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

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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