High-affinity binding to the GM-CSF receptor requires intact N-glycosylation sites in the extracellular domain of the β subunit

Abstract

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor consists of 2 glycoprotein subunits, GMR and GMRβ. GMR in isolation binds to GM-CSF with low affinity. GMRβ does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMR. Previously, it was found that N-glycosylation of GMR is essential for ligand binding. The present study investigated the role of N-glycosylation of the β subunit on GM-CSF receptor function. GMRβ has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants were constructed, converting asparagine in the target sites to aspartic acid or alanine. A single mutation at any of the 3 consensus N-glycosylation sites abolished high-affinity GM-CSF binding in transfected COS cells. Immunofluorescence and subcellular fractionation studies demonstrated that all of the GMRβ mutants were faithfully expressed on the cell surface. Reduction of apparent molecular weight of the triple mutant proteins was consistent with loss of N-glycosylation. Intact N-glycosylation sites of GMRβ in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.