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

FEBS Letters

Strosberg, A.Donny²  Conway, Shaun³  Guardiola-Lemaitre, Beatrice¹  Canning, Sarah J³  Barrett, Perry³  Morgan, Peter J³  Jockers, Ralf²  Drew, Janice E³  Delagrange, Phillipe¹ 

[1] Institut de Recherche Internationales Servier, 6 place des Pléiades, F-92415 Courbevoie Cedex, France;CNRS-UPR 0415 and Université Paris VII, Institut Cochin de Génétique Moleculaire, 22 rue Méchain, F-75014 Paris, France;Molecular Neuroendocrinology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK

关键词: Melatonin receptor;    2-[125I]Iodomelatonin binding;    Human kidney HEK293 cell;    Cyclic AMP inhibition;    G protein;    heterotrimeric guanine nucleotide-binding protein;    RT-PCR;    reverse-transcription polymerase chain reaction;    PBS;    phosphate buffered saline;    EGTA;    ethylene glycol-bis(β-aminoethyl ether) N;    N;    N′;    N′-tetraacetic acid;    PEG 8000;    polyethylene glycol (average molecular weight 8000);    IBMX;    3-isobutyl-1-methylxanthine;    TCA;    trichloroacetic acid;    M-MLV;    Moloney leukaemia virus;    DTT;    dithiothreitol;    dNTP;    deoxyribose nucleotide 5′-triphosphate;   

DOI  :  10.1016/S0014-5793(97)00315-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Mel_1a 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 Mel_1a 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.

【 授权许可】

Unknown