Yamashita Takahiro, Terakita Akihisa, Kai Toshihiro, Shichida Yoshinori
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
J Neurochem. 2008 Jul;106(2):850-9. doi: 10.1111/j.1471-4159.2008.05443.x. Epub 2008 Apr 28.
G protein-coupled receptors are classified into several families on the basis of their amino acid sequences and the members of the same family exhibit sequence similarity but those of different families do not. In family 1 GPCRs such as rhodopsin and adrenergic receptor, extensive studies have revealed the stimulus-dependent conformational change of the receptor: the rearrangement of transmembrane helices III and VI is essential for G protein activation. In contrast, in family 3 GPCRs such as metabotropic glutamate receptor (mGluR), the inter-protomer relocation upon ligand binding has been observed but there is much less information about the structural changes of the transmsmbrane helices and the cytoplasmic domains. Here we identified constitutively active mutation sites at the cytoplasmic borders of helices II and IV of mGluR8 and successfully inhibited the G protein activation ability by engineering disulfide cross-linking between these cytoplasmic regions. The analysis of all possible single substitution mutants of these residues revealed that some steric interactions around these sites would be important to keep the receptor protein inactive. These results provided the model that the conformational changes at the cytoplasmic ends of helices II and IV of mGluR are involved in the efficient G protein coupling.
G蛋白偶联受体根据其氨基酸序列被分为几个家族，同一家族的成员表现出序列相似性，而不同家族的成员则没有。在第1类GPCR中，如视紫红质和肾上腺素能受体，广泛的研究揭示了受体的刺激依赖性构象变化：跨膜螺旋III和VI的重排对于G蛋白激活至关重要。相比之下，在第3类GPCR中，如代谢型谷氨酸受体（mGluR），已观察到配体结合时原聚体间的重新定位，但关于跨膜螺旋和细胞质结构域的结构变化的信息要少得多。在这里，我们在mGluR8的螺旋II和IV的细胞质边界处鉴定出组成型活性突变位点，并通过在这些细胞质区域之间构建二硫键交联成功抑制了G蛋白激活能力。对这些残基的所有可能的单取代突变体的分析表明，这些位点周围的一些空间相互作用对于保持受体蛋白无活性很重要。这些结果提供了一个模型，即mGluR的螺旋II和IV的细胞质末端的构象变化参与了有效的G蛋白偶联。
