Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily
Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily
复制标题
DOI：
10.1074/jbc.m115.713446
复制
发表时间：
2016-03-25
影响因子：
4.8
通讯作者：
Martemyanov, Kirill A.
中科院分区：
文献类型：
--
作者：
Muntean, Brian S.;Martemyanov, Kirill A.
G蛋白信号调节因子(RGS)促进异源三聚体G蛋白失活，从而控制G蛋白偶联受体(GPCR)介导的反应的幅度和动力学。在神经系统中，RGS7和RGS9-2在视觉、奖赏处理和运动控制中起着至关重要的作用。RGS7和RGS9-2都属于RGS蛋白的R7亚家族，与R7结合蛋白(R7BP)形成大分子复合体。R7BP靶向质膜上的RGS蛋白，增强其GTP酶加速蛋白(GAP)活性，最终加速G蛋白信号的失活。然而，目前尚不清楚R7BP是仅作为膜锚定亚单位发挥作用，还是进一步调节RGS蛋白以增加其GAP活性。为了直接回答这个问题，我们利用了一种快速可逆的化学诱导蛋白质二聚化系统，使我们能够控制RGS在活细胞中的定位，而不是R7BP。为了监测G失活的动力学，我们将这一策略与在含有阿片受体的细胞系统中通过基于生物发光共振能量转移的分析测量GAP活性的变化相结合。这一方法被用来关联RGS定位的变化和R7BP存在或不存在时的活性。值得注意的是，我们观察到RGS活性通过膜招募以一种与方向无关的方式增强，而R7BP没有提供额外的贡献。这些发现认为，R7RGS蛋白与膜环境的结合对其GAP活性的调节提供了主要的直接贡献。
Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9-2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9-2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of G deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing -opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity.