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

G蛋白信号调节蛋白(RGS)是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B/R4亚家族，由短N末端和RGS结构域组成。生化研究表明，RGS8与GA0和GAI3特异性结合，是GAI家族的GTP酶激活蛋白。虽然RGS8对Gaq家族的亲和力很低，但我们最近发现RGS8以受体类型特异性的方式抑制GQ信号。然而，剪接变异体RGS8S显示出减弱的作用，其中N端的9个氨基酸被新的7个氨基酸取代。在这项研究中，我们重点研究了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。我们构建了与GA亲和力降低的RGS8点突变株(RGS8(L153F))，并对其…进行了研究非洲爪哇卵母细胞电生理分析对GQ偶联受体系统的更多影响。结果RGS8(L153F)不能有效地抑制任何GQ受体系统，表明RGS8的受体特异性抑制需要RGS结构域的功能。我们首次发现钙调蛋白以钙依赖的方式与RGS8的N末端结合。我们进一步研究了它与GQ受体的直接结合。我们发现RGS8直接与M1和M3的M_1和M_3受体的细胞内第三(I_3)环结合，而不与M_2受体结合，并且RGS8S的结合较弱。RGS8和M1之间的相互作用最强。然后我们鉴定了RGS8和M1受体之间的识别位点。我们制备了RGS8的N端缺失突变体，并检测了它们与M1受体的相互作用。虽然突变体缺乏5 AS与M1受体结合，但N末端9 AS的缺失导致结合活性降低，表明6-9 AS作为RGS8 N末端的重要性。此外，为了确定M1I3中的RGS8结合部位，我们还用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与所有三个部分结合，RGS8S与M1I3-N结合，弱与M1I3-M结合。因此，N端9AS是RGS8与钙调素和M1受体结合所必需的，这些相互作用在RGS8对受体的特异性抑制中起着重要作用。较少

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