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

J Recept Signal Transduct Res. Jul-Nov 1998;18(4-6):225-41. doi: 10.3109/10799899809047745.

Abstract

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.

MeSH terms

  • 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

Substances

  • HTR1B protein, human
  • Receptor, Serotonin, 5-HT1B
  • Receptors, Serotonin
  • Colforsin
  • Serotonin
  • Cyclic AMP
  • GTP-Binding Proteins