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. Ot her 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1d beta a nd 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous positi on in the seventh transmembrane domain. In the case of the human 5-HT1 D beta receptor, replacement of this T with asparagine (N), dramatical ly increases its ability to bind beta-adrenergic antagonists. To asses s whether other 5-HT1 receptors would behave similarly, we have used s ite-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 fo r propranolol and pindolol were significantly increased by 100-1000 fo ld; 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT 1E receptor displaying the lowest affinity. On the other hand, the aff inities for 5-HT were essentially unchanged as compared to the wild-ty pe receptors. All mutant receptors bound [I-125]iodocyanopindolol with high affinity, K-D values ranging between 0.04 nM (mutant 5-HT1D alph a) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concen tration range used for the mutant receptors. Therefore, the presence o f this key asparagine residue confers binding for beta-antagonists eve n in serotonin receptors which differ substantially in molecular struc ture. Even so, it appears likely that these ligands are recognized by the set of 5-HT1 receptors in a homologous orientation when interactin g with the ligand binding pocket.