Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer.
The insulin receptor (IR) is composed of two alpha-chains that bind ligands and two beta-chains that possess an intracellular tyrosine kinase activity. The IR is expressed in cells as two isoforms containing or not exon 11 (IRB and IRA, respectively). Several mRNA studies have demonstrated that the two isoforms are co-expressed in different tissues and in several cancer cells. IRA/IRB hybrid receptors, constituting of an alphabeta-chain from IRA and an alphabeta-chain from IRB, are likely to occur in cells co-expressing both isoforms, but their study has been hampered by the lack of specific tools. In previous work, we used BRET to study IR and IGF1R homodimers and heterodimers. Here, we have used BRET to characterize IRA/IRB hybrids. BRET saturation experiments showed that IRA/IRB hybrids are randomly formed in cells. Moreover, by co-transfecting HEK-293 cells with a luciferase-tagged kinase-dead version of one isoform and a wild-type untagged version of the other isoform, we showed that IRA/IRB hybrids can recruit, upon ligand stimulation, a YFP-tagged intracellular partner. Finally, using BRET, we have studied ligand-induced conformational changes within IRA/IRB hybrids. Dose-response experiments showed that hybrid receptors bind IGF-2 with the same affinity than IRA homodimers, whereas they bind IGF-1 with a lower affinity. Altogether, our data indicate that IRA/IRB hybrid receptors can form in cells co-expressing both IR isoforms, that they are capable of recruiting intracellular partners upon ligand stimulation, and that they have pharmacological properties more similar to those of IRA than those of IRB homodimers with regards to IGF-2.
Blanquart
Christophe
C
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.
Achi
Josepha
J
Issad
Tarik
T
D002460:Cell Line; D004735:Energy Transfer; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D049449:Luminescence; D008164:Luminescent Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*
D008024:Ligands; D008164:Luminescent Proteins; D007335:Insulin-Like Growth Factor II; D008156:Luciferases; D011972:Receptor, Insulin