The molecular mechanism for receptor specific suppression of Gq system by RGS8

G蛋白信号调节蛋白（RGS）包含一个调节异源三聚体G蛋白信号的大家族。RGS8是一种神经元特异性RGS蛋白，属于由短n端和RGS结构域组成的B/R4亚家族。生化研究表明，RGS8特异性结合Gao和Gai3，是Gai家族的GTPase激活蛋白。虽然RGS8对Gaq家族的亲和力较低，但我们最近发现RGS8以受体类型特异性的方式抑制Gq信号传导。然而，RGS8S，一个剪接变体，其中9个氨基酸（aa）在n端被新的7aa取代，显示出减弱的影响。本研究主要探讨RGS8对Gq系统受体特异性抑制的分子机制。我们首先研究了RGS8是否可能需要RGS8的GAP功能以受体类型特异性的方式抑制Gq信号。我们构建了一个对Ga亲和力降低的RGS8点突变体（RGS8(L153F)），并通过电生理分析研究了其对爪蟾卵母细胞Gq偶联受体系统的影响。结果表明，RGS8（L153F）不能有效抑制任何gq受体系统，表明RGS8抑制受体特异性需要RGS结构域的功能。接下来，我们研究了RGS8和Gq信号分子之间直接相互作用的可能性。我们首先发现钙调蛋白以Ca^<2+>依赖的方式结合到RGS8的特定n端。我们进一步研究了其与gq受体的直接结合。我们发现RGS8直接结合M1和M3毒蕈碱乙酰胆碱受体的第三胞内（i3）环，而不与M2受体结合，RGS8S的结合较弱。RGS8与M1的相互作用最强。然后我们对RGS8和M1受体之间的识别位点进行了表征。我们制作了RGS8的n端缺失突变体，并检测了它们与M1受体的相互作用。虽然缺乏5的突变体与M1受体结合，但n端9的缺失导致结合活性降低，这表明6-9作为RGS8的n端很重要。此外，为了确定RGS8在m1i3中的结合位点，我们还使用m1i3的三个部分（m1i3 - n, m1i3 -M, m1i3 - c）的gst融合蛋白进行了下拉实验。RGS8与这三个部分均结合，而RGS8S与m1i3 - n结合，与m1i3 -M结合较弱。因此，我们发现n端9对于RGS8与钙调素和M1受体的结合至关重要，并且这些相互作用在RGS8对受体的特异性抑制中发挥重要作用。少

RGS (regulator of G protein signaling) proteins comprise a large family that modulates heterotrimeric G protein signaling. RGS8 is a neuron-specific RGS protein, which belongs to the B/R4 subfamily composed of the short N-terminus and the RGS domain. Biochemical studies indicated that RGS8 specifically binds to Gao and Gai3, and that it functions as a GTPase activating protein for Gai family. Although RGS8 has low affinity to Gaq family, we recently found that RGS8 suppressed Gq signaling in a receptor type-specific manner. However, RGS8S, a splice variant in which 9 amino acids (aa) at the N-terminus is replaced with novel 7 aa, showed a diminished effect. In this study, we focused on the molecular mechanism for receptor specific suppression of Gq system by RGS8.We first examined whether RGS8 may require GAP function of RGS8 to suppress Gq signalling in a receptor type specific manner. We generated a point mutant of RGS8 with reduced affinity to Ga ( RGS8(L153F)), and investigated its … More effects on Gq -coupled receptor systems using electrophysiological analysis of Xenopus oocytes. Result that RGS8(L153F) could not efficiently inhibit any Gq-receptor systems, demonstrated that functions of the RGS domain are required for the receptor-specific suppression by RGS8.We next examined the possibility of the direct interaction between RGS8 and Gq signaling molecules. We first found that calmodulin binds to the specific N-terminus of RGS8 in a Ca^<2+>-dependent manner. We further studies the direct binding with Gq-receptors. We found that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors but not M2 receptor, and binding of RGS8S is weaker. The interaction between RGS8 and M1 is most strong. We then characterized the recognition sites between RGS8 and M1 receptor. We made N-terminal deletion mutants of RGS8 and examined their interaction with M1 receptor. Although a mutant lacking 5 as bound to M1 receptor, deletion of N-terminal 9 as resulted in the reduced binding activity, indicating the importance of 6-9 as of the N-terminus of RGS8. Further, to identify RGS8 binding sites in M 1i3, we also performed pull down assay using GST-fusion proteins of three parts of M 1i3 (M 1i3-N, M 1i3-M, M 1i3-C). RGS8 bound to all three parts, whereas RGS8S bound to M 1i3-N and weakly to M 1i3-M. Thus, it was revealed that the N-terminal 9 as was essential for binding of RGS8 to calmodulin and M1 receptor, and these interactions must play a significant role in receptor specific suppression by RGS8. Less