Molecular basis of G protein-coupled receptor function

GPCR同源二聚体的结构与功能研究 越来越多的证据表明，GPCRs能够形成二聚体和/或更高阶的寡聚复合体。A类受体是迄今为止最大的GPCRs亚家族，由670个成员组成。一些研究表明，A类受体同源二聚体/低聚物具有不同于单体受体物种的功能特性。要正确理解A类GPCRs如何在分子水平上发挥作用，需要确定控制这类受体二聚化/寡聚化的结构元件。 为了探索A家族GPCR二聚体组装和激活的结构机制，我们以大鼠M3毒扁豆碱受体(M3R)为模型系统。对在COS-7细胞中表达的半胱氨酸取代的突变体M3Rs的研究导致了几个突变体M3Rs的鉴定，它们在氧化条件下仅以交联二聚体的形式存在。交联残基位于跨膜区5(TM5)的底部和第三胞内环(I3环)的N端部分。尿素剥离膜的研究表明，M3R二硫键交联不需要异三聚体G蛋白的存在。分子模拟研究表明，交联数据与以TM5-TM5界面为特征的低能M3R二聚体的存在有很好的一致性。功能分析显示，在I3环(264C)的N端部分交联的M3R二聚体功能严重受损(与对照M3R相比，受体-G蛋白偶联减少了50%)。这些发现表明，激动剂诱导的M3R二聚体的激活需要I3环的N末端片段的构象变化。由于所有A家族的GPCR都有高度的结构同源性，我们的数据可能具有广泛的普遍相关性。 M3乙酰胆碱受体(M3R)高分辨X射线结构 不幸的是，目前还没有关于五种M受体亚型中任何一种的高分辨率结构信息。鉴于M3R亚型介导了许多重要的生理功能，我们与斯坦福大学的Brian Kobilka博士的团队合作，专注于获得该受体的X射线结构。这种结构清楚地识别了形成配体结合口袋的所有氨基酸。此外，M3R的细胞质一侧表现出明显的结构特征，这可能与其选择性地与GQ家族的G蛋白相互作用有关。高分辨率的M3R结构很可能有助于合理设计新的M3R亚型选择性毒鼠强药物。此外，由于所有五种M受体亚型都具有高度的序列保守性，这种新的结构信息应该与整个M受体家族高度相关。

GPCR homodimers: structure-function studies Accumulating evidence suggests that GPCRs are able to form dimers and/or higher-order oligomeric complexes. Class A receptors represent by far the largest subfamily of GPCRs, consisting of 670 members in humans. Several studies suggest that class A receptor homodimers/oligomers are endowed with functional properties that differ from the monomeric receptor species. A proper understanding of how class A GPCRs function at the molecular level requires the identification of the structural elements governing the dimerization/oligomerization of this class of receptors. To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M3 muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. Functional assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (50% reduction in receptor-G protein coupling, as compared to the control M3R). These findings indicate that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Since all family A GPCRs share a high degree of structural homology, our data are likely to be of broad general relevance. High-resolution X-ray structure of the M3 muscarinic acetylcholine receptor (M3R) Unfortunately, no high-resolution structural information is currently available for any of the five muscarinic receptor subtypes. Given the many important physiological functions mediated by the M3R subtype, we, in collaboration with Dr. Brian Kobilka's group at Stanford University, focused on obtaining an X-ray structure of this receptor. We recently succeeded in solving the structure of the rat M3R bound to the bronchodilator drug, tiotropium, an inverse muscarinic agonist that is widely used clinically for the treatment of chronic obstructive pulmonary disease (COPD). This structure clearly identifies all amino acids that form the ligand binding pocket. These residues are located on the inner surfaces of several TM helices and are 100% conserved among all five muscarinic receptor subtypes, explaining why it has been so difficult to develop subtype-selective muscarinic ligands targeted at this so-called orthosteric binding site. The M3R structure also reveals several features that appear to be specific for muscarinic receptors, including a large extracellular vestibule, which is less well conserved among the five muscarinic receptors, and a bend at the top of TM4. Moreover, the cytoplasmic side of the M3R displays distinct structural features that are probably related to its ability to selectively interact with G proteins of the Gq family. It is likely that the high-resolution M3R structure will facilitate the rational design of new, M3R subtype-selective muscarinic drugs. Moreover, since all five muscarinic receptor subtypes share a high degree of sequence conservation, this new structural information should be highly relevant for the entire muscarinic receptor family.