Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.).
Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.)
Mol Pharmacol. 2015 Jan;87(1):9-17. doi: 10.1124/mol.114.094722. Epub 2014 Oct 14.
G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein βγ subunits as well as activated Gαq subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gβγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M3-acetylcholine (M3-ACh) receptor as well as Gq-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M3-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gαq [GRK2(D110A)] and Gβγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gβγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signaling and desensitization.
G 蛋白偶联受体激酶 2（GRK2）是一种丝氨酸/苏氨酸激酶，在 G 蛋白偶联受体的脱敏中具有重要功能。基于其与 G 蛋白βγ亚基以及激活的 Gαq 亚基结合的能力，它可以被认为是 G 蛋白的效应物。GRK2 被招募到激活的受体上，这被认为是由 Gβγ与带负电荷的膜磷脂共同介导的。在目前的研究中，我们研究了 Gαq 在 GRK2 与激活的 Gq 蛋白偶联受体相互作用中的作用。因此，我们建立了新的荧光共振能量转移（FRET）基础测定法，以在单个活的人胚肾 293T 细胞中以高时空分辨率研究 GRK2 与 M3-乙酰胆碱（M3-ACh）受体以及 Gq 蛋白亚基的相互作用。用 10 μM 乙酰胆碱刺激 M3-ACh 受体导致 FRET 发生明显变化，这反映了相应蛋白质的相互作用。使用对 Gαq [GRK2(D110A)]和 Gβγ [GRK2(R587Q)]的结合亲和力降低的 GRK2 突变体来确定 GRK2 与 Gq 蛋白结合的特定作用。比较绝对 FRET 幅度表明，Gαq 增强了 GRK2-M3-ACh 受体相互作用的程度和稳定性，并且不仅 Gβγ而且 Gαq 都可以将 GRK2 靶向到膜上。这揭示了 Gαq 在将 GRK2 有效募集到 M3-ACh 受体中的重要作用。此外，Gαq 和 GRK2 之间的相互作用与 GRK2 和 M3-ACh 受体之间相互作用的延长以及这些受体募集的抑制蛋白增强有关，表明 Gαq 影响信号转导和脱敏。
