Transduction of cellular signals by G protein coupled

Transduction of cellular signals by G protein-coupled receptors (GPCRs) is stringently regulated to prevent the deleterious effects of unrestrained GPCR signaling. The rapid termination of signaling mediated by agonist-occupied GPCRs is referred to as homologous desensitization and involves the following: (i) Agonist-activated receptors selectively recruit a G protein-coupled receptor kinase (GRK) that phosphorylates particular serines and/or threonines in the receptor\'s intracellular loops or carboxyl-terminus (C-terminus). (ii) Phosphorylated receptors attract cytoplasmic βarrestins to the cell membrane where they bind to receptors, uncoupling them from their cognate Gα via competition and steric hindrance. (iii) βarrestins subsequently target the receptors to clathrin-coated pits for endocytosis by interacting with clathrin and clathrin adapter protein AP-2. Internalized receptors are either sorted for dephosphorylation and recycling back to the plasma membrane or trafficked into lysosomes for degradation. [13], [14], [15]. The stability of the receptor–βarrestin interaction distinguishes two KU-0063794 synthesis of GPCRs, termed class A and class B [15]. Class A GPCRs form transient complexes with βarrestin that dissociate at or near the plasma membrane after the receptors are directed to clathrin coated pits and consequently internalize without βarrestin. Class B receptors form stable complexes with βarrestins that internalize as a unit into endocytic vesicles and persist inside the cell. The stability of the receptor–βarrestin interaction appears to regulate the rate receptors resensitize as well as the spatial-temporal pattern of βarrestin-dependent signaling pathways [14], [15]. Recently, βarrestins have been shown to contribute to desensitization and internalization of the CRF1 and CRF2(b) receptors, the peripherally expressed splice variant of the CRF2 receptor [16]. There is no information, however, about the homologous regulation of the centrally expressed CRF2(a) receptor by phosphorylation, βarrestin recruitment, and internalization mechanisms. Since abnormal signaling by both CRF receptors in the extended amygdala and forebrain may contribute to the pathophysiology of human stress, anxiety, and depressive disorders, understanding control of CRF2 receptor function could provide important insight into the pathogenesis of posttraumatic stress disorder and affective illnesses. Thus, the primary goal of this study was to investigate the desensitization profile of CRF2(a) receptors following exposure to a variety of physiological ligands. We looked not only at the desensitization of cyclic AMP signaling by the CRF2(a) receptor but also at the three primary components of the desensitization process: (i) agonist-stimulated phosphorylation, (ii) βarrestin recruitment, and (iii) receptor internalization. In addition, because we have recently shown that Y79 cells co-express both CRF1 and CRF2(a) receptors [17], we were able to study potential regulatory interactions between CRF1 and CRF2(a) receptors in an endogenous setting. Results from our study reveal that desensitization of Gs-coupled CRF2(a) receptor signaling is unaffected by CRF1 receptor activity and the serine–threonine kinases, protein kinase A and casein kinases, but strongly dependent on agonist potency and βarrestin2 recruitment.