Molecular Regulation of Cardiovascular 7 TM Receptors

说明(申请人提供)：心血管功能的所有方面都由7TM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7TM受体的一个普遍机制是异源三聚体G蛋白信号的脱敏。经典地说，这是由一个两步过程介导的，在这个过程中，激活的受体被G蛋白偶联受体激酶(GRKs)磷酸化，导致B-arrestin分子的结合，从而立体地阻断G蛋白的进一步激活。在过去的几年里，已经很清楚，B-拦截素也可以作为多功能的内吞和信号适配器，可以激活更多的途径。其中包括MAP激酶，如ERK1/2和JNK3，以及趋化和抗细胞凋亡等生理结果，所有这些都在动脉粥样硬化、再狭窄和心肌肥厚的心血管功能调节中发挥重要作用。因此，这一建议有三个紧密联系的目标，主要涉及阐明B-arrestins和GRKs通过7TM受体介导信号的分子机制。我们的目标是阐明：1)通过发展siRNA技术和“敲除”小鼠胚胎成纤维细胞以减少或消除细胞中GRK的表达，并通过确定GRK磷酸化的位置及其对包括B2-肾上腺素能受体和Ang 111a受体在内的几种7TM受体的功能影响，阐明GRKs在介导7TM受体激活ERK和B-arrestin泛素化中的作用；2)B-arrestin 1和2在介导7TM受体激活ERK和B-arrestin泛素化过程中的作用；3)通过有限的蛋白水解法、色氨酸荧光和原子结构测定研究B-arrestin介导的信号转导的结构和生物物理基础。我们的假设是，不同GRK对受体不同部位的磷酸化导致结构和功能上不同的B-arrestins激活构象，从而介导不同的信号转导结果。由此产生的对新认识的GRK和α-arrestin介导的信号通路的分子基础的理解应该为心血管疾病的新疗法的发展指明方向。

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.