MEMBERS OF THE G-PROTEIN-COUPLED RECEPTOR KINASE FAMILY THAT PHOSPHORYLATE THE BETA(2)-ADRENERGIC RECEPTOR FACILITATE SEQUESTRATION

We recently reported that a beta(2)-adrenergic receptor (beta(2)AR) mu tant, Y326A, defective in its ability to sequester in response to agon ist stimulation was a poor substrate for G protein-coupled receptor ki nase (GRK)-mediated phosphorylation; however, its ability to be phosph orylated and sequestered could be restored by overexpressing GRK2 [Fer guson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphory late and rescue the sequestration of the Y326A mutant in HEK-293 cells . We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate th e Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestrati on of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated wit h the agonist-dependent rescue of sequestration. In contrast, overexpr ession of GRK4 and -6 led mainly to agonist-independent phosphorylatio n of the Y326A mutant accompanied by increased basal receptor sequestr ation. Our results demonstrate that phosphorylation per se, but not th e interaction with a specific GRK, is required to facilitate beta(2)AR sequestration.