The group had earlier seen that extracellular catalase, which breaks down H2O2, inhibited signaling through the GM-CSF receptor. They now find that ligand binding to this receptor increases the amount of extracellular H2O2. This occurs even when the cells are fixed before ligand addition or with a version of the receptor lacking intracellular signaling domains. H2O2 production was also evident with EGF binding to its receptor in the presence of an inhibitor of downstream signaling.
Extracellular destruction of H2O2 reduced signaling and cell survival downstream of GM-CSF binding. Addition of extracellular H2O2, by contrast, induced phosphorylation of downstream targets.
H2O2 makes a good second messenger—it is small, diffusible, and can be destroyed easily. It can also inactivate proteins such as phosphatases by oxidizing active site cysteine residues. How it is generated initially is a mystery. In intracellular ROS signaling the necessary electrons are supplied by NADPH oxidase, but outside the cell the only precedent is an unusual reaction between oxygen radicals and water catalyzed by antibodies. DeYulia and colleagues plan to mutate ligand and receptor residues to track down what parts of the proteins may be responsible for ROS generation.