Carboxyl-terminal and intracellular loop sites for CRF₁receptor phosphorylation and β-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses

Abstract

Oakley RH, Olivares-Reyes JA, Hudson CC, Flores-Vega F, Dautzenberg FM, Hauger RL. Carboxyl terminal and intracellular loop sites for CRF1 receptor phosphorylation and ␤-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses. Am J Physiol Regul Integr Comp Physiol 293: R209-R222, 2007. First published March 15, 2007; doi:10.1152/ajpregu.00099.2006.-The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for ␤-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to ␤-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. ␤-Arrestin-2 translocated significantly more rapidly than ␤-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust ␤-arrestin-2-CRF1 receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired ␤-arrestin-2 recruitment. Removal of a serine/ threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter ␤-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited ␤-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF 1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in ␤-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF 1 receptor conformation. Deficient ␤-arrestin-2-CRF 1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF 1 receptor signaling.corticotropin-releasing factor; G protein-coupled receptor kinase; receptor phosphorylation; internalization; stress adaptation THE MAGNITUDE AND DURATION of cellular signals transduced by G protein-coupled receptors (GPCRs) depend upon stringent regulation to prevent the deleterious effects of unrestrained receptor activation (12,26,27,31,45,50,55). Many studies have shown that agonist-induced GPCR signaling is rapidly attenuated by a mechanism termed "homologous desensitization." According to the classic model of homologous desensitization, agonist-activated receptors are phosphorylated by a family of G protein-coupled receptor...

Molecular Mechanisms of Corticotropin-Releasing Factor Receptor-Induced Calcium Signaling

“…The system has been further applied to studying CRF receptor internalization and ␤-arrestin recruitment (Oakley et al, 2007;Markovic et al, 2008), as well as Ca 2ϩ mobilization (Dautzenberg et al, 2004). To better understand the underlying mechanisms of this mobilization, we checked the involvement of the different potential Ca 2ϩ sources and the contribution of different signaling pathways.…”

Functional characterization of kurtz, a Drosophila non-visual arrestin, reveals conservation of GPCR desensitization mechanisms

“…In mammals, the arrestins are thought to contribute to stress adaptation due to interactions with CRF receptors (Oakley et al, 2007). Fig.…”

Sex differences in stress responses: a critical role for corticotropin-releasing factor

“…One cellular mechanism that compensates for excessive CRF release is receptor internalization. During internalization, β-arrestin2 binds to the CRF 1 receptor, initiating its trafficking from the plasma membrane to the cytosol where the receptor can no longer be activated [76][77][78][79][80]. In male rats, acute swim stressor exposure causes β-arrestin2 to bind to the CRF 1 receptor, an effect accompanied by CRF 1 receptor internalization in LC dendrites [81,82].…”