Influence of Gαq on the Dynamics of M3-Acetylcholine Receptor–G-Protein–Coupled Receptor Kinase 2 Interaction
Influence of Gαq on the Dynamics of M3-Acetylcholine Receptor–G-Protein–Coupled Receptor Kinase 2 Interaction
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DOI：
10.1124/mol.114.094722
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发表时间：
2015-01
影响因子：
3.6
通讯作者：
Valerie Wolters;C. Krasel;J. Brockmann;M. Bünemann
中科院分区：
文献类型：
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作者：
Valerie Wolters;C. Krasel;J. Brockmann;M. Bünemann
G 蛋白偶联受体激酶 2 (GRK2) 是一种丝氨酸/苏氨酸激酶，在 G 蛋白偶联受体脱敏中具有重要功能。基于其结合 G 蛋白 βγ 亚基以及激活的 Gαq 亚基的能力，它可以被视为 G 蛋白的效应子。众所周知，GRK2 向激活受体的募集是由 Gβγ 和带负电荷的膜磷脂共同介导的。在当前的研究中，我们探讨了 Gαq 在 GRK2 与激活的 Gq 蛋白偶联受体相互作用中的作用。因此，我们建立了新的基于 Förster 共振能量转移 (FRET) 的检测方法，以在单个活人胚胎肾 293T 细胞中研究 GRK2 与 M3-乙酰胆碱 (M3-ACh) 受体以及高时空分辨率的 Gq 蛋白亚基的相互作用。用 10 µM 乙酰胆碱刺激 M3-ACh 受体会导致 FRET 发生明显变化，这反映了各个蛋白质的相互作用。对 Gαq [GRK2(D110A)] 和 Gβγ [GRK2(R587Q)] 结合亲和力降低的 GRK2 突变体用于确定 GRK2 与 Gq 蛋白结合的特定作用。绝对 FRET 振幅的比较表明，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 靶向膜。这揭示了 Gαq 在 GRK2 有效招募 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对抑制蛋白的募集增强有关，表明 Gαq 影响信号传导和脱敏。
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.