Huang R R, Yu H, Strader C D, Fong T M
Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065.
Mol Pharmacol. 1994 Apr;45(4):690-5.
Previous studies have indicated that substitution of the third or fourth extracellular segment of the human neurokinin-1 receptor with the equivalent segment from the neurokinin-3 receptor affects the binding affinities of peptide agonists and/or nonpeptide antagonists. To elucidate the roles of the divergent residues within these domains in ligand binding, single-residue substitutions in these regions were analyzed. Neurokinin B affinity was increased by one single-residue substitution (E172K) in the third extracellular segment and one double-residue replacement (L279R+K280W) in the fourth extracellular segment, and the affinity for the antagonist L-703,606 was reduced by one single-residue substitution (Y272A) in the fourth extracellular segment. The effect of these three specific substitutions is consistent with the prediction of chimeric mutations. However, the substantial reduction in ligand binding affinity observed upon multiple-residue substitutions in the third extracellular segment (residues 176-183 or 187-195) has not been reproduced by eliminating potential electrostatic interactions or substituting with the corresponding residues from the neurokinin-3 receptor, suggesting that the reduction in ligand binding affinity observed with some chimeric receptors is not due to the loss of direct electrostatic interactions. These data indicate that other factors such as conformational effects may complicate the interpretation of data obtained with chimeric receptors, and they demonstrate the need to evaluate chimeric receptors along with single-residue substitutions in the same region to localize specific residues involved in ligand binding. Furthermore, the available data suggest that one major determinant of peptide selectivity in the neurokinin-1 receptor may be the conformational compatibility between a peptide and the receptor.
先前的研究表明，用人神经激肽-3受体的等效片段替换人神经激肽-1受体的第三或第四细胞外片段会影响肽激动剂和/或非肽拮抗剂的结合亲和力。为了阐明这些结构域中不同残基在配体结合中的作用，对这些区域中的单残基取代进行了分析。神经激肽B的亲和力在第三细胞外片段中的一个单残基取代（E172K）和第四细胞外片段中的一个双残基替换（L279R+K280W）后增加，而对拮抗剂L-703,606的亲和力在第四细胞外片段中的一个单残基取代（Y272A）后降低。这三个特定取代的效果与嵌合突变的预测一致。然而，在第三细胞外片段（残基176-183或187-195）中进行多残基取代后观察到的配体结合亲和力的大幅降低，在消除潜在的静电相互作用或用人神经激肽-3受体的相应残基进行取代时并未重现，这表明一些嵌合受体观察到的配体结合亲和力降低并非由于直接静电相互作用的丧失。这些数据表明，其他因素如构象效应可能会使对嵌合受体获得的数据的解释复杂化，并且它们证明需要在同一区域评估嵌合受体以及单残基取代，以定位参与配体结合的特定残基。此外，现有数据表明，神经激肽-1受体中肽选择性的一个主要决定因素可能是肽与受体之间的构象兼容性。
