title: Site-directed mutagenesis of the 5-HT1B receptor increases the affinity of 5-HT for the agonist low-affinity conformation and reduces the intrinsic activity of 5-HT.
abstract: The antagonist radioligand [3H]GR125743 and the agonist radioligand [3H]5-HT were used to investigate the pharmacological characteristics of the G protein uncoupled agonist low-affinity and G protein coupled agonist high-affinity conformations of the wild-type and mutant human 5-hydroxytryptamine 1B (5-HT1B) receptors. We found that substitution of phenylalanine 185 in transmembrane region IV by alanine or methionine resulted in a reduced number of receptors in the coupled conformation, as well as a reduced affinity of 5-HT for the uncoupled conformation. In contrast, substitution of phenylalanine 331 in transmembrane region VI by alanine increased the affinity of 5-HT for the uncoupled conformation 11-fold thus reducing the agonist low-affinity to agonist high-affinity (K(il)/K(ih)) ratio 5-fold. This reduced ratio was correlated with a significantly reduced intrinsic activity of 5-HT previously determined by its ability to inhibit forskolin-stimulated cAMP production. In conclusion, these results show that single amino acid substitutions can selectively change the affinity of 5-HT for the G protein uncoupled conformation of the 5-HT1B receptor and alter the intrinsic activity of the ligand.
authors.affiliation: Department of Neuroscience, Unit of Pharmacology, Uppsala University, Box 593, SE-751 24, Uppsala, Sweden. cgranas@mfi.ku.dk
authors.forename: C
authors.initials: C
authors.lastname: Grånäs
authors.forename: J
authors.initials: J
authors.lastname: Nordquist
authors.forename: N
authors.initials: N
authors.lastname: Mohell
authors.forename: D
authors.initials: D
authors.lastname: Larhammar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium
chemical_list: D001549:Benzamides; C470302:HTR1B protein, human; C107225:N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-(4-pyridyl)benzamide; D011725:Pyridines; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists; D014316:Tritium; D005576:Colforsin; D012701:Serotonin; D000242:Cyclic AMP