Han Sung-Jun, Hamdan Fadi F, Kim Soo-Kyung, Jacobson Kenneth A, Bloodworth Lanh M, Li Bo, Wess Jürgen
Molecular Signaling and Molecular Recognition Sections, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0810, USA.
J Biol Chem. 2005 Oct 14;280(41):34849-58. doi: 10.1074/jbc.M506711200. Epub 2005 Aug 10.
To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.
为了研究将G蛋白偶联受体（GPCRs）从静息状态转变为激活状态的构象变化，我们使用M(3)毒蕈碱型乙酰胆碱受体（一种典型的A类GPCR）作为模型系统。具体而言，我们采用了一种最近开发的原位二硫键交联策略，该策略允许在天然膜环境中存在的半胱氨酸取代的突变型M(3)毒蕈碱受体中形成二硫键。目前，关于GPCR配体在配体结合口袋紧邻区域诱导的构象变化知之甚少。为了解决这个问题，我们生成了11种半胱氨酸取代的突变型M(3)毒蕈碱受体，并在转染的COS-7细胞中对这些受体进行了表征。所有分析的突变型受体都含有一个位于跨膜结构域（TM）VII胞外侧段、靠近激动剂结合位点的内源性半胱氨酸残基（Cys-532(7.42)）。此外，所有突变型受体都含有第二个半胱氨酸残基，该残基被引入TM III的胞外侧段，位于Leu-142(3.27)-Asn-152(3.37)序列内。二硫键交联研究表明，毒蕈碱激动剂而非拮抗剂促进了S151(3.36)C与Cys-532之间二硫键的形成。M(3)毒蕈碱受体失活状态的三维模型表明，Cys-532和Ser-151在TM受体核心的中心彼此相对。因此我们的交联数据支持这样一个概念，即激动剂激活使TM VII和TM III的胞外侧段彼此更靠近。这种结构变化可能代表了触发细胞内受体表面更显著结构重组的早期构象事件之一。据我们所知，这是首次直接证明由可扩散配体激活的GPCR结合位点紧邻区域发生的构象变化。
