Kunapuli P, Gurevich V V, Benovic J L
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1994 Apr 8;269(14):10209-12.
G protein-coupled receptor kinases (GRKs) play an important role in mediating agonist-specific desensitization of numerous G protein-coupled receptors. GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction. Phosphoamino acid determination and mutagenesis studies demonstrate that autophosphorylation of GRK5 occurs primarily at residues Ser-484 and Thr-485. Expression and characterization of a mutant GRK5 that does not autophosphorylate (S484A and T485A) reveals that the mutant has a approximately 15-20-fold reduced ability to phosphorylate the beta 2-adrenergic receptor and rhodopsin compared to wild type GRK5. These results suggest that phospholipid-stimulated autophosphorylation may represent a novel mechanism for membrane association and regulation of GRK5 activity.
G蛋白偶联受体激酶（GRKs）在介导众多G蛋白偶联受体的激动剂特异性脱敏过程中发挥着重要作用。GRK5是GRK家族最近鉴定出的成员，它会经历快速的磷脂刺激的自磷酸化，达到约2摩尔磷酸盐/摩尔GRK5的化学计量比。磷脂刺激自磷酸化的能力在很大程度上被一种含有GRK5最后102个氨基酸（氨基酸489 - 590）的谷胱甘肽S - 转移酶融合蛋白所阻断，这表明这是参与GRK5/磷脂相互作用的主要区域。磷酸氨基酸测定和诱变研究表明，GRK5的自磷酸化主要发生在丝氨酸484和苏氨酸485残基处。对不进行自磷酸化的突变型GRK5（S484A和T485A）的表达和特性分析表明，与野生型GRK5相比，该突变体使β2 - 肾上腺素能受体和视紫红质磷酸化的能力降低了约15 - 20倍。这些结果表明，磷脂刺激的自磷酸化可能代表了一种GRK5膜结合和活性调节的新机制。
