Tan Karen, Pogozheva Irina D, Yeo Giles S H, Hadaschik Dirk, Keogh Julia M, Haskell-Leuvano Carrie, O'Rahilly Stephen, Mosberg Henry I, Farooqi I Sadaf
University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke's Hospital, Box 289, Hills Road, Cambridge CB2 2QQ, United Kingdom.
Endocrinology. 2009 Jan;150(1):114-25. doi: 10.1210/en.2008-0721. Epub 2008 Sep 18.
Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the beta2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease.
黑皮质素4受体（MC4R）基因的突变是已知的单基因人类肥胖最常见的病因。对2000名重度早发性肥胖受试者的MC4R基因进行了测序。我们在总共94名先证者中检测到7种不同的无义突变和19种错义突变，其中一些先前已被其他人报道过。我们对11种新的与肥胖相关的错义突变进行了功能表征。这些突变体中的7种（L54P、E61K、I69T、S136P、M161T、T162I和I269N）表现出细胞表面转运受损、放射性配体[125I]NDP-MSH的最大结合水平降低以及对配体产生cAMP的能力降低。4种突变型MC4R（G55V、G55D、S136F和A303T）表现出与野生型受体相似的细胞表面表达和激动剂结合，但cAMP产生受损，这表明这些残基可能对受体激活所必需的构象重排至关重要。使用基于β2肾上腺素能受体晶体结构的MC4R模型对这些突变体进行同源建模，以深入了解受体功能障碍的可能结构基础。跨膜（TM）结构域1、3、6、7和外周螺旋8似乎参与了激动剂诱导的构象重排，这是配体结合与信号传导偶联所必需 的。我们得出结论，G55V、G55D、S136F和A303T突变可能分别加强了TM1与TM2、TM3与TM6以及TM7与螺旋8之间的螺旋-螺旋相互作用，从而在受体激活过程中阻止了这些螺旋的相对运动。对MC4R中自然发生的致病突变进行功能研究和结构建模相结合，可以提供有关MC4R激活及其在人类疾病中的功能障碍分子机制的有价值信息。
