Identification of the G Protein-coupled Receptor Kinase Phosphorylation Sites in the Human b2-Adrenergic Receptor*

Rapid desensitization of G protein-coupled receptors is mediated, at least in part, by their phosphorylation by the G protein-coupled receptor kinases (GRKs). However, only in the case of rhodopsin have the actual sites of receptor phosphorylation been unambiguously determined. Although previous studies have implicated the cytoplasmic tail of the b2-adrenergic receptor (b2AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown. Here we report the identification of the sites of GRK2and GRK5-mediated b2AR phosphorylation. The phosphorylation sites of both serine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the b2AR. Of the seven phosphorylatable residues within this peptide, six are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (;1.0 mol Pi/mol receptor). In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate between sites are observed for GRK2 and GRK5. Increasing the stoichiometry of GRK2-mediated b2AR phosphorylation from ;1.0 to 5.0 mol Pi/mol receptor increases the stoichiometry of phosphorylation of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the b2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.