Freeman J L, De La Cruz E M, Pollard T D, Lefkowitz R J, Pitcher J A
Howard Hughes Medical Institute and Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1998 Aug 7;273(32):20653-7. doi: 10.1074/jbc.273.32.20653.
G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). Here we report that the cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin has previously been shown to bind to the amino terminus of GRK5 (Pronin, A. N., and Benovic, J. L. (1997) J. Biol. Chem. 272, 3806-3812) and here we show calmodulin displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates.
G蛋白偶联受体激酶（GRKs）启动导致激动剂占据的G蛋白偶联受体（GPCRs）脱敏的信号通路。在此我们报告细胞骨架蛋白肌动蛋白结合并抑制GRK5。肌动蛋白直接抑制激酶活性，减少GRK5介导的膜结合GPCRs和可溶性底物的磷酸化。GRK5与肌动蛋白单体结合的解离常数（Kd）为0.6微摩尔，与肌动蛋白丝结合的Kd为0.2微摩尔。GRK5氨基末端附近6个氨基酸的突变消除了肌动蛋白介导的对GRK5的抑制。此前已证明钙调蛋白与GRK5的氨基末端结合（普罗宁，A.N.，和贝诺维奇，J.L.（1997年）《生物化学杂志》272，3806 - 3812），在此我们表明钙调蛋白将GRK5从肌动蛋白上置换下来。钙调蛋白抑制GRK5介导的GPCRs磷酸化，但不抑制诸如酪蛋白等可溶性底物的磷酸化。因此，在肌动蛋白存在的情况下，钙调蛋白通过优先允许可溶性底物而非膜结合底物的磷酸化来决定GRK5的底物特异性。
