EGF receptor–ligand interaction generates extracellular hydrogen peroxide that inhibits EGFR-associated protein tyrosine phosphatases
Section snippets
Materials and methods
Proteins. Recombinant human soluble EGFR (sEGFR; Research Diagnostics) was approximately 80% pure. Recombinant human EGF (R&D Systems) was >97% pure. Recombinant human granulocyte–macrophage-colony stimulating factor (GM-CSF) was a gift from Amgen, Inc. Bovine liver catalase was obtained from Sigma. Active recombinant human full-length PTP1B was obtained from Upstate Biotechnology.
H2O2quantitation. H2O2 generated by receptor–ligand interaction was quantitated using a PeroxiLuminol Kit (World
Results and discussion
On the basis of our previous finding that EGFR–ligand interaction could generate extracellular hydrogen peroxide (H2O2) [23], we sought to examine the role of H2O2 in modulating the activity of phosphatases involved with EGFR signaling. First, we utilized purified EGF ligand and the sEGFR that possesses the extracellular binding domain of the receptor. We quantitated H2O2 production by EGF–sEGFR interaction in vitro using a luminometry-based assay. The PeroxiLuminol assay system determines H2O2
Acknowledgments
The authors dedicate this paper to Dr. David W. Golde, our mentor and friend. This work was supported by grants from the Lebensfeld and Schultz Foundations.
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