Identification of Residues in Transmembrane Regions III and VI that Contribute to the Ligand Binding Site of the Serotonin 5‐HT6 Receptor

• Published in: Journal of neurochemistry 1998-11, Vol.71 (5), p.2169-2177
• Main Authors: Boess, Frank G., Monsma, Frederick J., Sleight, Andrew J.
• Format: Article
• Language: English
• Subjects: Adenylyl cyclase; Adenylyl Cyclases - metabolism; Amino Acid Substitution; Binding Sites - physiology; Binding, Competitive; Biological and medical sciences; Cell Line - metabolism; Cell receptors; Cell structures and functions; Cyclic AMP - metabolism; d‐Lysergic acid diethylamide; Ergopeptines; Fundamental and applied biological sciences. Psychology; Humans; Ligands; Lysergic Acid Diethylamide - metabolism; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Mutagenesis, Site-Directed; Mutation - genetics; Receptors, Serotonin - genetics; Receptors, Serotonin - metabolism; Serotonin; Serotonin - metabolism; Serotonin 5‐HT6 receptors; Serotonin Receptor Agonists - metabolism; Site‐directed mutagenesis
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.1998.71052169.x

: We have examined the ligand binding site of the serotonin 5‐HT6 receptor using site‐directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated d‐[3H]lysergic acid diethylamide ([3H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5‐HT and LSD to stimulate adenylyl cyclase was reduced by 3,600‐ and 500‐fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5‐HT and D106 is essential for high‐affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy‐terminal half of transmembrane region VI (Ala287 and Asn288) for leucine and serine resulted in a mutant receptor with increased affinities (seven‐ to 30‐fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5‐HT6 receptor ligand binding site.