From the Department of Neuroscience, The Scripps Research Institute Florida, Jupiter, Florida 33410.
From the Department of Neuroscience, The Scripps Research Institute Florida, Jupiter, Florida 33410.
J Biol Chem. 2013 Aug 30;288(35):25129-25142. doi: 10.1074/jbc.M113.462283. Epub 2013 Jul 15.
Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.
G 蛋白信号调节蛋白（RGS 蛋白）在 G 蛋白偶联受体（GPCR）信号转导的调节中发挥着重要作用。由于存在数以百计的 GPCR 和数十种 G 蛋白，了解 RGS 如何调节选择性 GPCR-G 蛋白信号转导非常重要。在纹状体神经元中，两种 RGS 蛋白 RGS7 和 RGS9-2 调节 μ-阿片受体（MOR）和多巴胺 D2 受体（D2R）的信号转导，并与药物成瘾、运动障碍和痛觉过敏有关。这两种蛋白与非典型 G 蛋白 β 亚基 Gβ5 和膜锚定蛋白 R7BP 形成三聚体复合物。在这项研究中，我们使用基于生物发光共振能量转移的测定法，在活细胞中检查了 RGS7 和 RGS9-2 复合物的 GTPase 加速蛋白（GAP）活性以及 Gα 和 GPCR 选择性，该测定法监测 G 蛋白亚基的解离。我们表明，RGS9-2/Gβ5 调节 Gi 和 Go，偏向 Go，但 RGS7/Gβ5 只能作为 Go 的 GAP。有趣的是，R7BP 增强了 RGS7 和 RGS9-2 对 Go 和 Gi 的 GAP 活性，并使 RGS7 能够调节 Gi 信号转导。无论是在存在还是不存在 R7BP 的情况下，RGS7 或 RGS9-2 对 Gz、Gs 或 Gq 都没有任何活性。我们也没有观察到 GPCR（MOR 和 D2R）对 R7 RGS 蛋白的 G 蛋白偏向性有任何影响。然而，RGS9-2 的 GAP 活性对 D2R 比对 MOR 表现出强烈的受体偏好。最后，RGS7 的 GAP 活性相对于 RGS9-2 高出四倍。这些发现说明了纹状体 RGS 蛋白建立 G 蛋白和 GPCR 选择性的原则。
