Regulation of alpha2A-adrenergic receptor signaling and trafficking by spinophili

描述（由申请人提供）：2a -肾上腺素能受体（AR）属于大G蛋白偶联受体（GPCR）超家族。？介导的许多生理和药理反应。2AAR将这种特殊亚型定位为潜在的发病机制候选者和治疗多种疾病（包括多动症、高血压和抑郁症）的有价值靶点。到目前为止，内源性机制是什么？aar的功能是在分子和细胞水平上调控的，仍然是难以捉摸的。除G蛋白外，GPCR相互作用伙伴在调节GPCR信号传导中起着关键作用，代表了一种全新的药物开发途径。我们之前的研究确定嗜脊髓蛋白是一种？2ar -相互作用的合作伙伴，管理的多个方面？通过拮抗阻滞蛋白功能，aar运输和信号传导（即受体表面保留、内化、信号激活、脱敏和激动剂敏感性）。此外，嗜脊髓蛋白的缺失改变了？小鼠aar诱发反应。的深刻的体内相关性？2aar -嗜脊髓蛋白的相互作用促使我们探索嗜脊髓蛋白调控？原生神经元中的aar运输和信号，在哪里？aar和嗜脊髓蛋白引发了它们的主要生理功能。我们已经产生了一种新的小鼠系，其中n端HA标记？2AAR的表达是由内源性小鼠？2AAR位点(HA-？aar撞击还是？2AAR HA/HA)，可通过HA抗体检测。利用这种独特的敲入系，结合嗜脊髓蛋白敲除小鼠（Sp-/-），以及互补的细胞和分子策略，我们将测试中心假设：嗜脊髓蛋白调节？原生神经元中的aar运输和信号传导是为了微调吗？2 aar响应能力。特异性Aim 1将确定嗜脊髓蛋白在激动剂诱导的？原生神经元的aar内吞作用。除了肾上腺素（一种内源性配体），我们还将研究？由可乐定和胍法辛引起的aar贩运(2 ？2-激动剂（临床上常用）。如果嗜脊髓蛋白介导的？aar表现出激动剂偏倚，这可能是激动剂选择性调节受体运输的潜在机制。特异性Aim 2将确定嗜脊髓蛋白在调节？在神经元中，aar诱发的电（Ca2+电流的抑制）和生化（ERK的激活）反应。特异性Aim 3将确定pka介导的嗜脊髓蛋白磷酸化在调节？天然神经元中的aar转运和信号传导。我们新收集的初步数据表明，PKA磷酸化的嗜脊髓蛋白破坏了？2 aar-spinophilin交互。我们将进一步确定pka介导的调节在？天然神经元中的aar转运和信号传导。我们对审稿人的疑问做出了回应，并纳入了大量额外的初步数据，这一修订后的建议得到了显著改进，将促进我们对内源性调控的理解。从而为未来的药物开发提供了新的见解，特别是为作用于？2AAR增强或减少受体-嗜脊髓蛋白相互作用，取决于期望的功能结果。G蛋白偶联受体（gpcr）是最大的表面受体家族的成员，代表了最丰富的治疗靶点。的吗?肾上腺素能受体(？2AAR)亚家族介导广泛的关键生理/药理学反应，包括降低血压，唤醒镇静，减少疼痛感知和改善工作记忆。利用遗传模型和分子和细胞策略，我们的研究旨在了解如何？2AAR的功能受到相互作用蛋白嗜脊髓蛋白的严格调控。从我们的研究中获得的信息将促进我们对潜在的内源性细胞机制的理解。从而为治疗注意力缺陷和多动障碍（ADHD）、抑郁症、高血压和其他使用？aar激动剂可能是合理的。

DESCRIPTION (provided by applicant): The ?2A-adrenergic receptor (AR) belongs to the large G protein-coupled receptor (GPCR) superfamily. The many physiological and pharmacological responses mediated by the ?2AAR positions this particular subtype as a potential candidate for pathogenesis and a valuable target for treatment of a variety of diseases including ADHD, hypertension and depression. To date, the endogenous mechanisms whereby ?2AAR functions are regulated at the molecular and cellular levels remain elusive. GPCR interacting partners other than G proteins play pivotal roles in modulating GPCR signaling, and represent an entirely new avenue for drug development. Our previous studies identified spinophilin as an ?2AAR-interacting partner that regulates multiple aspects of ?2AAR trafficking and signaling (i.e. receptor surface retention, internalization, signal activation, desensitization and agonist sensitivity) through antagonizing arrestin functions. Furthermore, the absence of spinophilin alters the intensity and sensitivity of ?2AAR-evoked responses in mouse. The profound in vivo relevance of the ?2AAR-spinophilin interaction motivates us to explore the molecular and cellular mechanisms by which spinophilin regulates ?2AAR trafficking and signaling in native neurons, where both the ?2AAR and spinophilin elicit their major physiological functions. We have generated a novel mouse line in which N-terminal HA tagged ?2AAR expression is driven by the endogenous mouse ?2AAR locus (HA-?2AAR knock-in or ?2AAR HA/HA) and can be detected by HA antibodies. Using this unique knock-in line, in combination with the spinophilin knockout mice (Sp-/-), as well as complementary cellular and molecular strategies, we will test the central hypothesis: spinophilin regulates both the kinetics and agonist-sensitivity of ?2AAR trafficking and signaling in native neurons in order to fine-tune ?2AAR responsiveness. Specific Aim 1 will determine the role of spinophilin in regulation of agonist-induced ?2AAR endocytosis in native neurons. In addition to epinephrine (an endogenous ligand), we will study ?2AAR trafficking induced by clonidine and guanfacine (two ?2-agonists commonly used in the clinic). It would be particularly significant if spinophilin-mediated regulation of the ?2AAR exhibits agonist-bias, which would represent a potential mechanism underlying agonist-selective regulation of receptor trafficking. Specific Aim 2 will determine the role of spinophilin in regulation of ?2AAR - evoked electric (inhibition of Ca2+ currents) and biochemical (activation of ERK) responses in neurons. Specific Aim 3 will determine the role of PKA-mediated phosphorylation of spinophilin in regulation of ?2AAR trafficking and signaling in native neurons. Our newly collected preliminary data demonstrated that PKA phosphorylation of spinophilin disrupts the ?2AAR-spinophilin interaction. We will further determine the functional relevance of this PKA-mediate modulation on ?2AAR trafficking and signaling in native neurons. This revised proposal, significantly improved by our responding to the queries of the reviewers and by the inclusion of considerable additional preliminary data, will advance our understanding of endogenous regulation of the ?2AAR by spinophilin, thus providing new insight for future drug development, and in particular, for the potential design of allosteric agents acting at the ?2AAR to enhance or diminish receptor-spinophilin interactions, depending on the desired functional outcome. PUBLIC HEALTH RELEVANCE G protein-coupled receptors (GPCRs) are members of the largest family of surface receptors and represent the most abundant class of therapeutic targets. The ?2A- adrenergic receptor (?2AAR) subfamily mediates a wide range of critical physiological/pharmacological responses which include lowering blood pressure, evoking sedation, reducing pain perception, and improving working memory. Using genetic models and molecular and cellular strategies, our studies aim to understand how ?2AAR functions are tightly regulated by the interacting protein, spinophilin. Information obtained from our studies will advance our understanding of endogenous cellular mechanisms underlying ?2AAR functions, and thus provide novel insight into therapeutic strategies aimed at the treatment of attention deficit and hyperactivity disorder (ADHD), depression, hypertension and other clinical settings where use of ?2AAR-agonists may be warranted.