MECHANISMS OF ALPHA2 ADRENERGIC RECEPTOR FUNCTION

α-2-肾上腺素能受体是细胞表面受体 当与儿茶酚胺结合时，信号通过细胞内部 G蛋白。α-2-AR在几乎每个器官系统中都有表达，并且 已知在心血管、肺、肾、 肝脏、新陈代谢和中枢神经系统功能。阿尔法-2-AR 也牵涉到许多病理过程，并且是 药物治疗的多个靶点 疾病。该项目的长期目标是了解 这些受体的分子结构与它们之间的关系 它们的功能。 这些目标将主要通过定点突变来实现。 编码野生型α-2-AR亚型(α-2C10， Alpha-2-C4和Alpha-2-C2)，然后在 哺乳动物细胞。这允许直接比较给定的函数 野生型和突变型受体之间的关系 结构/功能关系。在具体目标1中，G的作用 蛋白偶联受体激酶在α-2-Ars磷酸化中的作用 在短期内，将研究激动剂促进的脱敏作用。在……里面 特定目标2，α-2-AR的磷酸化结构域将是 通过评估功能性短期激动剂促进的地图 野生型和α-2-ARs的脱敏和受体磷酸化 在我们怀疑的区域有突变残基 磷酸化。在具体目标3中，阿尔法的分子特征- 2-AR亚型导致激动剂- 促进三者之间的脱敏和磷酸化 将确定亚型。《特定目标4》探讨了 受体隔离(内化)和下调，两个关键 在长期使用激动剂期间发生的事件促进了α-受体的调节。 2-Ars.在这里，受体贩运的亚细胞事件和 处理过程将使用免疫电子显微镜进行检查。具体而言 目的5，α-2-AR的分子决定因素和 将使用区域的定点突变来研究下调 被怀疑参与这些过程的站点包括 棕榈酰化、糖基化和磷酸化及其涉及的区域 G蛋白偶联。在特定的目标6中，α-2的域- 负责耦合到G1和G2的AR将由双方划定 缺失和嵌合替换突变。在具体目标7中 观察到的G2偶联差异的分子决定因素 将确定三个阿尔法-2-AR亚型。 这些研究的结果将有助于从根本上确定 水平α-2-AR如何进行信号转导，它们是如何 以及它们如何被治疗剂调节。

Alpha-2-adrenergic receptors (alpha-2-AR) are cell surface receptors which upon binding catecholamines signal to the interior of the cell via G proteins. Alpha-2-AR are expressed in virtually every organ system and are known to play important roles in cardiovascular, pulmonary, renal, hepatic, metabolic, and central nervous system functions. Alpha-2-ARs have also been implicated in a number of pathologic processes and are the targets for pharmacologic agents in the treatment of a number of diseases. The long-term objective of this project is to understand the relationships between the molecular structures of these receptors and their functions. These goals will be carried out primarily by site-directed mutagenesis of the cDNAs encoding for the wild-type alpha-2-AR subtypes (alpha-2C10, alpha-2-C4, and alpha-2-C2), followed by recombinant expression in mammalian cells. This allows for directly comparing a given function between wild-type and mutated receptor and delineating a structure/function relationship. In Specific Aim 1, the role of G protein coupled receptor kinases in the phosphorylation of alpha-2-ARs during short-term agonist promoted desensitization will be studied. In Specific Aim 2, the phosphorylation domains of the alpha-2-AR will be mapped by assessing functional short-term agonist promoted desensitization and receptor phosphorylation in wild-type and alpha-2-ARs with mutated residues in regions that we suspect are sites for phosphorylation. In Specific Aim 3, the molecular features of the alpha- 2-AR subtypes which are responsible for the differences in agonist- promoted desensitization and phosphorylation observed between the three subtypes will be determined. Specific Aim 4 explores the mechanism of receptor sequestration (internalization) and downregulation, two key events which occur during long-term agonist promoted regulation of alpha- 2-ARs. Here, the subcellular events of receptor trafficking and processing will be examined using immunoelectron microscopy. In Specific Aim 5, the molecular determinants of alpha-2-AR sequestration and downregulation will be studied using site-directed mutagenesis of regions suspected to be involved in these processes including sites for palmitoylation, glycosylation, and phosphorylation, and regions involved with G protein coupling. In Specific Aim 6, the domains of the alpha-2- AR responsible for coupling to G1 and G2 will be delineated by both deletion and chimeric substitution mutagenesis. In Specific Aim 7 the molecular determinants of the observed differences in G2 coupling between the three alpha-2-AR subtypes will be determined. The results of these studies will help to determine at a fundamental level how alpha-2-AR carry out their signal transduction, how they are regulated, and how they can be modulated by therapeutic agents.