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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Carboxyl-terminal and intracellular loop sites for CRF₁ receptor phosphorylation and -arrestin-2 recruitment: a mechanism regulating stress and anxiety responses

¹Xsira Pharmaceuticals, Morrisville, North Carolina; ²Departamento de Bioquimica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City, Mexico; ³Johnson & Johnson Pharmaceuticals Research & Development, CNS Drug Discovery, Beerse, Belgium; and ⁴Veterans Affairs Healthcare System and Department of Psychiatry, University of California, San Diego, La Jolla, California

Submitted 7 February 2006 ; accepted in final form 27 February 2007

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 CRF₁ 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 CRF₁ receptor conformation. Deficient -arrestin-2-CRF₁ receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF₁ receptor signaling.

corticotropin-releasing factor; G protein-coupled receptor kinase; receptor phosphorylation; internalization; stress adaptation

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