REGULATORY DOMAINS OF G-PROTEIN-COUPLED RECEPTORS

G蛋白是异源三聚体鸟嘌呤核苷酸结合蛋白， 调节多种细胞通路对激素的反应。 这些 蛋白质形成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。 激素受体 活化的G蛋白在结构上也是同源的， G蛋白的活性通过类似的机制。 的发现 癌基因如ras，也是一种GTP结合蛋白， 正常的G蛋白和mas，实际上是血管紧张素受体， G蛋白和它们的受体密切参与了细胞内 生长控制以及调节cAMP水平、离子通道和 PI周转，本身是多种细胞途径的调节剂。 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，视杆细胞G蛋白。 这道光- 活化过程直接类似于酶介导的活化， 其他G蛋白偶联受体 该提案描述了 通过定点突变修饰牛视蛋白cDNA， 来确定G蛋白偶联受体的结构域， 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相互作用的位点 激酶，一种使光解视紫红质磷酸化的视杆细胞蛋白 抑制蛋白在促进Gt表达中的作用 光解的、磷酸化的视紫红质的脱敏作用也将被 考察 视紫红质光转导系统作为一个优秀的 研究G蛋白偶联酶活性调节的模型系统 受体，因为容易纯化大量的所有 参与这个过程的蛋白质。 此外，GT表现为 外源性膜蛋白 因此，动力学研究很容易 测量这种蛋白质与视杆细胞的结合 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.