Abstract
An intron-containing gene encoding a novel human serotonin (5-HT) receptor was isolated from human genomic and cDNA libraries with probes directed to transmembrane regions of the adenylate cyclase stimulatory Drosophila serotonin receptor gene, 5-HTdrol. Membranes harvested from transiently transfected Cos-7 cells displayed high affinity (Kd = 8.5 nM), saturable (Bmax = 6.6 pmol/mg protein) [3H]5-HT binding. The rank order of potencies for serotonergic ligands to displace specific [3H]5-HT binding was: 5-carboxamido-tryptamine > methiothepin > metergoline > 5-HT > 8-hydroxy-2-(di-n-propylamino)tetralin > sumatriptan > ketanserin > zacopride. 5-HT produced a dose-dependent (EC50 = 992 nM) stimulation (approximately 20-fold) of cAMP accumulation in transiently transfected cells, and this response was antagonized by the nonselective 5-HT antagonist methiothepin. RNA for this gene was predominantly detected in the human brain and a subset of peripheral tissues including coronary artery and several tissues of the gastrointestinal tract. The molecular biological and pharmacological properties of this receptor suggest that it is the first member of a new serotonin receptor subfamily (5-HT7). The second messenger coupling, and tissue distribution indicate a possible identity to 5-HT receptors that mediate relaxant responses in certain isolated blood vessels.
Highlights
Cos-7 the primary structure and pharmacological profile of this recellsdisplayedhighaffinity (I& = 8.5 m),saturable (Bm== 6.6 pmol/mg protein)[SHJ5-HTbinding
Cloning and Sequencing-A human placenta genomic library (Stratagene, La Jolla, CA) was screened using oligonucleotides derived from the Drosophila serotonin receptor gene, 5-HTd, [13] as probes
Hybridization tor suggest that isitthe firstmember ofa new serotoninand Southern blot analysis were performedas previously described[14]
Summary
Cloning and Primary Amino Acid Sequence of the 5-HT7 Receptor-In order to identifyserotonin receptors that positively couple to adenylate cyclase, we used a lowhomology screening approach with probes directed to conserved TM domains (TM 111, V, VI, and VII) of a Drosophila 5-HT receptor, 5-HTdrol [13].We obtained two related genomic clones, one of which (clone hp78a) appeared to be a novel receptor, based. Clone hp78aFL did notnegatively couple to adenylate cyclase when transfected cos-7 cells were coincubated with both 1 w forskolin and 11.1~. Fetal brain cDNA library, and three partial but overlapping The ability of serotonin to activate adenylate cyclase has cDNA clones were isolated. This receptor gene containsa n open reading frameof 1335 bp (5-HTlA)(201,human atria(5-HT4)(211,NCB2O neuroblastoma and encodes for apredicted protein of 445 amino acids in hybrid cells (5-HT4-like) (22, 231, and the porcine vena cava length, having a relative molecular mass of -49 kDa (Fig. 1). The pharma- gene) by using gene-specific PCR primersand gene-specific cological profile of clone hp78aFL was determinedfrom nonlin- internal oligonucleotide probes, which failed to detect the ear analysis of competition of high affinity [3H]5-HT binding.
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