Regulation of alpha2A-adrenergic receptor signaling and trafficking by spinophili

嗜棘蛋白对 α2A 肾上腺素受体信号传导和运输的调节

• 批准号: 8196966
• 负责人: Qin Wang
• 金额: $ 35.89万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-27 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196966
• 关键词: Abbreviations; Absence of pain sensation; Adrenergic Receptor; Agonist; Antibodies; Anxiety; Arrestins; Attention deficit hyperactivity disorder; Biochemical; Cells; Characteristics; Clinic; Clinical; Clonidine; Cognition; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Dose; Drug Delivery Systems; Endocytosis; Epinephrine; Exhibits; Family; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic Models; Goals; Guanfacine; Health; Hypertension; Hypotension; Intervention; Kinetics; Knock-in Mouse; Knockout Mice; Knowledge; Ligands; Linear Models; Mediating; Mental Depression; Molecular; Mus; N-terminal; Neurons; Pain; Pathogenesis; Perception; Pharmacology; Phosphorylation; Physiological; Play; Positioning Attribute; Proteins; Published Comment; Receptor Mediated Signal Transduction; Receptor Signaling; Regulation; Role; Sedation procedure; Short-Term Memory; Signal Transduction; Specificity; Surface; System; Testing; Therapeutic; Time; base; desensitization; design; drug development; functional outcomes; improved; in vivo; insight; member; novel; receptor; receptor coupling; response; spinophilin; stoichiometry; therapeutic target; tool; trafficking

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.

描述（由申请人提供）：该？2A-肾上腺素能受体（2A-adrenergic receptor，AR）属于大G蛋白偶联受体（great G protein-coupled receptor，GPCR）超家族。许多生理和药理反应介导的？2AAR将这种特殊的亚型定位为发病机制的潜在候选者和治疗包括ADHD、高血压和抑郁症在内的多种疾病的有价值的靶点。到目前为止，内源性机制，其中？2AAR功能在分子和细胞水平上的调节仍然是难以捉摸的。除了G蛋白之外的GPCR相互作用伴侣在调节GPCR信号传导中起着关键作用，并且代表了药物开发的全新途径。我们以前的研究确定spinophilin作为一个？2AAR相互作用的合作伙伴，调节多个方面？2AAR通过拮抗arrestin功能运输和信号传导（即受体表面保留、内化、信号激活、脱敏和激动剂敏感性）。此外，没有spinophilin改变的强度和敏感性？2小鼠AAR诱发反应。在体内的深刻的相关性？2AAR与spinophilin的相互作用促使我们探索spinophilin调控的分子和细胞机制。2AAR贩运和信号在天然神经元，在这两个？2AAR和spinophilin的主要生理功能。我们已经产生了一种新的小鼠线，其中N-末端HA标记？2AAR的表达是由内源性小鼠？2AAR位点（HA-？2AAR敲入还是？2AAR HA/HA），并且可以通过HA抗体检测。使用这种独特的敲入线，在与spinophilin基因敲除小鼠（Sp-/-），以及互补的细胞和分子策略相结合，我们将测试中心的假设：spinophilin调节动力学和激动剂敏感性？2AAR运输和信号在天然神经元，以微调？2AAR响应。具体目标1将确定在调节激动剂诱导的？2天然神经元中的AAR内吞作用。除了肾上腺素（内源性配体），我们将研究？2可乐定和胍法辛（2？2-临床上常用的激动剂）。这将是特别重要的，如果spinophilin介导的调节？2AAR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。具体目标2将确定的作用，在调节？2AAR -神经元中诱发的电（Ca 2+电流的抑制）和生化（ERK的激活）反应。具体目标3将确定的作用PKA介导的磷酸化的spinophilin调节？2AAR在天然神经元中的运输和信号传导。我们新收集的初步数据表明，PKA磷酸化的spinophilin破坏？2AAR-spinophilin相互作用。我们将进一步确定这种PKA介导的调制功能的相关性？2AAR在天然神经元中的运输和信号传导。这一修订后的建议，大大改善了我们的答复的询问的审查，并列入了相当多的额外的初步数据，将推进我们的理解内源性调节的？2AAR的spinophilin，从而提供了新的见解，为未来的药物开发，特别是潜在的设计变构剂作用于？2AAR增强或减弱受体-spinophilin相互作用，这取决于所需的功能结果。G蛋白偶联受体（GPCR）是最大的表面受体家族成员，代表了最丰富的一类治疗靶点。什么？2A-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、引起镇静、减少疼痛感知和改善工作记忆。使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质spinophilin的严格调节。从我们的研究中获得的信息将促进我们对内源性细胞机制的理解？2AAR功能，从而提供了新的洞察治疗策略，旨在治疗注意缺陷多动障碍（ADHD），抑郁症，高血压和其他临床环境中使用？2AAR激动剂可能是必要的。