Molecular cloning and functional expression of 5-HT1E-like rat and human 5-hydroxytryptamine receptor genes.
Sequential polymerase chain reaction experiments were performed to amplify a unique sequence representing a guanine nucleotide-binding protein (G-protein)-coupled receptor from rat hypothalamic cDNA. Degenerate oligonucleotides corresponding to conserved amino acids from transmembrane domains III, V, and VI of known receptors [5-HT1A, 5-HT1C, and 5-HT2; 5-HT is serotonin (5-hydroxytryptamine)] were used as primers for the sequential reactions. The resulting product was subcloned and used to screen a rat genomic library to identify a full-length clone (MR77) containing an intronless open reading frame encoding a 366-amino acid seven-transmembrane domain protein. The human homolog was isolated, and its encoded protein had 93% overall amino acid identity with the rat sequence. Within the conserved transmembrane domains, the sequences exhibit approximately 52%, 59%, 65%, and 68% amino acid identity with the known rat 5-HT1A, rat 5-HT1B, rat 5-HT1D, and human 5-HT1E receptors, respectively. MR77 was subcloned into a eukaryotic expression vector system and expressed in CosM6 cells. Studies on broken cell preparations indicate that the expressed receptor exhibits 125I-labeled d-lysergic acid diethylamide (LSD) binding that can be displaced by serotonin but not by other biogenic amines. The specific binding is displaced by the selective 5-HT1D agonist sumatriptan but not by the mixed 5-HT1A/1D agonist 5-carboxyamidotryptamine. 125I-labeled LSD binding was competitively antagonized by the ergot alkaloids methysergide and ergotamine. HeLa cells transfected with the MR77 gene exhibited inhibition of adenylate cyclase in response to serotonin. MR77 is expressed at low levels throughout the brain, with the greatest expression in the cortex, hippocampus, and striatum. MR77 thus represents a 5-HT receptor of the 5-HT1 class, and we propose that, based on the pharmacological characterization, MR77 represents an additional 5-HT1E-like receptor.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T2 T30 T31 T32 T33 T34 T35 T36 T37 T38 T39 T3 T40 T41 T42 T43 T44 T45 T46 T4 T5 T6 T7 T8 T9
CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-N GENE-Y GENE-N GENE-Y GENE-N GENE-Y CHEMICAL GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N CHEMICAL GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
ergotamine adenylate serotonin 5-HT oligonucleotides amino acids 5-HT serotonin 5-hydroxytryptamine amino acid guanine nucleotide amino acid amino acid 5-hydroxytryptamine MR77 guanine nucleotide-binding protein (G-protein)-coupled receptor 5-HT1D 5-HT1A/1D MR77 adenylate cyclase MR77 125I-labeled d-lysergic acid diethylamide MR77 5-HT receptor 5-HT1 MR77 5-HT1E 5-HT1A 5-HT1C 5-HT2 MR77 intronless open reading frame LSD 366-amino acid seven-transmembrane domain protein rat 5-HT1A rat 5-HT1B rat 5-HT1D human 5-HT1E 5-HT1E rat and human 5-hydroxytryptamine receptor serotonin amines sumatriptan 5-carboxyamidotryptamine 125I-labeled LSD methysergide