Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta(2)-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 beta(2)-adrenergic receptor (beta(2)AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown, Here we report the identification of the sites of GRK2- and GRK5-mediated beta(2)AR 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 beta(2)AR. Of the seven phosphorylatable residues within this peptide, sin are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (similar to 1.0 mol P-i/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 beta(2)AR phosphorylation from similar to 1.0 to 5.0 mol P-i/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 GRB5 phosphoacceptor sites at the extreme carboxyl terminus of the beta(2)AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.