Molecular Regulation of Cardiovascular 7 TM Receptors

描述（由申请人提供）：心血管功能的所有方面均受7 TM受体家族的受体调节。它是所有受体家族中最大和最普遍的，包括儿茶酚胺、乙酰胆碱、血管紧张素、腺苷和内皮素的受体。调节7 TM受体的普遍机制是异源三聚体G蛋白信号转导的脱敏。典型地，这是由两步过程介导的，其中激活的受体被G蛋白偶联受体激酶（GRK）磷酸化，导致B-抑制蛋白分子的结合，其在空间上阻止G蛋白的进一步激活。在过去的几年中，已经清楚的是，B-抑制蛋白也可以作为多功能的内吞和信号转导衔接子，可以激活其他途径。这些包括MAP激酶，如ERK 1/2和JNK 3和生理结果，如趋化性和抗凋亡，所有这些在动脉粥样硬化、再狭窄和心脏肥大中调节心血管功能都非常重要。因此，该提议具有3个密切相关的目标，其主要关注于阐明B-抑制蛋白和GRKs通过7 TM受体介导信号传导的分子机制。我们的目标是阐明：1）GRK在介导7 TM受体信号传导中的作用，通过开发siRNA技术和“敲除”小鼠胚胎成纤维细胞以减少或消除细胞中的GRK表达;以及通过确定GRK磷酸化的位点及其对几种7 TM受体（包括B2-肾上腺素能受体和Ang 111 A受体）的功能性后果;（2）B-arrestin 1和2在7 TM受体介导ERK激活中的作用以及B-arrestin泛素化在此过程中的作用; 3）通过有限的蛋白水解研究α-抑制蛋白的“活化”构象，来研究β-抑制蛋白介导的信号传导的结构和生物物理基础，色氨酸荧光和原子结构测定。我们的假设是，不同的GRK受体上的不同位点的磷酸化导致结构和功能不同的激活构象的B-抑制蛋白介导不同的信号转导结果。由此产生的理解的分子基础上的新认识GRK和α-抑制蛋白介导的信号通路应该指出的方式发展的新的治疗心血管疾病。

DESCRIPTION (provided by applicant): All aspects of cardiovascular function are regulated by receptors of the 7TM receptor family. The largest and most ubiquitous of all the receptor families, it includes receptors for catecholamines, acetylcholine, angiotensin, adenosine and endothelins. A universal mechanism regulating 7TM receptors is desensitization of heterotrimeric G-protein signaling. Classically, this is mediated by a 2-step process in which activated receptors are phosphorylated by G-protein-coupled receptor kinases (GRKs) leading to the binding of a B-arrestin molecule, which sterically interdicts further activation of the G protein. Over the past several years it has become clear that B-arrestins can also serve as multifunctional endocytic and signaling adaptors, which can ateo activate additional pathways. These include MAP kinases, such as ERK1/2 and JNK3 and physiological outcomes such as chemotaxis and anti-apoptosis, all of which are of great importance in regulating cardiovascular function in atherosclerosis, restenosis, and cardiac hypertrophy. Accordingly, this proposal has 3 closely linked aims, which involve a primary focus on elucidating the molecular mechanisms by which B-arrestins and GRKs mediate signaling by 7TM receptors. Our goals are to elucidate: 1) the roles of GRKs in mediating 7TM receptor signaling, by developing siRNA techniques and 'knock-out" mouse embryo fibroblasts to reduce or eliminate GRK expression from cells; and by determining the sites of GRK phosphorylation and their functional consequences for several 7TM receptors including the B2-adrenergic receptor and Ang 111A receptor; 2) the roles of B-arrestin 1 and 2 in mediating ERK activation by 7TM receptors and of B-arrestin ubiquitination in this process; 3) the structural and biophysical basis of B-arrestin-mediated signaling by studying the "activated" conformations of a-arrestins by limited proteolysis, tryptophan fluorescence and atomic structure determination. Our hypothesis is that the phosphorylation of distinct sites on receptors by different GRKs leads to structurally and functionally distinct activated conformations of B-arrestins which mediate distinct signaling outcomes. The resulting understanding of the molecular basis of the newly appreciated GRK and a-arrestin-mediated signaling pathways should point the way toward development of novel therapeutics for cardiovascular diseases.