Clarification of the role of N-glycans on the common β-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 β-receptor in ligand-binding and receptor activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific α-subunits and a common β-receptor (βc), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the βc-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether βc N-glycosylation plays a role in receptor function, since all structural studies of human βc to date have utilized recombinant protein lacking N-glycosylation at Asn328. Here, by eliminating individual N-glycans in human βc and the related murine homolog, βIL-3, we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the β-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human βc domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn328.