Identification of the G Protein-coupled Receptor Kinase Phosphorylation Sites in the Human β2-Adrenergic Receptor

doi:10.1074/jbc.271.23.13796

Identification of the G Protein-coupled Receptor Kinase Phosphorylation Sites in the Human β₂-Adrenergic Receptor*

From the Departments of Biochemistry and Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

^‡To whom correspondence should be addressed:
Duke University Medical Center, Dept. of Medicine, Box 3821, Durham, NC 27710.
Tel.: 919-684-2974; Fax: 919-684-8875.

Abstract

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 β₂-adrenergic receptor (β₂AR) 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 β₂AR 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 β₂AR. Of the seven phosphorylatable residues within this peptide, six 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 (~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 β₂AR phosphorylation from ~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 GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the β₂AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

β₂AR

β₂-adrenergic receptor

C-tail

cytoplasmic tail

G_βγ

G protein βγ subunits

GRK

G protein-coupled receptor kinase

HPLC

high performance liquid chromatography.
↵²There are six identified subtypes of GRKs: rhodopsin kinase or GRK1; β-adrenergic receptor kinase 1 or GRK2; β-adrenergic receptor kinase 2 or GRK3; GRK4; GRK5; and GRK6.
- Received December 22, 1995.
- Revision received March 12, 1996.