Arrestin Regulation of CRF1 Signaling: Mechanisms for Stress Resilience and PTSD

抑制素对 CRF1 信号传导的调节：压力恢复和 PTSD 的机制

• 批准号: 8440713
• 负责人: RICHARD L HAUGER
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440713
• 关键词: 1-Phosphatidylinositol 3-Kinase; ADRBK2 gene; Acoustics; Acute; Adverse effects; Agonist; Antipsychotic Agents; Anxiety; Arrestins; BCL2L11 gene; Basal Ganglia; Behavior; Behavioral; Binding; Brain; CRF receptor type 1; CRF receptor type 2; Cell model; Cells; Cognitive; Corticotropin-Releasing Hormone; Corticotropin-Releasing Hormone Receptors; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Disease remission; Dopamine D2 Receptor; Endosomes; Environment; Event; Exhibits; Family; Fright; Functional disorder; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; GRK; GTP-Binding Proteins; Gene Deletion; Genes; Goals; Homeostasis; Knock-out; Knockout Mice; Lead; Life; Ligands; Maintenance; Mediating; Mediator of activation protein; Medical; Membrane; Memory; Metabolic; Molecular; Molecular Target; Motor; Mus; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacotherapy; Phosphorylation; Physiological; Play; Post-Traumatic Stress Disorders; Protein Binding; Protein Kinase A Inhibitor; Protein Kinase C Inhibitor; Proteins; Receptor Signaling; Recovery; Recurrence; Reflex action; Regulation; Reporting; Research; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Site-Directed Mutagenesis; Small Interfering RNA; Specificity; Stimulus; Stress; Symptoms; System; Testing; Therapeutic Index; Time; Trauma; Veterans; Wild Type Mouse; Yeasts; attenuation; biological adaptation to stress; clinical efficacy; combat; counterregulation; desensitization; design; drug discovery; improved; insight; neurotransmission; novel; preclinical study; prevent; public health relevance; receptor; receptor function; research study; response; scaffold; stress resilience; urocortin; yeast two hybrid system

DESCRIPTION (provided by applicant): The central CRF system is a critical mediator of behavioral, cognitive, HPA axis, and autonomic responses to stress and trauma. While central CRF system activation is necessary for survival during life-threatening challenges to homeostasis, rapid counterregulation of CRF neurotransmission is equally important for restoring normal physiological function upon threat termination. The CRF/urocortin family of stress ligands activates signaling of the two cloned CRF receptors CRF-R1 and CRF-R2 via Gs-coupled, Gq-coupled and G protein-independent pathways. Preclinical studies including our predators stress paradigm have shown that CRF-R1 signal transduction plays a vital role in the induction and maintenance of a number of behaviors associated with PTSD including stress-induced hyperarousal, hypervigiliance, stimuli avoidance, startle hyperreactivity, persistent anxiety, and consolidation of contextual fear memories. Combat Veterans with PTSD have markers of abnormal CRF system functioning, including elevated cerebrospinal CRF levels and abnormal HPA axis function. Our research has shown that CRF-R1 signaling via the cyclic AMP-protein kinase A pathway is strongly counterregulated by a GRK-¿arrestin2 desensitization mechanism, which inhibits G protein binding and internalizes the membrane receptor into cytosolic endosomes. We have demonstrated that a ¿arrestin2 gene deletion in CRF-R1-expressing cells induces a 5-fold increase in the maximum for CRF-stimulated cyclic AMP accumulation compared to normal Gs-coupled CRF-R1 signaling in wild-type and ¿arrestin1 knockout cells. Our preliminary behavioral experiments found that ¿arrestin2 knockout mice exhibit greater and more prolonged startle hyperreactivity in response to central CRF administration compared to CRF-enhanced startle behavior in controls. We hypothesize that ¿arrestin2 regulation of CRF-R1 signaling is necessary for acute and long-term stress recovery. Conversely, we hypothesize that a ¿arrestin2 deficiency will result in persistent hyperarousal, hypervigiliance, anxiety, and trauma memory due to dysregulated CRF-R1 signal transduction. We will test our hypotheses in the following manner: In Specific Aim #1, we will examine molecular regulation of G protein-coupled and G protein-independent CRF-R1 signaling pathways by ¿arrestin2 mechanisms. We anticipate that ¿arrestin2 will restrain and desensitize Gs- and possibly Gq-coupled signaling after CRF-R1 is phosphorylated by GRK3. We will also determine if ¿arrestin2 will promote alternative CRF-R1 signaling via the PI-3 kinase-Akt cascade independent of G protein coupling. In addition, we will use PDZ scaffold arrays and yeast two-hybrid screens to identify cellular proteins modulating ¿arrestin2 translocation/binding to CRF-R1 and ¿arrestin2-regulated CRF-R1 pathways. In Specific Aim #2, we will determine if ¿arrestin2 counter regulation of G protein-coupled CRF-R1 signaling is critical for stress recovery. We will complete complementary experiments determining the magnitude and time course of CRF- or predator stress-induced anxiety-like defensive behavior in ¿arrestin2 knockout and wild-type mice. We predict that ¿arrestin2 knockout mice will exhibit abnormally enhanced and prolonged anxiety responses to CRF and stress, due to unrestrained Gs-and/or Gq-coupled CRF-R1 signaling. The major goal of our study is to generate important insight into the role of aberrant ¿arrestin2 regulation of CRF-R1 signaling in PTSD pathophysiology. We will also direct our research toward identifying novel molecular targets for drug discovery that will lead to new PTSD treatments with higher efficacy and therapeutic index.

