Molecular cloning, pharmacological properties and tissue distribution of the porcine 5-HT1B receptor

1 Using a combination of RT-PCR and inverse-PCR techniques, we amplified, c loned and sequenced a full-length porcine 5-HT1B receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an op en reading frame of 390 amino acids showing a 95% similarity with the human 5-HT1B receptor. 2 The recombinant porcine 5-HT1B cDNA was expressed in monkey Cos-7 cells a nd its pharmacological profile was determined by radioligand binding assay using [H-3]-GR125743. The affinities of several agonists (L694247 > ergotam ine greater than or equal to 5-carboxamidotryptamine = dihydroergotamine= 5 -HT > CP122638 = zolmitriptan > sumatriptan) and putative antagonists (GR12 7935 > methiothepin > SB224289 > > ritanserin > ketanserin greater than or equal to BRL15572) correlated highly with those described for the recombina nt human 5-HT1B receptor. 3 In membranes obtained from cells co-expressing the porcine 5-HT1B recepto r and a mutant G(alphaO)Cys(351)Ile protein, 5-HT and zolmitriptan increase d, while the 5-HT1B receptor antagonist SB224289 decreased basal [S-35]-GTP gammaS binding, thus showing inverse agonism. The potency of zolmitriptan in the [S-35]-GTP gammaS binding assay (pEC(50): 7.64 +/- 0.04) agreed with its affinity in displacing the antagonist [H-3]-GR125743 (pK(i): 7.36 +/- 0.07). 4 The 5-HT1B 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 -HT1B receptor mRNA in pyramidal cells. 5 In conclusion, we have cloned and established the amino acid sequence, li gand binding profile and location of the porcine 5-HT1B receptor. This info rmation may be useful in exploring the role of 5-HT1B receptor in pathophys iological processes relevant for novel drug discovery in diseases such as m igraine.