A SINGLE-POINT MUTATION INCREASES THE AFFINITY OF SEROTONIN 5-HT1D-ALPHA, 5-HT1D-BETA, 5-HT1E AND 5-HT1F RECEPTORS FOR BETA-ADRENERGIC ANTAGONISTS

The serotonin 5-HT1B and 5-HT1A receptors bind certain beta-adrenergic antagonists, such as propranolol and pindolol, with high affinity. Other 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1d beta and 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous position in the seventh transmembrane domain. In the case of the human 5-HT1D beta receptor, replacement of this T with asparagine (N), dramatically increases its ability to bind beta-adrenergic antagonists. To assess whether other 5-HT1 receptors would behave similarly, we have used site-directed mutagenesis to replace the T or A in 5-HT1d alpha, 5-HT1E and 5-HT1F receptors with N. Both the wild-type and mutant genes were expressed transiently in COS-7 cells and radioligand binding studies were performed by using [H-3]5-HT and [I-125]iodocyanopindolol. Using [H-3]5-HT, we found that the affinities of all the mutant receptors for propranolol and pindolol were significantly increased by 100-1000 fold; 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT1E receptor displaying the lowest affinity. On the other hand, the affinities for 5-HT were essentially unchanged as compared to the wild-type receptors. All mutant receptors bound [I-125]iodocyanopindolol with high affinity, K-D values ranging between 0.04 nM (mutant 5-HT1D alpha) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concentration range used for the mutant receptors. Therefore, the presence of this key asparagine residue confers binding for beta-antagonists even in serotonin receptors which differ substantially in molecular structure. Even so, it appears likely that these ligands are recognized by the set of 5-HT1 receptors in a homologous orientation when interacting with the ligand binding pocket.