描述(由申请人提供)： 中枢CRF系统是行为、认知、HPA轴和自主神经对应激和创伤反应的重要中介。虽然中枢CRF系统的激活是在威胁生命的动态平衡挑战中生存所必需的，但快速的CRF神经传递的反向调节对于在威胁终止时恢复正常的生理功能同样重要。CRF/urocortin应激配体家族通过Gs偶联、Gq偶联和G蛋白非依赖性途径激活两个克隆的CRF受体CRF-R1和CRF-R2的信号。包括我们的捕食者应激范式在内的临床前研究表明，CRF-R1信号转导在PTSD相关行为的诱导和维持中起着至关重要的作用，这些行为包括应激诱导的过度觉醒、过度活力、刺激回避、惊恐高反应、持续性焦虑和情景恐惧记忆的巩固。患有创伤后应激障碍的退伍军人有CRF系统功能异常的标志，包括脑脊髓CRF水平升高和HPA轴功能异常。我们的研究表明，通过环磷酸腺苷-蛋白激酶A途径的CRF-R1信号被GRK-arrestin2脱敏机制强烈地逆转，该脱敏机制抑制G蛋白结合，并将膜受体内化到胞浆内小体。我们已经证明，在表达CRF-R1的细胞中，与野生型和arrestin1基因敲除细胞中正常的Gs偶联CRF-R1信号相比，表达CRF-R1的细胞中CRF-R1基因缺失导致CRF刺激的循环AMP积聚的最大值增加5倍。我们的初步行为实验发现，与对照组中CRF增强的惊吓行为相比，2基因敲除小鼠对中枢CRF注射的反应表现出更大、更长时间的惊吓高反应。我们假设，阻止CRF-R1信号的调节对于急性和长期的应激恢复是必要的。相反地，我们假设拦阻蛋白2缺乏会导致持续性 CRF-R1信号转导失调引起的过度觉醒、精力旺盛、焦虑和创伤记忆。我们将通过以下方式验证我们的假设：在特定的目标#1中，我们将研究G蛋白偶联和G蛋白非依赖的CRF-R1信号通路的分子调控。我们预计，在CRF-R1被GRK3磷酸化后，arrestin2将抑制和脱敏Gs-以及可能的Gq偶联信号。我们还将确定arrestin2是否会通过不依赖G蛋白偶联的PI-3激酶-Akt级联来促进替代的CRF-R1信号。此外，我们将使用PDZ支架阵列和酵母双杂交筛选来鉴定细胞蛋白质，这些蛋白质调节arrestin2转位/结合到CRF-R1，并调控CRF-R1途径。在特定的目标#2中，我们将确定抑制素2对G蛋白偶联的CRF-R1信号的反向调节是否对应激恢复至关重要。我们将完成补充实验，确定CRF或捕食者应激诱导的焦虑样防御行为在2基因敲除小鼠和野生型小鼠中的幅度和时间过程。我们预测，由于不受限制的Gs和/或Gq偶联的CRF-R1信号，2基因敲除小鼠的受阻将表现出对CRF和压力的异常增强和持续的焦虑反应。我们研究的主要目的是深入了解CRF-R1信号的异常抑制在创伤后应激障碍的病理生理学中的作用。我们还将引导我们的研究方向，为药物发现寻找新的分子靶点，从而导致具有更高疗效和治疗指数的新PTSD治疗方法。