Monitoring the activation state of insulin/insulin-like growth factor-1 hybrid receptors using bioluminescence resonance energy transfer

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 alpha beta-IRA chain associated with an alpha beta-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 alpha beta-Rluc moiety by the wild-type alpha beta 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.