Identification of residues in transmembrane regions III and VI that contribute to the ligand binding site of the serotonin 5-HT6 receptor.
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.
Boess
F G
FG
Pharma Division, Preclinical Research, F. Hoffmann-LaRoche Ltd., Basel, Switzerland.
Monsma
F J
FJ
Sleight
A J
AJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D002460:Cell Line / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008238:Lysergic Acid Diethylamide / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000378:metabolism
D008024:Ligands; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; D012701:Serotonin; D008238:Lysergic Acid Diethylamide; D000242:Cyclic AMP; D000262:Adenylyl Cyclases