Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer

We have developed a procedure based on bioluminescence resonance energy tra nsfer (BRET) to monitor the activation state of the insulin receptor in vit ro. Human insulin receptor cDNA was fused to either Renilla luciferase (Rlu c) or enhanced yellow fluorescent protein (EYFP) coding sequences. Fusion i nsulin receptors were partially purified by wheat-germ lectin chromatograph y from human embryonic kidney 293 cells cotransfected with these constructs . The conformational change induced by insulin on its receptor could be det ected as an energy transfer (BRET signal) between Rluc and EYFP. BRET signa l parallels insulin-induced autophosphorylation of the fusion receptor. Dos e-dependent effects of insulin, insulin-like growth factor 1, and epidermal growth factor on BRET signal are in agreement with known pharmacological p roperties of these ligands. Moreover, antibodies that activate or inhibit t he auto phosphorylation of the receptor have similar effects on BRET signal . This method allows for rapid analysis of the effects of agonists on insul in receptor activity and could therefore be used in a high-throughput scree ning test for discovery of molecules with insulin-like properties.