Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht(1F) 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-ht(1F)) 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-ht(1F) receptor sequences from other species, including human. The recombinant porcine 5-ht(1F) 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 =5HT>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-ht(1F) receptor (Spearman correlation coefficient; r(s)=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-ht(1F) receptor. Using RT-PCR technique, the expression of porcine 5-ht(1F) 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-ht(1F) receptor as well as the distribution of its mRNA. This information may be helpful in exploring the role of 5-ht(1F) receptor in physiological processes and diseases, such as migraine.
Bhalla
Pankaj
P
Department of Pharmacology, Erasmus University Medical Centre Rotterdam, Post Box 1738, 3000 DR, Rotterdam, The Netherlands.
Sharma
Hari S
HS
Wurch
Thierry
T
Pauwels
Petrus J
PJ
Saxena
Pramod R
PR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012702:Serotonin Antagonists / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000378:metabolism; D013552:Swine; D012668:Trigeminal Ganglion / Q000378:metabolism*; D000096642:Receptor, Serotonin, 5-HT1F
D018076:DNA, Complementary; D012333:RNA, Messenger; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D000096642:Receptor, Serotonin, 5-HT1F