Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT(1B) receptor.

Abstract

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.

• Authors: P Bhalla, H S Sharma, X Ma, T Wurch, P J Pauwels, P R Saxena
• Journal: British journal of pharmacology (Br J Pharmacol) Vol. 133 Issue 6 Pg. 891-901 (Jul 2001) ISSN: 0007-1188 [Print] England
• PMID: 11454663 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Benzamides
• DNA, Complementary
• N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-(4-pyridyl)benzamide
• Pyridines
• RNA, Messenger
• Receptor, Serotonin, 5-HT1B
• Receptors, Serotonin
• Recombinant Proteins
• Serotonin Antagonists
• Serotonin Receptor Agonists
• Sulfur Radioisotopes
• Tritium
• Guanosine 5'-O-(3-Thiotriphosphate)

Topics

• Amino Acid Sequence
• Animals
• Base Sequence
• Benzamides (metabolism)
• Binding, Competitive (drug effects)
• COS Cells
• Cerebral Cortex (metabolism)
• Cloning, Molecular
• DNA, Complementary (chemistry, genetics)
• Dose-Response Relationship, Drug
• Gene Expression
• Guanosine 5'-O-(3-Thiotriphosphate) (metabolism)
• In Situ Hybridization
• Molecular Sequence Data
• Pyridines (metabolism)
• RNA, Messenger (genetics, metabolism)
• Radioligand Assay
• Receptor, Serotonin, 5-HT1B
• Receptors, Serotonin (drug effects, genetics, metabolism)
• Recombinant Proteins (drug effects, genetics, metabolism)
• Sequence Alignment
• Sequence Analysis, DNA
• Sequence Homology, Amino Acid
• Sequence Homology, Nucleic Acid
• Serotonin Antagonists (pharmacology)
• Serotonin Receptor Agonists (pharmacology)
• Sulfur Radioisotopes
• Swine
• Tissue Distribution
• Tritium