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.
Only 20% of PTSD patients achieve sustained remission with current medications. Cutting-edge research has successfully targeted ?arrestin2-regulated GPCR pathways to develop drugs with high selectivity, specificity and efficacy for treating medical illnesses with minimal side effects. ?arrestin2 regulation of CRF1 receptor signal transduction prevents over-activation of the CRF system that coordinates behavioral, cognitive, autonomic and HPA stress responses. PTSD patients exhibit brain CRF hypersecretion and HPA dysregulation. We have shown that a ?arrestin2 deficiency magnifies Gs-coupled CRF-R1 signaling in cells and CRF-treated ?arrestin2 knockout mice exhibit startle hyperreactivity, a cardinal symptom of PTSD. We posit that unrestrained CRF-R1 signaling due to deficient ?arrestin2 regulation is important for onset and chronicity of PTSD. Our goal is to find novel molecular targets for PTSD drug discovery by elucidating ?arrestin2-regulated CRF-R1 signaling pathways involved in trauma-induced anxiety and stress responses.