Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor.
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.
Bhalla
P
P
Department of Pharmacology, Erasmus University Medical Centre Rotterdam, Rotterdam, The Netherlands.
Sharma
H S
HS
Wurch
T
T
Pauwels
P J
PJ
Saxena
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D011725:Pyridines / Q000378:metabolism; D051381:Rats; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D001549:Benzamides; C107225:N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-(4-pyridyl)benzamide; D011725:Pyridines; D044346:Receptor, Serotonin, 5-HT1D; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate)