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: 8760302
• 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/8760302
• 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; Functional disorder; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; GTP-Binding Proteins; Gene Deletion; Genes; Goals; Health; 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; Selective Serotonin Reuptake Inhibitor; 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; fear memory; improved; insight; neurotransmission; novel; preclinical study; prevent; 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）： 中央CRF系统是对应激和创伤的行为、认知、HPA轴和自主反应的关键介导者。 虽然中央CRF系统激活是必要的生存在危及生命的挑战，稳态，CRF神经传递的快速反调节是同样重要的威胁终止后恢复正常的生理功能。 应激配体的CRF/尿皮质素家族通过GS偶联、Gq偶联和G蛋白非依赖性途径激活两种克隆的CRF受体CRF-R1和CRF-R2的信号传导。 包括捕食者应激范式在内的临床前研究表明，CRF-R1信号转导在诱导和维持许多与PTSD相关的行为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、过度警觉、刺激回避、惊吓过度反应、持续焦虑和巩固背景恐惧记忆。 患有PTSD的退伍军人有CRF系统功能异常的标志物，包括脑脊液CRF水平升高和HPA轴功能异常。 我们的研究表明，通过环AMP-蛋白激酶A途径的CRF-R1信号传导受到GRK-抑制蛋白2脱敏机制的强烈反调节，该机制抑制G蛋白结合并将膜受体内化到细胞溶质内体中。 我们已经证明，与野生型和<$arrestin1敲除细胞中正常的GS偶联CRF-R1信号传导相比，在表达CRF-R1的细胞中<$arrestin2基因缺失诱导CRF刺激的环AMP积累的最大值增加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，Arrestin 2基因敲除小鼠对中央CRF给药的反应表现出更大和更长的惊吓高反应性。 我们假设，CRF-R1信号的抑制蛋白2调节对于急性和长期应激恢复是必要的。 相反，我们假设，抑制蛋白2缺乏将导致持续性的 CRF-R1信号转导失调引起的过度觉醒、过度警觉、焦虑和创伤记忆。 我们将以以下方式测试我们的假设：在具体目标#1中，我们将通过抑制蛋白2机制研究G蛋白偶联和G蛋白非依赖性CRF-R1信号通路的分子调控。 我们预计，在GRK 3磷酸化CRF-R1后，抑制蛋白2将抑制和脱敏Gs-和可能的Gq-偶联信号传导。 我们还将确定抑制蛋白2是否会通过PI-3激酶-Akt级联反应独立于G蛋白偶联促进替代CRF-R1信号传导。 此外，我们将使用PDZ支架阵列和酵母双杂交筛选来鉴定调节抑制蛋白2易位/结合CRF-R1和抑制蛋白2调节的CRF-R1途径的细胞蛋白。 在具体目标#2中，我们将确定G蛋白偶联的CRF-R1信号传导的抑制蛋白2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arrestin 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信号传导，arrestin 2敲除小鼠将表现出对CRF和应激的异常增强和延长的焦虑反应。 我们研究的主要目标是对CRF-R1信号传导的异常抑制蛋白2调节在PTSD病理生理学中的作用产生重要的见解。 我们还将指导我们的研究，以确定药物发现的新分子靶点，这将导致新的PTSD治疗具有更高的疗效和治疗指数。