Abstract
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 αβ-IRA chain associated with an αβ-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 αβ-Rluc moiety by the wild-type αβ 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.
Footnotes
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This work was supported by Association pour la Recherche sur le Cancer Grant 3781 and by the Ligue contre le Cancer, Comité de Paris Grant R04/75/75. C.G.-Y. holds a grant from the Fondation pour la Recherche Médicale.
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ABBREVIATIONS: IGF, insulin-like growth factor; TK, tyrosine kinase; IR, insulin receptor; IGF1R, insulin-like growth factor-1 receptor; BRET, bioluminescence resonance energy transfer; Rluc, R. reniformis luciferase; YFP, yellow fluorescent protein; PTP1B, protein tyrosine phosphatase 1B; IRA, insulin receptor isoform A; HEK, human embryonic kidney; KD, kinase dead; DMEM, Dulbecco's modified Eagle's medium; WGL, wheat germ lectin; MOPS, 3-(N-morpholino)propanesulfonic acid; mBU, milliBRET unit; AG1024, 3-bromo-5-t-butyl-4-hydroxy-benzylidene malonitrile.
- Received May 22, 2006.
- Accepted August 22, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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