Molecular Regulation of Cardiovascular 7 TM Receptors

描述（由申请人提供）：心血管功能的各个方面都由7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，这是由两步过程介导的，在这个过程中，活化的受体被g蛋白偶联受体激酶（GRKs）磷酸化，导致一个抑制蛋白（arr）分子的结合，该分子在空间上阻断g蛋白的进一步激活。最近，人们已经清楚地发现，¿arrs也可以作为多功能内吞和信号转接器，它还可以激活参与趋化性和抗凋亡等生理结果的其他途径，从而调节动脉粥样硬化、再狭窄和心脏肥大的心血管功能。此外，对于一些受体，包括- 1和- 2肾上腺素能受体（ARs）和血管紧张素1A受体（AT1AR）等，可以发现配体不成比例地激活g蛋白或- arr介导的信号传导。“偏置配体”。这类化合物具有潜在的独特治疗特性。因此，本研究有三个密切相关的目标，包括重点阐明2arrs介导7TMRs信号传导的分子机制。我们的目标是：1)利用全球细胞磷酸化蛋白质组学和系统生物学的方法阐明心血管7tmr（如AT1AR和¿2-AR）下游的arr介导的信号网络的全部范围；2)通过高通量和靶向筛选为¿2- ar开发¿2- ar偏配体；3)通过对受体磷酸化位点的分析、抗体构象特异性抗原结合片段（fab）的开发和晶体学技术，阐明这种信号传导的分子和生物物理基础。我们的假设是，不同的GRKs对受体上不同位点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。通过了解功能信号的后果和结构基础的arr-biased agonism，我们将奠定基础，开发一类新的治疗药物。

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.