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 l east in part, by their phosphorylation by the G protein-coupled recept or kinases (GRKs), However, only in the case of rhodopsin have the act ual sites of receptor phosphorylation been unambiguously determined, A lthough previous studies have implicated the cytoplasmic tail of the b eta(2)-adrenergic receptor (beta(2)AR) as the site of GRK-mediated pho sphorylation, the identities of the phosphorylated residues were unkno wn, Here we report the identification of the sites of GRK2- and GRK5-m ediated beta(2)AR phosphorylation. The phosphorylation sites of both s erine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the beta(2)AR. Of the seve n phosphorylatable residues within this peptide, sin are phosphorylate d by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) a nd four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser -407) at equivalent phosphorylation stoichiometries (similar to 1.0 mo l P-i/mol receptor), In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate b etween sites are observed for GRK2 and GRK5. Increasing the stoichiome try of GRK2-mediated beta(2)AR phosphorylation from similar to 1.0 to 5.0 mol P-i/mol receptor increases the stoichiometry of phosphorylatio n of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GR B5 phosphoacceptor sites at the extreme carboxyl terminus of the beta( 2)AR is highly reminiscent of GRK1-mediated phosphorylation of rhodops in.