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: 8597932
• 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/8597932
• 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）： 中央 CRF 系统是行为、认知、HPA 轴以及对压力和创伤的自主反应的关键调节者。 虽然中枢 CRF 系统的激活对于在威胁生命的稳态挑战中生存是必要的，但 CRF 神经传递的快速反调节对于在威胁终止时恢复正常的生理功能同样重要。 CRF/尿皮质素应激配体家族通过 Gs 偶联、Gq 偶联和 G 蛋白独立途径激活两个克隆 CRF 受体 CRF-R1 和 CRF-R2 的信号传导。 包括我们的捕食者压力范例在内的临床前研究表明，CRF-R1 信号转导在许多与 PTSD 相关的行为的诱导和维持中发挥着至关重要的作用，包括压力引起的过度警觉、过度警觉、避免刺激、惊吓过度反应、持续焦虑和情境恐惧记忆的巩固。 患有 PTSD 的退伍军人具有 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 AMP-蛋白激酶 A 途径的 CRF-R1 信号传导受到 GRK-¿arrestin2 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胞质内体中。 我们已经证明，与野生型和 ¿arrestin1 敲除细胞中正常 Gs 偶联的 CRF-R1 信号传导相比，表达 CRF-R1 的细胞中 ¿arrestin2 基因缺失会导致 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，arrestin2 敲除小鼠对中枢 CRF 给药表现出更大、更持久的惊吓过度反应。 我们假设，arrestin2 对 CRF-R1 信号传导的调节对于急性和长期压力恢复是必要的。 相反，我们假设“arrestin2”缺陷将导致持续的 由于 CRF-R1 信号转导失调而导致的过度警觉、过度警觉、焦虑和创伤记忆。 我们将通过以下方式测试我们的假设：在特定目标#1中，我们将通过“arrestin2”机制检查G蛋白偶联和G蛋白独立的CRF-R1信号通路的分子调节。 我们预计，在 CRF-R1 被 GRK3 磷酸化后，arrestin2 将抑制 Gs 和可能的 Gq 偶联信号传导并使之脱敏。 我们还将确定 ¿arrestin2 是否会通过独立于 G 蛋白偶联的 PI-3 激酶-Akt 级联促进替代 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 ¡arrestin2 易位/结合 CRF-R1 和 ¡arrestin2 调节的 CRF-R1 途径的细胞蛋白。 在具体目标#2 中，我们将确定 ¿arrestin2 对 G 蛋白偶联 CRF-R1 信号传导的反向调节对于应激恢复是否至关重要。 我们将完成补充实验，确定arrestin2敲除小鼠和野生型小鼠中CRF或捕食者应激诱导的焦虑样防御行为的程度和时间过程。 我们预测，由于 Gs 和/或 Gq 耦合的 CRF-R1 信号传导不受限制，arrestin2 敲除小鼠将对 CRF 和压力表现出异常增强和延长的焦虑反应。 我们研究的主要目标是深入了解 PTSD 病理生理学中 CRF-R1 信号传导的异常 ¿arrestin2 调节的作用。 我们还将研究方向确定用于药物发现的新分子靶标，这将带来具有更高功效和治疗指数的新 PTSD 治疗方法。