Yang Y K, Fong T M, Dickinson C J, Mao C, Li J Y, Tota M R, Mosley R, Van Der Ploeg L H, Gantz I
Departments of General Surgery and Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Biochemistry. 2000 Dec 5;39(48):14900-11. doi: 10.1021/bi001684q.
To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.
为阐明配体与人黑皮质素-4受体（hMC4R）相互作用的分子基础，进行了激动剂构效关系研究和受体点突变实验。[Nle(4), D-Phe(7)]-α-黑素细胞刺激素（NDP-MSH）的构效关系研究确定D-Phe7-Arg8-Trp9为在hMC4R上具有完全激动剂效力的最小NDP-MSH片段。为确定可能与该三肽序列中的氨基酸相互作用的受体残基，对24个hMC4R跨膜（TM）残基进行了突变（选择特定受体残基进行突变的原理在结果部分概述）。TM3残基D122和D126以及TM6残基F261和H264的突变使NDP-MSH的结合亲和力降低了5倍或更多，从而确定这些受体残基为可能参与所寻求的配体-受体相互作用的位点。通过检测取代的NDP-MSH肽在受体突变体上的结合亲和力和效力，发现黑皮质素核心肽残基Arg8在分子水平上与hMC4R TM3残基D122相互作用。还观察到TM3突变会降低hMC4R拮抗剂的结合。值得注意的是，TM3残基D126突变为丙氨酸使内源性黑皮质素拮抗剂的C末端衍生物AGRP（87-132）的结合亲和力降低了8倍，同时突变D122A/D126A则完全消除了AGRP（87-132）的结合。此外，TM3残基D122或D126的突变降低了hMC4R拮抗剂SHU 9119的结合亲和力。这些结果为hMC4R配体结合的分子决定因素提供了进一步的见解。
