Desensitization of β2-Adrenergic Receptors with Mutations of the Proposed G Protein-coupled Receptor Kinase Phosphorylation Sites*

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the β2-adrenergic receptor (βAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796–13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed βARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants βARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 μmepinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89–94% after 5 min of 10 μmepinephrine stimulation and 96–98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant βARs relative to the WT in the extent of 10 μmepinephrine-induced internalization (77–82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA− mutant βARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivoGRK-dependent desensitization of the βAR.