Mutagenesis of the human 5-HT1B receptor: differences from the closely related 5-HT1A receptor and the role of residue F331 in signal transduction.
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.
Grånäs
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Department of Neuroscience, Uppsala University, Sweden.
Nordvall
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Larhammar
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D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin / Q000737:chemistry / Q000502:physiology*; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship
C470302:HTR1B protein, human; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin; D005576:Colforsin; D012701:Serotonin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins