A
cDNA encoding the full-length
5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig
5-HT(1D) receptor coding sequence
oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine
5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine
5-HT(1D) receptor cDNA revealed an open reading frame of 1134
nucleotides encoding a
polypeptide of 377
amino acids having 92% homology with the human
5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine
5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14
serotonin ligands were highly correlated with those obtained with the human
5-HT(1D) receptor. Nonetheless, a selective antagonist at the human
5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine
5-HT(1D) receptor and rat G(alphail)Cys(351) Ile
protein, it was shown that
5-HT and
zolmitriptan increased, while
ketanserin decreased basal [(35)S]-
GTPgammaS binding. The potency of
zolmitriptan in the [(35)S]-
GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the
cDNA sequence and pharmacology of the cloned porcine
5-HT(1D) receptor. This information would be useful in exploring the role of divergent
amino acid residues in the receptor-
ligand interaction as well as the role of
5-HT(1D) receptor in pathophysiological processes relevant for novel
drug discovery in diseases such as
migraine.