Huang R R, Vicario P P, Strader C D, Fong T M
Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Biochemistry. 1995 Aug 8;34(31):10048-55. doi: 10.1021/bi00031a029.
Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and location of these receptor residues suggest that the distance between bound peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thus receptor subtype selectivity. While many single-residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists.
已确定人类神经激肽-2受体的几个残基对于肽激动剂和非肽拮抗剂的结合至关重要。第一和第二细胞外区段以及第二跨膜区段中的氨基酸取代导致肽亲和力大幅降低，而不影响拮抗剂SR48968的亲和力。这些效应与在神经激肽-1受体中同源残基所观察到的效应相同，表明这三个区域参与了两种受体亚型与高亲和力肽的结合。另一方面，第四至第七跨膜区段中的一些保守残基仅对于一种受体亚型的肽结合是必需的，而非两种受体亚型。这些受体残基的保守性质和位置表明，结合肽与螺旋4 - 7之间的距离因受体亚型而异。配体与给定受体之间的构象兼容性可能决定了结合亲和力的大小，进而决定了受体亚型选择性。虽然许多单残基取代不影响拮抗剂SR48968的结合亲和力，但在第六和第七跨膜区段中发现的两个双突变体大幅降低了其亲和力。因此，受体残基协同参与SR48968的结合。这些结果证明了在研究和确认受体残基在配体结合中的作用时结合单残基取代的有用性。最后，激动剂和拮抗剂结合位点的重叠性质与某些残基取代会改变肽激动剂和非肽拮抗剂两者结合亲和力的观察结果一致。
