Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109.
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109.
Proc Natl Acad Sci U S A. 2019 Aug 6;116(32):15895-15900. doi: 10.1073/pnas.1818547116. Epub 2019 Jul 23.
G protein-coupled receptor (GPCR) kinases (GRKs) are responsible for initiating desensitization of activated GPCRs. GRK5 is potently inhibited by the calcium-sensing protein calmodulin (CaM), which leads to nuclear translocation of GRK5 and promotion of cardiac hypertrophy. Herein, we report the architecture of the Ca·CaM-GRK5 complex determined by small-angle X-ray scattering and negative-stain electron microscopy. Ca·CaM binds primarily to the small lobe of the kinase domain of GRK5 near elements critical for receptor interaction and membrane association, thereby inhibiting receptor phosphorylation while activating the kinase for phosphorylation of soluble substrates. To define the role of each lobe of Ca·CaM, we utilized the natural product malbrancheamide as a chemical probe to show that the C-terminal lobe of Ca·CaM regulates membrane binding while the N-terminal lobe regulates receptor phosphorylation and kinase domain activation. In cells, malbrancheamide attenuated GRK5 nuclear translocation and effectively blocked the hypertrophic response, demonstrating the utility of this natural product and its derivatives in probing Ca·CaM-dependent hypertrophy.
G 蛋白偶联受体激酶（GRKs）负责启动激活的 GPCR 脱敏。钙敏感受蛋白钙调蛋白（CaM）能强烈抑制 GRK5，导致 GRK5 核转位并促进心肌肥厚。在此，我们通过小角度 X 射线散射和负染电子显微镜报告了 Ca·CaM-GRK5 复合物的结构。Ca·CaM 主要与 GRK5 激酶结构域的小 lobe 结合，该 lobe 靠近对受体相互作用和膜结合至关重要的元件，从而抑制受体磷酸化，同时激活激酶对可溶性底物的磷酸化。为了确定 Ca·CaM 的每个 lobe 的作用，我们利用天然产物马布兰酰胺作为化学探针，表明 Ca·CaM 的 C 末端 lobe 调节膜结合，而 N 末端 lobe 调节受体磷酸化和激酶结构域激活。在细胞中，马布兰酰胺减弱了 GRK5 的核转位，并有效地阻止了肥厚反应，证明了这种天然产物及其衍生物在探测 Ca·CaM 依赖性肥厚中的应用。
