Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT1D receptor

1 A cDNA encoding the full-length 5-HT1D receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT1D receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). The 5' and 3' ends of the porcine 5-HT1D receptor cDNA were verified by inverse PCR. 2 Sequence analysis of porcine 5-HT1D receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT1D receptor and 88-90% homology with other species homologues. 3 The porcine 5-HT1D receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [H-3]-5-CT or [H-3]-GR125743 on Cos-7 cell membranes showed that pK, values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT1D receptor. Nonetheless, a selective antagonist at the human 5-HT1D receptor, BRL15572, only poorly recognized the porcine homologue. 4 Using membranes from cells co-expressing the porcine 5-HT1D receptor and rat G(alpha 1l)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [S-35]-GTP gammaS binding. The potency of zolmitriptan in the [S-35]-GTP gammaS binding assay (pEC(50): 8.46+/-0.08) agreed with its affinity in displacing the radioligands [H-3]-5-CT and [H-3]-GR125743 (pK(i): 8.38 +/- 0.15 and 8.67 +/- 0.08, respectively). 5 In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT1D 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-HT1D receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.