Site-directed mutagenesis of the 5-HT1B receptor increases the affinity of5-HT for the agonist low-affinity conformation and reduces the intrinsic activity of 5-HT

The antagonist radioligand [H-3]GR125743 and the agonist radioligand [H-3]5 -HT were used to investigate the pharmacological characteristics of the G p rotein uncoupled agonist low-affinity and G protein coupled agonist high-af finity 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 numb er of receptors in the coupled conformation, as well as a reduced affinity of 5-HT for the uncoupled conformation. In contrast, substitution of phenyl alanine 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 ra tio was correlated with a significantly reduced intrinsic activity of 5-HT previously determined by its ability to inhibit forskolin-stimulated cAMP p roduction. In conclusion, these results show that single amino acid substit utions can selectively change the affinity of 5-HT for the G protein uncoup led conformation of the 5-HT1B receptor and alter the intrinsic activity of the ligand. (C) 2001 Elsevier Science B.V. All rights reserved.