Molecular cloning, functional expression, and pharmacological characterization of a novel serotonin receptor (5-hydroxytryptamine2F) from rat stomach fundus.
Using the polymerase chain reaction amplification technique in conjunction with conventional cloning techniques, we have isolated a novel member of the serotonin [5-hydroxytryptamine (5-HT)] 1C/2 receptor subfamily (designated 5-HT2F) from rat stomach fundus. Two DNA fragments were amplified from cDNA synthesized from rat stomach fundus poly(A)+ RNA using the polymerase chain reaction technique with degenerate oligonucleotide primers derived from sequence comparisons of the second, third, and sixth putative transmembrane domains of known 5-HT receptors. These fragments were used as hybridization probes to isolate full length cDNA clones from rat stomach fundus cDNA libraries. Full length cDNA clones contained one open reading frame encoding a 479-amino acid protein with seven hydrophobic domains, characteristic of members of the guanine nucleotide-binding protein-coupled receptor superfamily. Within these seven putative transmembrane domains, the 5-HT2F receptor shared greatest homology with the rat 5-HT1C and 5-HT2 receptor subtypes (70% and 68%, respectively). Cell lines stably expressing the 5-HT2F receptor were established and demonstrated functional coupling to phosphatidylinositol hydrolysis upon 5-HT stimulation analagous to that observed for the 5-HT1C and 5-HT2 receptors. Membranes from the stably transfected cell lines (but not the untransfected parental lines) exhibited high affinity (Kd = 7.9 nM), saturable binding of [3H]5-HT. Maximum binding ranged from 0.1 to 2.4 pmol/mg of protein, depending on the clonal isolate. Using [3H]5-HT as the basis for a radioligand binding assay, the relative affinities of several tryptamine and piperazine derivatives for the cloned 5-HT2F receptor correlated with their relative potencies to contract the rat stomach fundus. These data suggest a probable relationship between this novel 5-HT2F receptor and the serotonin contractile receptor of the rat stomach fundus.
Kursar
J D
JD
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285.
Nelson
D L
DL
Wainscott
D B
DB
Cohen
M L
ML
Baez
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D008024:Ligands; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010716:Phosphatidylinositols / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011985:Receptors, Serotonin / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D016415:Sequence Alignment; D015398:Signal Transduction; D013270:Stomach / Q000737:chemistry*; D014162:Transfection
D008024:Ligands; D009838:Oligodeoxyribonucleotides; D010716:Phosphatidylinositols; D012333:RNA, Messenger; D011985:Receptors, Serotonin