Wess J, Bonner T I, Brann M R
Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Mol Pharmacol. 1990 Dec;38(6):872-7.
The cloning and expression of five mammalian muscarinic receptor genes (m1-m5) have shown that the individual receptor subtypes differ in their functional and ligand-binding properties. To study the role of the carboxyl terminal receptor domains in this pharmacological diversity, we constructed chimeric m2/m3 receptors in which a region comprising part of transmembrane domain VI, the third extracellular loop, transmembrane region VII, and the cytoplasmic tail (collectively referred to as C-terminal domains) was exchanged between the human m2 and the rat m3 receptor. The ability of the cloned receptors to mediate stimulation of phosphoinositide hydrolysis and to bind subtype-selective muscarinic ligands was studied after their transient expression in COS-7 cells. Whereas wild-type m3 strongly stimulated phosphoinositide breakdown, wild-type m2 gave only a poor response. Exchange of the C-terminal domains between m2 and m3 had no significant effect on the magnitude of these responses. In N-[3H]methylscopolamine competition binding studies, the muscarinic antagonists AF-DX 116 and methoctramine showed 11- and 23-fold higher affinities, respectively, for m2 than for m3, whereas hexahydro-silad-ifenidol (HHSiD) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) displayed the reverse selectivity profile, having approximately 10-fold higher affinities for m3. In comparison with wild-type m3, the mutant m3 receptor containing the C-terminal domains of m2 displayed 2.5- and 8-fold higher affinities for AF-DX 116 and methoctramine but 7- and 3-fold lower affinities for HHSiD and 4-DAMP, respectively. The mutant m2 receptor with the C-terminal domains of m3 showed 2-3-fold lower affinities for AF-DX 116 and methoctramine but 2-3-fold higher affinities for HHSiD and 4-DAMP, as compared with wild-type m2. These data suggest that the C-terminal domains of the muscarinic receptors are not involved in conferring selectivity of coupling to phosphoinositide hydrolysis but contain major structural determinants of antagonist binding selectivity.
五个哺乳动物毒蕈碱受体基因（m1 - m5）的克隆和表达表明，各个受体亚型在功能和配体结合特性上存在差异。为了研究羧基末端受体结构域在这种药理学多样性中的作用，我们构建了嵌合的m2/m3受体，其中包含跨膜结构域VI的一部分、第三个细胞外环、跨膜区域VII和细胞质尾巴（统称为C末端结构域）的区域在人m2和大鼠m3受体之间进行了交换。在COS - 7细胞中瞬时表达后，研究了克隆受体介导磷酸肌醇水解刺激和结合亚型选择性毒蕈碱配体的能力。野生型m3强烈刺激磷酸肌醇分解，而野生型m2的反应则很差。m2和m3之间C末端结构域的交换对这些反应的幅度没有显著影响。在N - [³H]甲基东莨菪碱竞争结合研究中，毒蕈碱拮抗剂AF - DX 116和甲奥克太明对m2的亲和力分别比对m3高11倍和23倍，而六氢硅代二苯乙内酰脲（HHSiD）和4 - 二苯基乙酰氧基 - N - 甲基哌啶甲碘化物（4 - DAMP）则表现出相反的选择性，对m3的亲和力高约10倍。与野生型m3相比，含有m2 C末端结构域的突变型m3受体对AF - DX 116和甲奥克太明的亲和力分别高2.5倍和8倍，但对HHSiD和4 - DAMP的亲和力分别低7倍和3倍。与野生型m2相比，具有m3 C末端结构域的突变型m2受体对AF - DX 116和甲奥克太明的亲和力低2 - 3倍，但对HHSiD和4 - DAMP的亲和力高2 - 3倍。这些数据表明，毒蕈碱受体的C末端结构域不参与赋予与磷酸肌醇水解偶联的选择性，但包含拮抗剂结合选择性的主要结构决定因素。
