DataSheet2_Functional optimization of light-activatable Opto-GPCRs: Illuminating the importance of the proximal C-terminus in G-protein specificity.PDF

Introduction: G-protein coupled receptors (GPCRs) are the largest family of human receptors that transmit signals from natural ligands and pharmaceutical drugs into essentially every physiological process. One main characteristic of G-protein coupled receptors is their ability to specifically couple with different families of G-proteins, thereby triggering specific downstream signaling pathways. While an abundance of structural information is available on G-protein coupled receptorn interactions with G-proteins, little is known about the G-protein coupled receptor domains functionally mediating G-protein specificity, in particular the proximal C-terminus, the structure which cannot be predicted with high confidentiality due to its flexibility.Methods: In this study, we exploited OptoGPCR chimeras between lightgated G-protein coupled receptors (opsins) and ligand-gated G-protein coupled receptors to systematically investigate the involvement of the C-terminus steering G-protein specificity. We employed rhodopsin-beta2-adrenoceptor and melanopsin-mGluR6 chimeras in second messenger assays and developed structural models of the chimeras.Results: We discovered a dominant role of the proximal C-terminus, dictating G-protein selectivity in the melanopsin-mGluR6 chimera, whereas it is the intracellular loop 3, which steers G-protein tropism in the rhodopsin-beta2-adrenoceptor. From the functional results and structural predictions, melanopsin and mGluR6 use a different mechanism to bovine rhodopsin and b2AR to couple to a selective G-protein.Discussion: Collectively, this work adds knowledge to the G-protein coupled receptor domains mediating G-protein selectivity, ultimately paving the way to optogenetically elicited specific G-protein signaling on demand.

引言：G蛋白偶联受体（G-protein coupled receptors，GPCRs）是人类最大的受体家族，它们从天然配体和药物中传递信号至几乎所有生理过程。G蛋白偶联受体的一个主要特征是它们能够与不同的G蛋白家族特异性结合，从而触发特定的下游信号通路。尽管关于G蛋白偶联受体与G蛋白相互作用的结构信息丰富，但对于功能上介导G蛋白特异性的G蛋白偶联受体结构域，特别是邻近的C端，由于其灵活性，其结构无法以高置信度进行预测。方法：在本研究中，我们利用光门控G蛋白偶联受体（视蛋白）与配体门控G蛋白偶联受体之间的OptoGPCR嵌合体，系统地研究了C端在引导G蛋白特异性中的作用。我们采用视蛋白-β2肾上腺素受体和黑视蛋白-mGluR6嵌合体进行第二信使实验，并开发了嵌合体的结构模型。结果：我们发现邻近的C端在黑视蛋白-mGluR6嵌合体中起着主导作用，决定了G蛋白的选择性，而在视蛋白-β2肾上腺素受体中，是细胞内环3引导了G蛋白的趋化性。从功能结果和结构预测来看，黑视蛋白和mGluR6与牛视蛋白和β2AR连接至选择性G蛋白的机制不同。讨论：总的来说，这项工作为介导G蛋白选择性的G蛋白偶联受体结构域增添了知识，最终为按需诱导的光遗传学G蛋白信号通路铺平了道路。