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.

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.