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

The human granulocyte-macrophage colony stimulating factor(GM-CSF)receptor consists of 2 glycoprotein subunits, GMR alpha and GMR beta, GMR alpha in i solation binds to GM-CSF with low affinity, GMR beta does not bind GM-CSF b y itself, but forms a high-affinity receptor in association with GMR alpha, Previously, it was found that N-glycosylation of GMR alpha is essential fo r ligand binding, The present study investigated the role of N-glycosylatio n of the beta subunit on GM-CSF receptor function. GMR beta has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and As n346, Single mutants and triple mutants were constructed, converting aspara gine 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 fract ionation studies demonstrated that all of the GMR beta mutants were faithfu lly expressed on the cell surface, Reduction of apparent molecular weight o f the triple mutant proteins was consistent with loss of N-glycosylation. I ntact N-glycosylation sites of GMR beta in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM -CSF binding. (Blood, 2000;95:3357-3362) (C) 2000 by The American Society c t Hematology.