Mutagenesis of the human 5-HT1B receptor: differences from the closely related 5-HT1A receptor and the role of residue F331 in signal transduction

• Published in: Journal of receptors and signal transduction Vol. 18; no. 4-6; p. 225
• Main Authors: Grånäs, C, Nordvall, G, Larhammar, D
• Format: Journal Article
• Language: English
• Published: England 01.01.1998
• Subjects: Animals; CHO Cells; Colforsin - pharmacology; Cricetinae; Cyclic AMP - biosynthesis; GTP-Binding Proteins - physiology; Humans; Mutagenesis, Site-Directed; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin - chemistry; Receptors, Serotonin - physiology; Serotonin - metabolism; Signal Transduction; Structure-Activity Relationship
• ISSN: 1079-9893; 1532-4281
• DOI: 10.3109/10799899809047745

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.