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 [3H]5-HT and [125I]iodocyanopindolol. Using [3H]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 [125I]iodocyanopindolol with high affinity, KD 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Adham
N
N
Synaptic Pharmaceutical Corporation, Paramus, N.J. 07652.
Tamm
J A
JA
Salon
J A
JA
Vaysse
P J
PJ
Weinshank
R L
RL
Branchek
T A
TA
D000319:Adrenergic beta-Antagonists / Q000493:pharmacokinetics*; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D020120:Iodocyanopindolol; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010869:Pindolol / Q000031:analogs & derivatives; D017354:Point Mutation; D011433:Propranolol / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000502:physiology
D000319:Adrenergic beta-Antagonists; D008024:Ligands; D011985:Receptors, Serotonin; D020120:Iodocyanopindolol; D011433:Propranolol; D010869:Pindolol