REGULATORY DOMAINS OF G-PROTEIN COUPLED RECEPTORS

G蛋白偶联受体是一个多基因家族的成员， 调节细胞对各种 细胞外信号包括激素、趋化剂和 感官刺激这些事件在控制生长方面很重要， 真核细胞的分化和代谢。G的激活 蛋白质通过这些受体导致产生多个第二 信使，刺激磷酸化级联反应和调节 离子通道的数量这些受体的缺陷，它们的G蛋白或 效应酶与良性和恶性肿瘤的控制有关， 恶性肿瘤形成以及许多代谢紊乱。为 例如，视紫红质中自然发生的点突变 几种形式的视网膜色素变性疾病， 哺乳动物视网膜的部分损坏导致失明。 视紫红质是哺乳动物视杆细胞的光感受器，已被用作 一个结构模型，用于研究分子之间的相互作用， G蛋白偶联受体及其G蛋白与 受体脱敏，这涉及失去敏感性的 受体与其原始刺激的反应。这项研究的主要焦点是 建议利用牛视紫红质作为研究的模型系统 G蛋白偶联受体，G 蛋白质和激酶和抑制蛋白参与受体 脱敏。为了实现这一目标， 将通过定点突变在牛视蛋白的cDNA中产生缺失， 预测参与的特定结构域的诱变 这些重要的信号转导过程。随后将 这些改变的蛋白质在HEK-293细胞中的瞬时表达， 产生大量的突变体视蛋白质， 用视杆细胞G蛋白Gt和视紫红质重建 激酶、蛋白激酶C和抑制蛋白。视紫红质中的结构域， 感光细胞与其G蛋白相互作用的关键 使用与GT结合的光依赖性GTP γ S来鉴定。 观察到的突变之间的相互作用的协同性 视紫红质及其G蛋白也将被检查。监管领域 视紫红质激酶和抑制蛋白的光依赖性结合所需 将通过磷酸化和结合测定来鉴定。 的 羧基末端的富含丝氨酸/苏氨酸的结构域将突变为 鉴定那些最有可能被磷酸化的位点， 视紫红质激酶和蛋白激酶C，以及那些最关键的 用于抑制蛋白的结合。更完整地理解 视紫红质内的重叠结构域控制 G蛋白的激活和脱敏过程将有助于 定义由G缺陷引起的疾病的潜在机制 蛋白偶联受体信号通路。

G protein-coupled receptors are members of a multigene family of cell surface receptors that regulate cellular responses to a variety of~extracellular signals including hormones, chemotactic agents and sensory stimuli. These events are important in the control of growth, differentiation and metabolism in eucaryotic cells. The activation of G proteins by these receptors results in the generation of multiple second messengers, stimulation of phosphorylation cascades and regulation of a number of ion channels. Defects in these receptors, their G proteins or effector enzymes have been implicated in the control of both benign and malignant tumor formation as well as a number of metabolic disorders. For example, naturally occurring point mutations in rhodopsin are responsible for several forms of the disease retinitis pigmentosa which results in a partial destruction of the mammalian retina causing blindness. Rhodopsin, the photoreceptor of the mammalian rod cell, has been used as a structural model for investigating the molecular interactions between G protein-coupled receptors and their G proteins and the process of receptor desensitization, which involves a loss of sensitivity of the receptor to its original stimulus. The primary focus of this research proposal is to utilize bovine rhodopsin as a model system for studying the molecular interactions between G protein-coupled receptors, G proteins and the kinases and arrestins involved in receptor desensitization. In order to achieve this goal, point mutations and deletions will be generated in the cDNA for bovine opsin by site-directed mutagenesis of specific structural domains predicted to be involved in these important signal transduction processes. This will be followed by transient expression of these altered proteins in HEK-293 cells for generating large quantities of the mutant opsin proteins and functional reconstitution with the rod cell G protein, Gt, as well as rhodopsin kinase, protein kinase C and arrestin. Domains in rhodopsin which are critical for the interaction of the photoreceptor with its G protein will be identified using light-dependent GTPgammaS binding to Gt. The effect of mutations on the observed cooperativity of the interaction between rhodopsin and its G protein will also be examined. The regulatory domains required for the light-dependent binding of rhodopsin kinase and arrestin will be identified by phosphorylation and binding assays. The serine/threonine-rich domain of the carboxy-terminus will be mutated to identify those sites that are most likely to be phosphorylated by rhodopsin kinase and protein kinase C and those that are most critical for the binding of arrestin. A more complete understanding of the mechanisms by which overlapping domains within rhodopsin control the processes of G protein activation and desensitization will aid in defining potential mechanisms of disease resulting from defects in G protein-coupled receptor signaling pathways.