Abstract
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 eitherRenilla 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.
Footnotes
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This work was supported the Center National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, and by a Roche-Pharma–Association de Langue Française d'Etude du Diabète et des Maladies Métaboliques) research grant.
- Abbreviations:
- BRET
- bioluminescence resonance energy transfer
- EYFP
- enhanced yellow fluorescent protein
- Rluc
- Renilla luciferase
- HEK
- human embryonic kidney
- MOPS
- 4-morpholinepropanesulfonic acid
- WGL
- wheat-germ lectin
- mBU
- milliBRET unit
- IR
- insulin receptor
- IGF
- insulin-like growth factor
- EGF
- epidermal growth factor
- Received May 7, 2001.
- Accepted June 15, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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