Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT(1B) receptor.
Using a combination of RT - PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT(1B) receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT(1B) receptor. The recombinant porcine 5-HT(1B) cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [(3)H]-GR125743. The affinities of several agonists (L694247>ergotamine > or =5-carboxamidotryptamine=dihydroergotamine=5-HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>>ritanserin>ketanserin > or =BRL15572) correlated highly with those described for the recombinant human 5-HT(1B) receptor. In membranes obtained from cells co-expressing the porcine 5-HT(1B) receptor and a mutant G(alphao)Cys(351)Ile protein, 5-HT and zolmitriptan increased, while the 5-HT(1B) receptor antagonist SB224289 decreased basal [(35)S]-GTPgammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 7.64+/-0.04) agreed with its affinity in displacing the antagonist [(3)H]-GR125743 (pK(i): 7.36+/-0.07). The 5-HT(1B) receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT(1B) receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT(1B) receptor. This information may be useful in exploring the role of 5-HT(1B) 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
Ma
X
X
Wurch
T
T
Pauwels
P J
PJ
Saxena
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D002540:Cerebral Cortex / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D011725:Pyridines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012702:Serotonin Antagonists / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013552:Swine; D014018:Tissue Distribution; D014316:Tritium
D001549:Benzamides; D018076:DNA, Complementary; C107225:N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-(4-pyridyl)benzamide; D011725:Pyridines; D012333:RNA, Messenger; D044302:Receptor, Serotonin, 5-HT1B; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D013462:Sulfur Radioisotopes; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate)