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MOLECULAR PHARMACOLOGY 52:515-523 (1997).

Interaction of Tryptamine and Ergoline Compounds with Threonine 196 in the Ligand Binding Site of the 5-Hydroxytryptamine₆ Receptor

Frank G. Boess, Frederick J. Monsma, Jr., Valerie Meyer, Catherine Zwingelstein, and Andrew J. Sleight

Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland

We examined the ligand-binding site of the 5-hydroxytryptamine₆ (5-HT₆) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of transmembrane region V are important for ligand binding in several bioamine receptors. In the 5-HT₆ receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [³H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [³H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30-fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mianserin, changed very little or increased. This indicates that in wild-type 5-HT₆ receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT_2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT₆ receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT₆ receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT₆ receptor.