰牛视蛋白基因的研究 确定与G蛋白偶联受体相关的结构域 用于G蛋白的结合和激活。此外，突变 将进行分析以确定与视紫红质的相互作用部位 一种能磷酸化光解视紫红质的杆状细胞蛋白--激酶 降低了它激活GT的能力。芦荟素在促进剂中的作用 光解、磷酸化视紫红质的脱敏作用也将 检查过了。视紫红质光传导系统是一种极好的 研究G蛋白偶联活性调控的模型系统 受体因为易于大量纯化所有的 参与这一过程的蛋白质。此外，GT的行为类似于 外源性膜蛋白。因此动力学研究可以很容易地 用来测量这种蛋白质与杆状细胞的结合 光感受器及其随后的激活。

G-proteins are heterotrimeric guanine nucleotide binding proteins that regulate multiple cellular pathways in response to hormones. These proteins form a multigene family that possess similar modes of activation related to their overall structural homology. The hormone receptors that activate G-proteins are also structurally homologous and appear to regulate the activity of G-proteins by similar mechanisms. The discovery of oncogenes such as ras, also a GTP-binding protein with many similarities to normal G-proteins, and mas, actually the angiotensin receptor, suggests that G-proteins and their receptors are intimately involved in cellular growth control as well as in the regulation f cAMP levels, ion channels and PI turnover, itself a regulator of multiple cellular pathways. Rhodopsin, the photoreceptor of the mammalian rod cell regulates the activity of a cGMP phosphodiesterase via Gt, the rod cell G-protein. This light- activated process is directly analogous to hormone-mediated activation of other G-protein-coupled receptors. This proposal describes the modification of the bovine opsin cDNA by site-directed mutagenesis in order to define domains of the G-protein-coupled receptor that are responsible for the binding and activation of G-proteins. In addition mutational analysis will be performed to define sites of interaction with rhodopsin kinase, a rod cell protein that phosphorylates photolyzed rhodopsin reducing its ability to activate Gt. The role of arrestin in promoting desensitization of photolyzed, phosphorylated rhodopsin will also be examined. The rhodopsin phototransduction system serves as an excellent model system for studying the regulation of activity of G-protein-coupled receptors because of the ease of purification of large quantities of all of the proteins involved in this process. In addition, Gt behaves as an extrinsic membrane protein. Therefore kinetic studies can easily be performed to measure the binding of this protein to the rod cell photoreceptor and its subsequent activation.