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: 8011736
• 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/8011736
• 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(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。？2AAR介导的许多生理和药理学反应使这个特殊的亚型成为潜在的发病机制候选者，并成为治疗包括ADHD、高血压和抑郁症在内的各种疾病的有价值的靶点。到目前为止，2AAR功能在分子和细胞水平上被调控的内源性机制仍然难以捉摸。G蛋白以外的GPCR相互作用伙伴在调节GPCR信号转导中起着关键作用，为药物开发开辟了一条全新的途径。我们以前的研究发现，Spinophlin是一个与2AAR相互作用的伙伴，通过拮抗arrestin功能来调节？2AAR运输和信号传递的多个方面(即受体表面保留、内化、信号激活、脱敏和激动剂敏感性)。此外，亲刺素的缺乏改变了小鼠？2AAR诱发反应的强度和敏感性。？2AAR-SPIN亲和素相互作用在体内的深刻相关性促使我们探索SPIN调节天然神经元中？2AAR转运和信号转导的分子和细胞机制，其中？2AAR和SPIN都是其主要的生理功能。我们建立了一种新的小鼠品系，其N端HA标记的2AAR表达是由内源性小鼠2AAR基因(HA-2AAR基因敲入或HA/HA)驱动的，并可被HA抗体检测到。利用这种独特的敲除系，结合亲刺蛋白基因敲除小鼠(Sp-/-)，以及互补的细胞和分子策略，我们将检验中心假设：亲刺蛋白调节天然神经元中？2AAR运输和信号的动力学和激动剂敏感性，以微调？2AAR的反应性。具体目标1将确定亲刺蛋白在调节激动剂诱导的天然神经元内吞？2AAR中的作用。除了肾上腺素(一种内源性配体)外，我们还将研究可乐定和胍法辛(临床上常用的两种？2-激动剂)诱导的？2AAR转运。如果亲刺素介导的？2AAR调节表现出激动剂偏向，这将是一种潜在的激动剂选择性调节受体运输的机制，这将是特别重要的。具体目标2将确定亲脊髓蛋白在调节神经元中？2AAR诱发的电(抑制钙电流)和生化(激活ERK)反应中的作用。特异靶3将确定PKA介导的亲刺素的磷酸化在调节天然神经元中？2AAR转运和信号转导中的作用。我们最新收集的初步数据表明，亲刺蛋白的PKA磷酸化破坏了？2AAR-亲刺蛋白的相互作用。我们将进一步确定这种PKA介导的对天然神经元中？2AAR运输和信号转导的功能相关性。这一修订后的建议，通过我们对评审者的提问的回应和大量额外的初步数据的包含，将促进我们对亲刺蛋白对？2AAR的内源性调节的理解，从而为未来的药物开发提供新的见解，特别是为潜在的作用于？2AAR的变构药物的设计提供新的见解，以增强或减弱受体-亲刺蛋白的相互作用，取决于所需的功能结果。与公共健康相关的G蛋白偶联受体(GPCRs)是最大的表面受体家族成员，代表着最丰富的治疗靶点。？2a-肾上腺素能受体(？2AAR)亚家族介导一系列重要的生理/药理反应，包括降低血压、引起镇静、减少痛觉和改善工作记忆。利用遗传模型以及分子和细胞策略，我们的研究旨在了解？2AAR功能是如何受到相互作用的蛋白质--亲刺蛋白的严格调控的。从我们的研究中获得的信息将促进我们对？2AAR功能的内源性细胞机制的理解，从而为旨在治疗注意力缺陷和多动障碍(ADHD)、抑郁症、高血压和其他可能需要使用？2AAR激动剂的临床环境的治疗策略提供新的见解。