MECHANISMS OF ALPHA2 ADRENERGIC RECEPTOR FUNCTION

α-2-肾上腺素能受体（α-2-AR）是细胞表面受体 其在结合儿茶酚胺后通过以下途径向细胞内部发出信号 G蛋白。 α-2-AR几乎在每个器官系统中表达， 已知在心血管、肺、肾 肝脏、代谢和中枢神经系统功能。 α-2-AR 也与许多病理过程有关， 药物治疗的靶点 疾病 本项目的长期目标是了解 这些受体的分子结构之间的关系， 它们的功能。 这些目标将主要通过定点诱变来实现 在编码野生型α-2-AR亚型（α-2C10， α-2-C4和α-2-C2），然后在大肠杆菌中重组表达。 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 α-2-AR磷酸化中的蛋白偶联受体激酶 在短期激动剂促进脱敏期间将进行研究。 在 具体目标2，α-2-AR的磷酸化结构域将被 通过评估功能性短期激动剂促进的 野生型和α-2-AR的脱敏和受体磷酸化 在我们怀疑是 磷酸化 在具体目标3中，α- 2-AR亚型，负责激动剂的差异- 促进脱敏和磷酸化之间观察到的三个 将确定子类型。 具体目标4探讨了 受体隔离（内化）和下调，两个关键 在长期激动剂促进α- 2-AR。 在这里，受体运输的亚细胞事件和 将使用免疫电子显微镜检查处理。 在特定 目的5，α-2-AR螯合和 下调将使用区域的定点突变来研究 被怀疑参与这些过程，包括网站， 棕榈酰化，糖基化和磷酸化，以及涉及的区域 G蛋白偶联。 在具体目标6中，α-2- 负责与G1和G2偶联的AR将由以下两种描述： 缺失和嵌合取代诱变。 第七章具体目标 G2偶联中观察到的差异的分子决定因素 将确定三种α-2-AR亚型。 这些研究的结果将有助于确定一个基本的 水平α-2-AR如何进行其信号转导， 调节，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.