Transduction of cellular signals by
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. , , . The stability of the receptor–βarrestin interaction distinguishes two ARCA EGFP sale of GPCRs, termed class A and class B . 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 , .
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 . 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 , 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.
Methods and materials
Discussion The data reported herein establishes that the human CRF2(a) receptor is stringently regulated by a rapid and strong desensitization mechanism following activation by the urocortins or related stresscopin peptides but not CRF. Furthermore, UCN2 and UCN3 at saturating concentrations stimulated similar high levels of CRF2(a) receptor phosphorylation and maximal βarrestin2 recruitment while CRF2(a) receptors homologously desensitized and internalized via the dynamin/clathrin-mediated pathway to a greater degree in response to UCN2 compared to UCN3. In contrast, CRF induced very low levels of CRF2(a) receptor phosphorylation, βarrestin2 recruitment, and internalization. These findings indicate the CRF2(a) receptor adopts distinct conformations depending on the bound agonist that can modulate its βarrestin2 recruitment, subcellular trafficking and functional regulation, perhaps through differences in picketing and fencing of the cell membrane by the actin cytoskeleton known to be involved in synaptic plasticity . We also discovered that deletion of the βarrestin2 gene in MEF cells significantly upregulated Gs-coupled CRF2(a) receptor signaling activated by UCN2 and unrestrained by rapid homologous desensitization. Likewise, isoproterenol-stimulated cyclic AMP accumulation is abnormally high and prolonged in βarrestin2 knockout MEF cells expressing β2-adrenergic adrenergic receptors , and UCN2-induced CRF2(b) receptor desensitization is greatly impaired when βarrestin2 protein expression is knocked down by siRNA in HEK293 cells . Neither protein kinase A nor casein kinase mechanisms, however, contributed to CRF2(a) receptor phosphorylation and desensitization. Overall, our study suggests that the rate and magnitude of homologous desensitization of Gs-coupled signaling by the CRF2(a) receptor is governed by the affinity and selectivity of agonist-induced conformations for triggering GRK-dependent phosphorylation, βarrestin2 translocation, and dynamin/clathrin-dependent internalization.