A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer.

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. This autophosphorylation stimulates the tyrosine kinase activity of the receptor toward intracellular substrates involved in the transmission of the signal. The discovery of pharmacological agents that specifically activate the tyrosine kinase activity of the insulin receptor will be of great importance for the treatment of insulin-resistant or insulin-deficient patients. We have developed a procedure based on Bioluminescence Resonance Energy Transfer (BRET) to monitor the activation state of the insulin receptor. Human insulin receptor cDNA, was fused to either Renilla luciferase or yellow fluorescent protein coding sequences. Fusion insulin receptors were partially purified by wheat-germ lectin chromatography from HEK-293 cells co-transfected with these constructs. The conformational change induced by insulin on its receptor could be detected as an energy transfer (BRET signal) between Renilla luciferase and yellow fluorescent protein. BRET signal paralleled insulin-induced autophosphorylation of the fusion receptor. Dose-dependent effects of insulin, insulin-like growth factor 1 and epidermal growth factor on BRET signal were in agreement with known pharmacological properties of these ligands. Moreover, an antibody, which activated the autophosphorylation of the receptor, had similar effects on BRET signal. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and could, therefore, be used in high-throughput screening for the discovery of molecules with insulin-like properties.