Chai Biao-Xin, Pogozheva Irina D, Lai Yu-Mei, Li Ji-Yao, Neubig Richard R, Mosberg Henry I, Gantz Ira
Department of Surgery, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.
Biochemistry. 2005 Mar 8;44(9):3418-31. doi: 10.1021/bi0478704.
The molecular interactions between human melanocortin receptor-1 and -4 (hMC1R and hMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP), were assessed by studying the effects of site-directed mutations on the binding affinity of (125)I-ASIP[90-132(L89Y)] and (125)I-AGRP(86-132). Mutations of homologous residues from transmembrane helices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMC1R and D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R and binding of the ASIP fragment to hMC1R. However, the mutations in TMH2 (E94A in hMC1R and E100A in hMC4R), TMH7 (F280A in hMC1R and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H in hMC1R) only significantly affected binding of the ASIP fragment. The dependence of agonist binding on the dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A, and C279A mutants and a biphasic curve for hMC1R, suggesting the presence of at least one structurally and functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models of complexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculated using constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deduced hydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derived from our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the core ligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7 relative to its position in the AGRP-hMC4R model, while the N-terminal loop and two central disulfides of the antagonists interact with EL2 of the receptors.
通过研究定点突变对（125）I-ASIP[90-132（L89Y）]和（125）I-AGRP（86-132）结合亲和力的影响，评估了人类黑皮质素受体-1和-4（hMC1R和hMC4R）与其内源性拮抗剂刺鼠信号蛋白（ASIP）和刺鼠相关蛋白（AGRP）之间的分子相互作用。跨膜螺旋（TMHs）3和6以及细胞外环（EL）3同源残基的突变（hMC1R中的D121A、T124A、F257A和F277M以及hMC4R中的D126A、I129A、F261A和M281F）损害了两种拮抗剂与hMC4R的结合以及ASIP片段与hMC1R的结合。然而，TMH2（hMC1R中的E94A和hMC4R中的E100A）、TMH7（hMC1R中的F280A和hMC4R中的F284A）和EL2（hMC1R中的Y183S、H184S和D184H）中的突变仅显著影响ASIP片段的结合。激动剂结合对二硫苏糖醇浓度的依赖性对于野生型hMC4R及其C40A、C271A和C279A突变体呈单相曲线，而对于hMC1R呈双相曲线，这表明在野生型受体和hMC4R突变体中均存在至少一个结构和功能上必不可少的二硫键。利用视紫红质和AGRP片段的实验结构、一组推导的氢键，并辅以两个提议的二硫键和受体-配体接触（源自我们的诱变数据）作为约束条件，计算了两种受体与ASIP片段以及hMC4R与AGRP片段的复合物模型。在与两种受体复合的ASIP片段模型中，位于TMHs 3和6之间的核心配体三肽Arg-Phe-Phe相对于其在AGRP-hMC4R模型中的位置向TMHs 2和7移动，而拮抗剂的N端环和两个中央二硫键与受体的EL2相互作用。
