Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht1F receptor

Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht1F) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht1F receptor sequences from other species, including human. The recombinant porcine 5-ht1F receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 (N-methyl-3 [pyrrolidin 2(R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan=5-HT>eletriptan>sumatriptan>frovatriptan= avitriptan>dihydroergotamine>zolmitriptan>5- carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl 4′-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1′-methyl-5-[2′-methyl-4′ (5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4′-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-(S,R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht1F receptor (Spearman correlation coefficient; rs=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht1F receptor. Using RT-PCR technique, the expression of porcine 5-ht1F receptor mRNA was observed in cerebral cortex, trigeminal ganglion and several blood vessels, but not in skeletal muscles. In conclusion, we have cloned and established the amino acid sequence and ligand binding profile of the porcine 5-ht1F receptor as well as the distribution of its mRNA. This information may be helpful in exploring the role of 5-ht1F receptor in physiological processes and diseases, such as migraine.

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