Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3357-3362
High-affinity binding to the GM-CSF receptor requires intact
N-glycosylation sites in the extracellular domain of the 
subunit
Linghao Niu,
Mark L. Heaney,
Juan Carlos Vera, and
David W. Golde
From the Program in Molecular Pharmacology and Therapeutics and
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New
York, NY.
The human granulocyte-macrophage colony-stimulating factor (GM-CSF)
receptor consists of 2 glycoprotein subunits, GMR
and GMR
. GMR
in isolation binds to GM-CSF with low affinity. GMR 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 subunit on GM-CSF receptor function.
GMR 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 GMR 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 GMR in the
extracellular domain are not required for cell surface targeting but
are essential for high-affinity GM-CSF binding.
