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Vol. 60, Issue 4, 640-645, October 2001
Unité Propre de Recherche 415-Centre National de la
Recherche Scientifique, Institut Cochin de Génétique
Moléculaire, Paris, France.
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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