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.

Amino Acid Sequence, Animals, Base Sequence, Benzamides/metabolism, CHO Cells, COS Cells, Cloning, Molecular, Cricetinae, Guanosine 5'-O-(3-Thiotriphosphate)/metabolism, Guinea Pigs, Humans, Molecular Sequence Data, Pyridines/metabolism, Rats, Receptor, Serotonin, 5-HT1D, Receptors, Serotonin/chemistry/*genetics/metabolism, Recombinant Proteins/metabolism, Swine
dx.doi.org/10.1038/sj.bjp.0703645, hdl.handle.net/1765/12889
British Journal of Pharmacology
Erasmus MC: University Medical Center Rotterdam

Bhalla, P.L, Sharma, H.S, Wurch, T, Pauwels, P.J, & Saxena, P.R. (2000). Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor. British Journal of Pharmacology, 949–957. doi:10.1038/sj.bjp.0703645