In Vivo Implications of Agonist Selective Activation of Delta Opioid Receptor

描述(申请人提供)：我目前是高级博士后研究员，主要研究阿片受体药理学。我打算在接下来的两年内过渡到初级教员的职位，而我目前的指导职位就是为了实现这一目标而构建的。我在疼痛和成瘾的体内模型方面有广泛的经验，在评估受体结合、功能和体外定位的技术方面也有丰富的经验。我目前在加州大学洛杉矶分校(UCLA)著名阿片研究人员克里斯托弗·埃文斯博士的实验室工作。在加州大学洛杉矶分校期间，我将通过学习原代细胞培养、电生理学和阵列方面的新技术来提高我的技能。我和我的导师还计划了几项策略，以促进我向教员职位的过渡，包括预算责任、教学和课程工作。加州大学洛杉矶分校是一所著名的机构，拥有许多知名的研究人员，在这个指导阶段，我将发展重要的合作，我希望这些合作能持续到我的独立。此外，该大学还拥有几个最先进的核心设施，通过这些设施，我将增加我的研究机会。我的研究重点是配体定向阿片受体信号在体内的影响。这种受体的激动剂正在开发用于临床，因为激活4种受体可以缓解疼痛，减少焦虑和抑郁。然而，这些化合物临床应用的一个主要限制是，4种激动剂的子集也会产生惊厥，其机制尚不清楚。这种激动剂选择性行为可能是由于4受体的不同运输和信号传递，我最近的工作表明，内在化激动剂SNC80会产生非内在化激动剂ARM390没有观察到的惊厥。2-arrestins是受体内化的主要介质，也是随后的受体信号转导的中介。这项建议的目的之一是利用2-arrestin 1和2基因敲除小鼠，确定2-arrestins在SNC80和ARM390诱导的行为中的作用。此外，为了表征调控这种功能选择性的不同信号机制，将使用邻近连接分析和磷蛋白阵列来确定SNC80和ARM390形成的不同信号复合体。此外，我还发现，在炎性疼痛模型中重复使用这些激动剂也会导致不同的耐受性。SNC80产生受体下调和对所有激动剂诱导的效应的普遍耐受性。相比之下，对ARM390耐受的动物表现出完整的4个受体，只有镇痛耐受-其机制尚不清楚。这项应用的另一个目的是通过寻找长期使用后背根神经节内受体-离子通道偶联的变化来表征这种差异耐受性。我还将研究2-arrestins在这两种耐受中的作用，并使用DNA微阵列探索这些差异的可能机制。这项工作具有重要的治疗意义，并将加深我们对体内阿片受体运输和信号转导的理解。 与公共卫生相关：激活Delta阿片受体可以缓解疼痛，减少焦虑和抑郁；因此，该受体的激动剂正在开发用于临床。然而，阻止这些化合物发展的一个因素是，一些Delta激动剂也会产生惊厥，目前还无法预测一种新的激动剂是否会具有这种特性。这项建议的目标是确定解释与同一受体结合的激动剂之间差异的机制。最终，这项工作将增加增量阿片受体作为一种新的药物治疗的潜力。

DESCRIPTION (provided by applicant): I am currently a senior level postdoctoral fellow with a primary research focus in opioid receptor pharmacology. I intend to transition to a junior faculty position in the next two years, and my current mentored position has been structured to accomplish this goal. I have a wide-range of experience with in vivo models of pain and addiction, as well as extensive experience with techniques to assess receptor binding, function, and localization ex vivo. I am currently working in the laboratory of Dr. Christopher Evans, a prominent opioid researcher, at the University of California Los Angeles (UCLA). During my time at UCLA, I will enhance my skill set by learning new techniques in primary cell culture, electrophysiology, and arrays. My mentor and I have also planned several strategies to facilitate my transition to a faculty position, including budgetary responsibility, teaching, and course work. UCLA is a renowned institution with a number of high profile researchers, and during this mentored phase I will develop important collaborations that I hope to sustain into my independence. In addition, the university also has several state-of-the-art core facilities through which I will enhance my research opportunities. My research focus is on the in vivo consequences of ligand directed signaling at the delta (4) opioid receptor. Agonists for this receptor are being developed for clinical use, as activation of 4 receptors relieves pain, and reduces anxiety and depression. However, a major limitation to the clinical use of these compounds is that a subset of 4 agonists also produce convulsions, the mechanism of which is unknown. This agonist-selective behavior could be due to differential trafficking and signaling of the 4 receptor, and my recent work shows that the internalizing agonist, SNC80, produces convulsions which are not observed with the non-internalizing agonist, ARM390. 2-arrestins are major mediators of receptor internalization, and also mediate subsequent receptor signaling. One of the aims of this proposal is to determine the role of 2-arrestins in SNC80 and ARM390-induced behaviors, using 2-arrestin 1 and 2 knockout mice. In addition, to characterize the different signaling mechanisms regulating this functional selectivity, the distinct signaling complexes formed by SNC80 and ARM390 will be determined using proximity ligation assays and phosphoprotein arrays. Furthermore, I have found that repeated use of these agonists in an inflammatory pain model also results in differential tolerance. SNC80 produces receptor down regulation and generalized tolerance to all agonist-induced effects. In contrast, ARM390-tolerant animals show intact 4 receptors, and analgesic tolerance only - the mechanism of which is unknown. A further aim of this application is to characterize this differential tolerance, by looking for changes in receptor-ion channel coupling within the dorsal root ganglia following chronic use. I will also examine the role of 2-arrestins in these two types of tolerance, and explore possible mechanisms underlying these differences using DNA microarrays. This work has important therapeutic implications, and will enhance our understanding of in vivo opioid receptor trafficking and signaling. PUBLIC HEALTH RELEVANCE: Delta opioid receptor activation produces pain-relief, and reduces anxiety and depression; and for this reason agonist to this receptor are being developed for clinical use. However, a deterrent to the development of these compounds is that some delta agonists also produce convulsions, and at the moment there is no way to predict whether a novel agonist will have this property. The goal of this proposal is to determine the mechanism that accounts for this difference between agonists that bind to the same receptor. Ultimately, this work would increase the potential for the delta opioid receptor as a novel drug therapy.