title: Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and beta-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses.
abstract: The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF(1)) receptor phosphorylation is required for beta-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to beta-arrestin recruitment of motifs in the CRF(1) receptor carboxyl terminus and third intracellular loop. beta-Arrestin-2 translocated significantly more rapidly than beta-arrestin-1 to agonist-activated membrane CRF(1) receptors in multiple cell lines. Although CRF(1) receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF(1) receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF(1) receptor internalization. To investigate molecular determinants mediating the robust beta-arrestin-2-CRF(1) receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF(1) receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired beta-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF(1) receptor phosphorylation but did not alter beta-arrestin-2 recruitment. Phosphorylation was abolished in a CRF(1) receptor possessing both mutations. Surprisingly, this mutant still recruited beta-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 beta-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 beta-arrestin-2-CRF(1) receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF(1) receptor signaling.
authors.affiliation: Department of Psychiatry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0603, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Oakley
authors.forename: J Alberto
authors.initials: JA
authors.lastname: Olivares-Reyes
authors.forename: Christine C
authors.initials: CC
authors.lastname: Hudson
authors.forename: Fabiola
authors.initials: F
authors.lastname: Flores-Vega
authors.forename: Frank M
authors.initials: FM
authors.lastname: Dautzenberg
authors.forename: Richard L
authors.initials: RL
authors.lastname: Hauger
mesh_terms: D000595:Amino Acid Sequence; D001007:Anxiety / Q000503:physiopathology*; D019390:Arrestins / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D000939:Epitopes / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D010766:Phosphorylation; D021381:Protein Transport; D018019:Receptors, Corticotropin-Releasing Hormone / Q000819:agonists / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D013315:Stress, Psychological / Q000503:physiopathology*; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D000939:Epitopes; D018019:Receptors, Corticotropin-Releasing Hormone; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1; D000242:Cyclic AMP