Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer.
We have developed a procedure based on bioluminescence resonance energy transfer (BRET) to monitor the activation state of the insulin receptor in vitro. Human insulin receptor cDNA was fused to either Renilla luciferase (Rluc) or enhanced yellow fluorescent protein (EYFP) coding sequences. Fusion insulin receptors were partially purified by wheat-germ lectin chromatography from human embryonic kidney 293 cells cotransfected with these constructs. The conformational change induced by insulin on its receptor could be detected as an energy transfer (BRET signal) between Rluc and EYFP. BRET signal parallels insulin-induced autophosphorylation of the fusion receptor. Dose-dependent effects of insulin, insulin-like growth factor 1, and epidermal growth factor on BRET signal are in agreement with known pharmacological properties of these ligands. Moreover, antibodies that activate or inhibit the autophosphorylation of the receptor have similar effects on BRET signal. This method allows for rapid analysis of the effects of agonists on insulin receptor activity and could therefore be used in a high-throughput screening test for discovery of molecules with insulin-like properties.
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Unité Propre de Recherche 415-Centre National de la Recherche Scientifique, Institut Cochin de Génétique Moléculaire, Paris, France.
Pernet
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Issad
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D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D004815:Epidermal Growth Factor / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008163:Luminescent Measurements; D011972:Receptor, Insulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D000906:Antibodies; D007328:Insulin; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin