Molecular Regulation of Cardiovascular 7 TM Receptors

描述(申请人提供)：心血管功能的所有方面都由七种跨膜受体(7TMR)家族的受体调节。它是所有受体家族中最大的，包括儿茶酚胺、乙酰胆碱、血管紧张素、腺苷和内皮素的受体。调节这些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，这是一个两步过程，激活的受体被G蛋白偶联受体激酶(GRK)磷酸化，导致与精氨酸(ARR)分子的结合，这在空间上阻断了G蛋白的进一步激活。最近发现，ARRs还可以作为多功能的内吞和信号转接子，它还可以激活参与趋化和抗细胞凋亡等生理结果的额外通路，从而调节动脉粥样硬化、再狭窄和心肌肥厚的心血管功能。此外，对于几种受体，包括肾上腺素能受体(ARs)和血管紧张素受体(AT1AR)等，可以发现不成比例地激活G蛋白或ARR介导的信号的配体--即“有偏见的配体”。这类化合物具有潜在的独特治疗特性。因此，这项建议有三个紧密联系的目标，其中包括重点阐明2ARR介导7TMR信号传递的分子机制。我们的目标是：1)利用全球细胞磷蛋白质组学和系统生物学的方法，全面阐明ARR介导的心血管7TMR下游的信号网络，如AT1AR和2-AR；2)通过高通量和靶向筛选，为2-AR开发偏向ARR的配体；以及3)通过分析受体磷酸化位点、开发构象特异的抗体抗原结合片段(FAB)和结晶学技术，从配体特异性构象变化的角度阐明这种信号转导的分子和生物物理基础。我们的假设是，不同GRK对受体不同部位的磷酸化导致受体和受体在结构和功能上不同的激活构象，从而介导不同的信号转导结果。通过了解ARR偏向激动剂的功能信号后果和结构基础，我们将为开发一类新的治疗剂奠定基础。

DESCRIPTION (provided by applicant): All aspects of cardiovascular function are regulated by receptors of the seven transmembrane receptor (7TMR) family. The largest of all the receptor families, it includes receptors for catecholamines, acetylcholine, angiotensin, adenosine and endothelins. A universal mechanism regulating these receptors is desensitization of heterotrimeric G-protein signaling. Classically, this is mediated by a two-step process in which activated receptors are phosphorylated by G-protein-coupled receptor kinases (GRKs) leading to the binding of a ¿- arrestin (¿ arr) molecule, which sterically interdicts further activation of the G-protein. More recently it has become clear that ¿ arrs can also serve as multifunctional endocytic and signaling adaptors, which can also activate additional pathways involved in such physiological outcomes as chemotaxis and anti-apoptosis, thus regulating cardiovascular function in atherosclerosis, restenosis, and cardiac hypertrophy. Moreover, for several receptors, including the ¿1 and ¿2-adrenergic receptors (ARs) and angiotensin 1A receptor (AT1AR), amongst others, ligands can be found which disproportionately activate either G-protein or ¿ arr mediated signaling - i.e. "biased ligands". Such compounds have potentially unique therapeutic properties. Accordingly, this proposal has three closely linked aims, which involve a focus on elucidating the molecular mechanisms by which 2arrs mediate signaling by 7TMRs. Our goals are: 1) to elucidate the full extent of ¿ arr mediated signaling networks downstream of cardiovascular 7TMRs such as the AT1AR and ¿ 2-AR using the approaches of global cellular phosphoproteomics and systems biology; 2) to develop ¿ arr-biased ligands for the ¿ 2-AR via high throughput and targeted screening; and 3)to elucidate the molecular and biophysical basis of such signaling in terms of ligand specific conformational alterations in ¿ arrs via analysis of receptor phosphorylation sites, development of conformationally specific antigen binding fragments of antibodies (Fabs) and crystallographic techniques. Our hypothesis is that the phosphorylation of distinct sites on receptors by different GRKs leads to structurally and functionally distinct activated conformations of the receptors and ¿ arrs which mediate distinct signaling outcomes. By understanding the functional signaling consequences and structural basis of ¿ arr-biased agonism we will lay the basis for the development of a novel class of therapeutic agents.