Fredericks Z L, Pitcher J A, Lefkowitz R J
Department of Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1996 Jun 7;271(23):13796-803. doi: 10.1074/jbc.271.23.13796.
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 beta2-adrenergic receptor (beta2AR) 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 beta2AR 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 beta2AR. 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 (approximately 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 beta2AR phosphorylation from approximately 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 beta2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin.
G蛋白偶联受体的快速脱敏至少部分是由G蛋白偶联受体激酶（GRK）使其磷酸化介导的。然而，只有视紫红质的受体磷酸化实际位点已被明确确定。尽管先前的研究表明β2 - 肾上腺素能受体（β2AR）的胞质尾是GRK介导的磷酸化位点，但磷酸化残基的具体身份尚不清楚。在此，我们报告了GRK2和GRK5介导的β2AR磷酸化位点的鉴定。这两种丝氨酸/苏氨酸激酶的磷酸化位点仅位于β2AR极端羧基末端的一个40个氨基酸的肽段中。在该肽段内的七个可磷酸化残基中，六个被GRK5磷酸化（苏氨酸-384、苏氨酸-393、丝氨酸-396、丝氨酸-401、丝氨酸-407和丝氨酸-411），四个被GRK2磷酸化（苏氨酸-384、丝氨酸-396、丝氨酸-401和丝氨酸-407），磷酸化化学计量比相当（约1.0摩尔磷酸根/摩尔受体）。除了GRK5对苏氨酸-393和丝氨酸-411的特异性磷酸化外，还观察到GRK2和GRK5在位点之间磷酸分布的差异。将GRK2介导的β2AR磷酸化化学计量比从约1.0摩尔磷酸根/摩尔受体增加到5.0摩尔磷酸根/摩尔受体，会增加苏氨酸-384、丝氨酸-396、丝氨酸-401和丝氨酸-407的磷酸化化学计量比，而不是增加磷酸受体位点的数量。β2AR极端羧基末端多个GRK2和GRK5磷酸受体位点的位置与GRK1介导的视紫红质磷酸化非常相似。
