Receptor-ligand interactions of the human 5-HT1B receptor : A site-directed mutagenesis study

Abstract: The 5-hydroxytryptamine (5-HT, serotonin)1B receptor is one of thirteen G protein-coupled 5-HT receptors. 5-HT is known to influence temperature regulation, pain, cognition and sexual and feeding behaviours. The presynaptic 5-HT1B receptor, which is broadly distributed throughout brain of mammals, regulates 5-HT release and may be involved in depression and anxiety disorders. In this study a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis was used to investigate the receptor-ligand interactions of the 5-HT1B receptor. Mutant 5-HT1B receptors were constructed and the importance of specific amino acid residues in agonist and antagonist binding as well as G protein coupling was investigated by radioligand binding and functional assays on receptors stably expressed in Chinese hamster ovary cells. Several amino acid residues have been identified that are important for ligand selectivity and may contribute to the pharmacological differences between the various 5-HT receptor subtypes. Most importantly, tryptophan 327 was shown to be crucial for binding the antimigraine drug sumatriptan. Interestingly, 5-HT was found to interact differently with amino acid residues that are conserved between closely related 5-HT receptor subtypes.A number of amino acids were found to be involved in the conformational changes necessary for receptor activation. In addition, 5-HT displayed increased affinity for the inactive conformation of several of the mutants with reduced functional activity. Methiothepin which is an inverse agonist and inhibits basal receptor activity was found to have higher affinity for the inactive conformation of the receptor than for the active conformation.The results in this study show that the roles of conserved amino acids in ligand binding may differ between closely related receptor subtypes and must be tested individually. Identification of the amino acids that endow each 5-HT receptor subtype with unique pharmacological properties may facilitate the development of new selective drugs. The results in this study contribute to the knowledge of ligand-receptor interactions in the 5-HT1B receptor and of the G protein coupled receptors in general.