Synergistic Regulation of b2-Adrenergic Receptor Sequestration: Intracellular Complement of b-Adrenergic Receptor Kinase and b-Arrestin Determine Kinetics of Internalization

Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the b-adrenergic receptor kinase (bARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the b2-adrenergic receptor (b2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that bARK and arrestin proteins (b-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the b2AR to be phosphorylated by bARK or to interact normally with b-arrestin substantially reduces agonist-mediated sequestration. To evaluate bARK and b-arrestin regulation of b2AR sequestration, we examined the relationship between bARK and/or b-arrestin expression and b2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous b-arrestin expression and b2AR sequestration, whereas HEK 293 had the highest. Overexpression of b-arrestin, but not bARK, in COS cells increased the extent of wild-type b2AR sequestration to levels observed in HEK 293 cells. However, a bARK phosphorylation-impaired b2AR mutant (Y326A) required the simultaneous overexpression of both bARK and b-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of bARK and b-arrestin expression. Moreover, an agonist-mediated translocation of wild-type b2AR and endogenous b-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular bARK and arrestin proteins synergistically regulate b2AR sequestration but also that b-arrestins directly regulate b2AR trafficking as well as desensitization.