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

RGS8受体特异性抑制Gq系统的分子机制

基本信息

批准号：
15570132
负责人：
SAITOH Osamu
金额：
$ 2.37万
依托单位：
Nagahama Institute of Bio-Science and Technology (2004)Tokyo Metropolitan Organization for Medical Research (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570132/
关键词：
RGS G protein receptor Gq intracellular Ca^<2+>cerebellum Purkinje cells Gタンパク

项目摘要

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

RGS（G蛋白信号传导调节子）蛋白包括调节异源三聚体G蛋白信号传导的大家族。RGS 8是一种神经元特异性的RGS蛋白，属于B/R4亚家族，由短的N-末端和RGS结构域组成。生物化学研究表明RGS 8与Gao和Gai 3特异性结合，是Gai家族的GT3激活蛋白。虽然RGS 8与Gaq家族的亲和力较低，但我们最近发现RGS 8以受体类型特异性的方式抑制Gq信号传导。然而，RGS 8 S，一种剪接变体，其中N-末端的9个氨基酸（aa）被新的7个aa取代，显示出减弱的作用。本研究旨在探讨RGS 8受体特异性抑制Gq系统的分子机制。首先，我们研究了RGS 8是否需要GAP功能才能以受体类型特异性的方式抑制Gq信号。我们产生了RGS 8的点突变体，其对Ga的亲和力降低（RGS 8（L153 F）），并研究了其对Ga的亲和力。 ...更多信息用非洲爪蟾卵母细胞的电生理学分析对Gq偶联受体系统的影响。结果表明RGS 8（L153 F）不能有效抑制任何Gq受体系统，说明RGS 8的受体特异性抑制需要RGS结构域的功能。我们进一步研究了RGS 8与Gq信号分子直接相互作用的可能性。我们首次发现钙调素以Ca^2+依赖性方式与RGS 8的特异性N端结合。我们进一步研究了与Gq受体的直接结合。我们发现，RGS 8直接结合M1和M3毒蕈碱乙酰胆碱受体的第三胞内（i3）环，但不结合M2受体，而RGS 8 S的结合较弱。RGS 8与M1的相互作用最强。然后，我们表征了RGS 8和M1受体之间的识别位点。我们制作了RGS 8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RGS 8的N-末端6-9 as的重要性。此外，为了鉴定M1 i3中的RGS 8结合位点，我们还使用M1 i3的三个部分（M1 i3-N、M1 i3-M、M1 i3-C）的GST融合蛋白进行下拉测定。RGS 8与所有三个部分结合，而RGS 8 S与M113-N结合并且与M113-M弱结合。因此，揭示了N-末端9as对于RGS 8与钙调蛋白和M1受体的结合是必不可少的，并且这些相互作用在RGS 8的受体特异性抑制中一定起重要作用。少