SYNERGISTIC REGULATION OF BETA(2)-ADRENERGIC RECEPTOR SEQUESTRATION -INTRACELLULAR COMPLEMENT OF BETA-ADRENERGIC-RECEPTOR KINASE AND BETA-ARRESTIN DETERMINE KINETICS OF INTERNALIZATION

Two of the common mechanisms regulating G protein-coupled receptor (GP CR) signal transduction are phosphorylation and sequestration (interna lization). Agonist-mediated receptor phosphorylation by the beta-adren ergic receptor kinase (beta ARK) facilitates subsequent interaction wi th an arrestin protein, resulting in receptor desensitization. Studies of the beta(2)-adrenergic receptor (beta(2)AR) receptor in human embr yonic kidney (HEK) 293 cells indicate that beta ARK and arrestin prote ins (beta-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the beta(2)AR to be phosphorylated by beta ARK or to intera ct normally with beta-arrestin substantially reduces agonist-mediated sequestration. To evaluate beta ARK and beta-arrestin regulation of be ta(2)AR sequestration, we examined the relationship between beta ARK a nd/or beta-arrestin expression and beta(2)AR sequestration in a variet y of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous beta-arrestin expressi on and beta(2)AR sequestration, whereas HEK 293 had the highest. Overe xpression of beta-arrestin, but not beta ARK, in COS cells increased t he extent of wild-type beta(2)AR sequestration to levels observed in H EK 293 cells. However, a beta ARK phosphorylation-impaired beta(2)AR m utant (Y326A) required the simultaneous overexpression of both beta AR K and beta-arrestin for this to occur. Among all cell lines, sequestra tion correlated best with the product of beta ARK and beta-arrestin ex pression. Moreover, an agonist-mediated translocation of wild-type bet a(2)AR and endogenous beta-arrestin 2 to endocytic vesicles prepared f rom CHO fibroblasts was observed. These data suggest not only that the complement of cellular beta ARK and arrestin proteins synergistically regulate beta(2)AR sequestration but also that beta-arrestins directl y regulate beta(2)AR trafficking as well as desensitization.