Yang Y k, Dickinson C, Haskell-Luevano C, Gantz I
Department of Internal Medicine, University of Michigan Medical School and Veterans Administration Medical Center, Ann Arbor, Michigan 48109-0682, USA.
J Biol Chem. 1997 Sep 12;272(37):23000-10. doi: 10.1074/jbc.272.37.23000.
The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G-protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and alpha-, beta-, and gamma- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent alpha-MSH analog [Nle4,D-Phe7]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists alpha-MSH, gamma-MSH, and Ac-Nle4-cyclic-[Asp5,His6,D-Phe7,Arg8,Trp9,Lys10]NH2 (MT-II). Mutagenesis of acidic receptor residues Glu94 in TM2 and Asp117 or Asp121 in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe175, Phe196, and Phe257, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand-receptor interactions between these melanocortins and the hMC1R.
黑皮质素-1受体（MC1R）是一种七跨膜（TM）G蛋白偶联受体，其天然配体为黑皮质素肽、促肾上腺皮质激素以及α-、β-和γ-黑素细胞刺激激素（MSH）。为了验证之前构建的长效、超强效α-MSH类似物[Nle4,D-Phe7]MSH（NDP-MSH）与人类MC1R之间分子相互作用的三维模型，我们研究了定点受体诱变对NDP-MSH结合亲和力和效力的影响。此外，我们还研究了这些相同突变对结构相关激动剂α-MSH、γ-MSH和Ac-Nle4-环-[Asp5,His6,D-Phe7,Arg8,Trp9,Lys10]NH2（MT-II）结合亲和力和效力的影响。跨膜区2中的酸性受体残基Glu94以及跨膜区3中的Asp117或Asp121发生诱变，会显著改变所有四种激动剂的结合亲和力和效力，这表明这些受体残基对所有配体-受体相互作用都很重要。这些酸性残基同时突变（突变体构建体D117A/D121A或E94A/D117A/D121A）或引入单个正电荷（突变体构建体D121K）时，激动剂效力与亲和力出现不成比例的变化，这也表明这些残基参与受体激活。此外，芳香族受体残基Phe175、Phe196和Phe257的单个突变以及多个跨膜区4、5和6的酪氨酸和苯丙氨酸残基的同时突变结果表明，芳香-芳香配体-受体相互作用也参与这些黑皮质素与MC1R的结合。这些实验似乎已经确定了这些黑皮质素与hMC1R之间配体-受体相互作用中一些关键的受体残基。
