Identification of Mel1a melatonin receptors in the human embryonic kidney cell line HEK293: evidence of G protein‐coupled melatonin receptors which do not mediate the inhibition of stimulated cyclic AMP levels

• Published in: FEBS letters 1997-04, Vol.407 (1), p.121-126
• Main Authors: Conway, Shaun, Drew, Janice E, Canning, Sarah J, Barrett, Perry, Jockers, Ralf, Strosberg, A.Donny, Guardiola-Lemaitre, Beatrice, Delagrange, Phillipe, Morgan, Peter J
• Format: Article
• Language: English
• Subjects: 2-[125I]Iodomelatonin binding; 3-isobutyl-1-methylxanthine; Binding, Competitive; Cell Line; Colforsin - pharmacology; Cyclic AMP - metabolism; Cyclic AMP inhibition; deoxyribose nucleotide 5′-triphosphate; dithiothreitol; dNTP; DTT; EGTA; ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid; G protein; Genomic Library; GTP-Binding Proteins - metabolism; heterotrimeric guanine nucleotide-binding protein; Human kidney HEK293 cell; Humans; IBMX; Kidney - cytology; Kidney - embryology; Kidney - metabolism; M-MLV; Melatonin - analogs & derivatives; Melatonin - metabolism; Melatonin receptor; Moloney leukaemia virus; PBS; PEG 8000; phosphate buffered saline; polyethylene glycol (average molecular weight 8000); Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Receptors, Melatonin; reverse-transcription polymerase chain reaction; RNA, Messenger - genetics; RT-PCR; Sequence Analysis, DNA; Serotonin - analogs & derivatives; Serotonin - metabolism; Signal Transduction; TCA; trichloroacetic acid; Tryptamines - metabolism
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(97)00315-3

Binding assays using 2‐[125I]iodomelatonin revealed high‐affinity, guanosine 5′‐O‐(3‐thiotriphosphate) sensitive, melatonin binding sites (B max 1.1 fmol/mg protein) in the human embryonic kidney cell line HEK293. Competition studies using the selective melatonin receptor antagonist luzindole and RT‐PCR techniques identified these sites as human Mel1a melatonin receptors. Challenge of HEK293 cells with 1 μM melatonin had no effect on forskolin stimulated cyclic AMP levels, whereas in HEK293 cells engineered to stably over‐express the human Mel1a melatonin receptor (B max>400 fmol/mg protein) melatonin dose‐dependently inhibited stimulated cyclic AMP levels (IC50 7.7 pM). These data may indicate that certain tissues, expressing low levels of G protein‐coupled melatonin receptors, do not display melatonin mediated inhibition of cAMP.