Monitoring the activation state of the insulin-like growth factor-1 receptor and its interaction with protein tyrosine phosphatase 1B using bioluminescence resonance energy transfer.
We have developed two bioluminescence resonance energy transfer (BRET)-based approaches to monitor 1) ligand-induced conformational changes within partially purified insulin-like growth factor-1 (IGF-1) receptors (IGF1R) and 2) IGF1R interaction with a substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B-D181A) in living cells. In the first assay, human IGF1R fused to Renilla reniformis luciferase (Rluc) or yellow fluorescent protein (YFP) were cotransfected in human embryonic kidney (HEK)-293 cells. The chimeric receptors were then partially purified by wheat germ lectin chromatography, and BRET measurements were performed in vitro. In the second assay, BRET measurements were performed on living HEK-293 cells cotransfected with IGF1R-Rluc and YFP-PTP1B-D181A. Ligand-induced conformational changes within the IGF1R and interaction of the IGF1R with PTP1B could be detected as an energy transfer between Rluc and YFP. Dose-response experiments with IGF-1, IGF-2, and insulin demonstrated that the effects of these ligands on BRET correlate well with their known pharmacological properties toward the IGF1R. Inhibition of IGF1R autophosphorylation by the tyrphostin AG1024 (3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile) resulted in the inhibition of IGF1-induced BRET signal between the IGF1R and PTP1B. In addition, an anti-IGF1R antibody known to inhibit the biological effects of IGF-1 inhibited ligand-induced BRET signal within the IGF1R, as well as between IGF1R and PTP1B. This inhibition of BRET signal paralleled the inhibition of the ligand-induced autophosphorylation of the IGF1R by this antibody. In conclusion, these BRET-based assays permit 1) the rapid evaluation of the effects of agonists or inhibitory molecules on IGF1R activation and 2) the analysis of the regulation of IGF1R-PTP1B interaction in living cells.
Department of Cell Biology, Institut Cochin, 22 Rue Méchain, 75014 Paris, France.
Christophe
C
Blanquart
Nicolas
N
Boute
Danièle
D
Lacasa
Tarik
T
Issad
D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004735:Energy Transfer; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D007328:Insulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D017526:Receptor, IGF Type 1; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases