Investigating the requirement of MRAP2 for ghrelin function

项目摘要 Ghrelin是一种循环促食欲激素，主要由胃在禁食期间分泌， 饥饿对大脑Ghrelin通过作用于其受体生长因子来调节食物摄入和能量消耗。 激素促分泌素受体1a（GHSR1a）。Ghrelin对摄食和能量平衡的影响 是通过AGRP/NPY神经元的激活和生长激素释放肽的全局或中枢缺失来介导的 受体可以防止饮食引起的肥胖。由于这个原因，GHSR1a是一个有前途的治疗靶点， 为此目的，已经开发了肥胖症和生长素释放肽受体拮抗剂，然而，它们的滥交 或低生物利用度使它们不能用于临床。具有改进的药理学活性的新化合物 化学性质和更好的体内功效可能通过使用更相关的药物发现来确定 战略布局改善筛选策略将需要更好地了解GHSR1a调节。我们 最近发现，生长激素释放肽的促食欲作用在黑皮质素受体 辅助蛋白2（MRAP 2）缺失。我们的初步结果表明，MRAP 2与 GHSR1a和强烈增强ghrelin刺激的受体下游信号传导。此外，我们显示 MRAP 2在ghrelin反应性AGRP/NPY神经元中表达。与这些发现一致，我们发现 来自MRAP 2 KO小鼠的AGRP神经元不能响应饥饿而激活。建议的实验 该项目将利用几种允许靶向调节MRAP 2的动物模型 在特定神经元中的生长素释放肽信号传导的表达和测量。本提案的目标是：（1）确定 MRAP 2在促进AGRP神经元的ghrelin信号和饥饿反应中的作用; 2）鉴定 MRAP 2通过其增强GHSR 1a信号传导的功效的机制。目标1中的研究将测试 这一假说认为AGRP神经元中MRAP 2表达的调节改变了饥饿感和ghrelin的作用 在大脑里。目的2中的研究将检验MRAP 2增强GHSR 1a信号传导的假设， 在体内和体外，通过干扰受体的脱敏。这项研究很重要，因为 成功完成将提供关于MRAP 2在以下方面的作用和作用机制的基本信息： AGRP神经元，因为它涉及ghrelin功能，推进我们对AGRP神经元调节的理解， 下丘脑控制能量平衡。这项研究是创新的，因为完成将产生新的 关于辅助蛋白调节G蛋白偶联受体的知识，并将MRAP 2鉴定为 AGRP神经元中的新型能量传感器。我们还将使用我们的创新小鼠模型， 在实验室中，以组织特异性方式准确检测和遗传调节MRAP 2的表达。 最终，该项目的完成将为GHSR1a的监管提供重要见解， 这种受体的强制性辅助蛋白，并使设计药物发现策略的目标生长激素释放肽 这将比以前的尝试更具生理相关性，更有可能成功。

PROJECT SUMMARY Ghrelin is a circulating orexigenic hormone mainly secreted by the stomach during fasting periods to signal hunger to the brain. Ghrelin regulates food intake and energy expenditure by acting at its receptor the Growth Hormone Secretagogue Receptor 1a (GHSR1a). The effect of ghrelin on food intake and energy homeostasis are mediated centrally through the activation of AGRP/NPY neurons, and global or central deletion of the ghrelin receptor protects from diet-induced obesity. For this reason, GHSR1a is a promising target for the treatment of obesity and antagonists of the ghrelin receptor have been developed for this purpose, however, their promiscuity or low bioavailabilty precludes them from being used in the clinic. New compounds with improved pharmaco- chemical properties and better in-vivo efficacy could likely be identified by using more relevant drug discovery strategies. Improvement of screening strategies will require a better understanding of GHSR1a regulation. We have recently discovered that the orexigenic effect of ghrelin is lost in mice in which the Melanocortin Receptor Accessory Protein 2 (MRAP2) was deleted. Our preliminary results demonstrate that MRAP2 interacts with GHSR1a and strongly potentiates ghrelin-stimulated signaling downstream of the receptor. Additionally, we show that MRAP2 is expressed in the ghrelin responsive AGRP/NPY neurons. Consistent with those findings, we show that AGRP neurons from MRAP2 KO mice fail to activate in response to starvation. Experiments proposed in this project will take advantage of several animal models that allow the targeted modulation of MRAP2 expression and the measurement of ghrelin signaling in specific neurons. The goal of this proposal is to 1) Identify the role of MRAP2 in promoting ghrelin signaling and starvation response of AGRP neurons; 2) Identify the mechanisms through which MRAP2 enhances to efficacy of GHSR1a signaling. The studies in aim 1 will test the hypothesis that the modulation of MRAP2 expression in AGRP neurons alters hunger sensing and ghrelin actions in the brain. The studies in aim 2 will test the hypothesis that MRAP2 potentiates GHSR1a signaling, both in-vivo and in-vitro, by interfering with the desensitization of the receptor. This research is significant because successful completion will provide fundamental information on the role and mechanism of action of MRAP2 in AGRP neurons as it pertains to ghrelin functions, advance our understanding of AGRP neurons regulation and hypothalamic control of energy homeostasis. This research is innovative because completion will generate novel knowledge on the regulation of G-Protein Coupled receptors by accessory proteins and identify MRAP2 as a novel energy sensor in AGRP neurons. We will also be using innovative mouse model generated in our laboratory to accurately detect and genetically modulate the expression of MRAP2 in a tissue specific manner. Ultimately, the completion of this project will provide critical insights in the regulation of GHSR1a, identify an obligatory accessory protein of this receptor and enable the design of drug discovery strategies targeting ghrelin signaling that will be far more physiologically relevant and more likely to succeed than previous attempts.