Granulocyte-macrophage colony-stimulating factor signals for increased glucose transport via phosphatidylinositol 3-kinase- and hydrogen peroxide-dependent mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent Km for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the a subunit of the GM-CSF receptor (alphaGMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity aGMR and in human cells expressing the high affinity alphabetaGMR complex. We identified a Src homology 3 domain-binding motif in aGMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to aGMR was obtained by co-immunoprecipitation with antibodies to aGMR and p85, and an aGMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of aGMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H2O2), generated by ligand or antibody binding to aGMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-aGMR antibody-stimulated glucose uptake in alphaGMR-expressing oocytes, and H2O2 activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to aGMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H2O2 generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated a subunit of the GM-CSF receptor.