Novel Interactions Between G protein coupled receptors and Receptor Activity Modifying Proteins and Their Control of Angiogenic Cues

摘要 G蛋白偶联受体（GPCR）代表最大类的细胞受体。GPCR信号 涉及配体的激活，导致通过它们的变化将信号传导到细胞内部。 结构这些受体介导生理过程，如神经传递，细胞分化， 生长和免疫反应。由于这种控制，GPCR已成为药物的主要靶点 的发现据估计，约60%的所有临床处方的外源性药物靶向GPCR。随着这一成功 目标明确，人们普遍承认，对它们的监管非常复杂。另外存在 几个已知的GPCR相互作用蛋白家族。研究最多的GPCR相互作用伙伴的例子 调节受体药理学的是受体活性修饰蛋白（RAMP 1、-2和-3）。这些 在集中于降钙素基因相关肽（CGRP）的实验中鉴定了蛋白质。这是 确定功能性CGRP受体需要降钙素受体样受体的表达 (CLR)蛋白质伴侣RAMP 1。随后，当RAMP 2与RAMP 2共表达时， 在受体的形成中，不是由CGRP激活，而是由一种称为肾上腺髓质素的相关肽激活。 （上午）。自这些早期研究以来，9种GPCR已被证明与RAMP家族成员相互作用。 Caron实验室对RAMP：GPCR相互作用及其对细胞功能的影响有着长期的兴趣。 淋巴管生成、心脏发育及其在妊娠期间的参与。我们实验室以前的研究 已经表明，某种受体作为AM的“诱饵”受体，因此提供了良好的- 调节AM介导的LEC增殖的控制。根据这个诱饵的活动，表达模式，和我的 初步研究，我建议调查的假设，这种“诱饵”受体也调节亲， 新血管的血管生成发芽，并且这种活性进一步受到新型GPCR：RAMP的调节 交互.我将利用体外方法来证实两种新的GPCR之间的相互作用：RAMP 伙伴此外，我将使用我们目前在跖骨和基质胶塞测定中拥有的KO小鼠， 检查这些GPCR和RAMP的遗传缺失对血管生成的影响。最后，我会适应 一种独特的筛选模式，在高通量平台上询问“可药物化”的GPCR组， 阐明以前未知的GPCR：RAMP相互作用。这对于开发治疗方法至关重要， 可能针对各种RAMP相互作用受体。这项提案的结果将证实我的初步建议。 存在至少两种新的GPCR：RAMP相互作用的数据（目标1）。它将决定，体外和体内 体内，无论“诱饵”活性和/或RAMP影响血管生成（目的2）。最后，它将提供独特的 深入了解以前未表征的G蛋白信号转导修饰，并提供新的药物靶点（目的3）。 这将允许一个研究计划的发展，我最终可以建立自己的研究。

Abstract G-protein coupled receptors (GPCRs) represent the largest class of cellular receptors. Signaling by GPCRs involves activation by ligands, resulting in signal transduction to the interior of the cell through changes in their structure. These receptors mediate physiological processes such as neurotransmission, cellular differentiation, growth, and immune responses. Because of this control GPCRs have emerged as major targets for the drug discovery. It is estimated that ~60% of all clinically prescribed xenobiotics target GPCRs. With this successful targeting, it has become widely acknowledged that their regulation is very complex. Additionally, there are several known families of GPCR interacting proteins. The most studied example of GPCR-interacting partners that regulate receptor pharmacology is the receptor activity modifying proteins (RAMP1, -2 and -3). These proteins were identified in experiments focused on the calcitonin gene-related peptide (CGRP). It was determined that the functional CGRP receptor required the expression of the calcitonin receptor-like receptor (CLR) with the protein partner RAMP1. Subsequently, when CLR was co-expressed with RAMP2, this resulted in the formation of receptors activated not by CGRP, but rather by a related peptide known as adrenomedullin (AM). Since these early studies nine GPCRs have been shown to interact with the RAMP family members. The Caron lab has a long standing interest in RAMP: GPCR interactions and their functional consequences on lymphangiogenesis, cardiac development, and their involvement during pregnancy. Previous studies in our lab have shown that a certain receptor serves as a “decoy” receptor for AM which consequently provides fine- tuned control of AM-mediated proliferation of LECs. Based on this decoy activity, expression patterns, and my preliminary studies, I propose to investigate the hypothesis that this “decoy” receptor also modulates the pro- angiogenic sprouting of new blood vessels and that this activity is further regulated by novel GPCR: RAMP interactions. I will utilize in vitro approaches to confirm the interaction between two new GPCR: RAMP partners. Furthermore, I will use KO mice we currently possess in the metatarsal and matrigel plug assays to examine the effects of the genetic depletion of these GPCRs and RAMPs on angiogenesis. Finally, I will adapt a unique screening paradigm to interrogate the “druggable” GPCR-ome in a high-throughput platform to elucidate previously unknown GPCR: RAMP interactions. This is essential for developing therapeutics that might target the various RAMP-interacting receptors. Results from this proposal will confirm my preliminary data of the existence of at least two new GPCR: RAMP interactions (Aim 1). It will determine, ex vivo and in vivo, whether the “decoy” activities and/or RAMPs influence angiogenesis (Aim 2). Lastly, it will provide unique insight into previously uncharacterized G protein signaling modifications and provide new drug targets (Aim 3). This will allow for the development of a research program upon which I can eventually build my own research.