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Monitoring the Activation State of Insulin/Insulin-Like Growth Factor-1 Hybrid Receptors Using Bioluminescence Resonance Energy Transfer

Institut Cochin, Département de Biologie Cellulaire, Paris, France; Institut National de la Santé et de la Recherche Médicale, U567, Paris, France; Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, Paris, France; and Université Paris Descartes, Facultéde Médecine René Descartes, UM 3, Paris, France

In cells expressing both the insulin receptor isoform A (IRA) and the insulin-like growth factor-1 receptor (IGF1R), the presence of hybrid receptors, made up of an -IRA chain associated with an -IGF1R chain, has been demonstrated. These heterodimers are found in normal cells, and they also seem to play crucial roles in a number of cancers. However, they remain difficult to study, due to the concomitant presence of IRA and IGF1R homodimers. Using bioluminescence resonance energy transfer (BRET), we have developed assays to specifically monitor the activation state of IRA/IGF1R hybrids, both in vitro and in living cells. The first assay allowed the study of ligand-induced conformational changes within hybrid receptors purified from cells cotransfected with one type of receptor fused to Renilla reniformis luciferase (Rluc), and the other type of receptor fused to yellow fluorescent protein (YFP). In these conditions, only hybrid receptors were BRET-competent. In the second assay, the activation state of IRA/IGF1R hybrids was monitored in real time, in living cells, by cotransfection of kinase-dead versions of IRA-Rluc or IGF1R-Rluc, wild-type untagged IRA or IGF1R, and a YFP-tagged soluble version of the substrate-trapping mutant of protein tyrosine phosphatase 1B (YFP-PTP1B-D181A-Cter). In hybrid receptors, trans-phosphorylation of the kinase-dead -Rluc moiety by the wild-type moiety induced the recruitment of YFP-PTP1B-D181A-Cter, resulting in a hybrid-specific ligand-induced BRET signal. Therefore, both methods allow monitoring of the activity of IRA/IGF1R hybrid receptor and could be used to detect molecules of therapeutic interest for the treatment of cancer.

Address correspondence to: Dr. Tarik Issad, Department of Cell Biology, Institut Cochin, 22 Rue Méchain, 75014 Paris, France. E-mail: issad{at}cochin.inserm.fr

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