Identification of an amino acid residue important for binding of methiothepin and sumatriptan to the human 5-HT1B receptor

Site-directed mutagenesis of the human 5-HT1B receptor was performed to inv estigate the role of the amino acid residues cysteine 326 and tryptophan 32 7 in transmembrane region VI and aspartic acid 352 in transmembrane region VII in ligand binding. Binding studies were performed with the antagonist r adioligand [H-3]GR125743 on mutant and wild-type receptors stably expressed in Chinese hamster ovary cells (CHO)-K1 cells. Substitution of tryptophan 327 by alanine resulted in decreased affinities of all ligands tested. The most prominent changes in affinity were observed for the antagonist methiot hepin and the antimigraine drug sumatriptan, which were reduced approximate ly 300- and 60-fold, respectively. Nevertheless, the affinity of 5-HT remai ned the same. Replacement of the aspartic acid 352 by alanine reduced high- affinity binding of 5-HT. Substitution of cysteine 326 by alanine had minor effects on ligand binding. Some of these results agree with the results fr om mutagenesis studies of the corresponding amino acids in other receptors. However, some notable differences also emerge showing that functional role s of individual amino acid residues must be tested experimentally in each r eceptor subtype. (C) 1999 Elsevier Science B.V. All rights reserved.