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, GMRalpha and GMRbeta. GMRalpha in isolation binds to GM-CSF with low affinity. GMRbeta does not bind GM-CSF by itself, but forms a high-affinity receptor in association with GMRalpha. Previously, it was found that N-glycosylation of GMRalpha is essential for ligand binding. The present study investigated the role of N-glycosylation of the beta subunit on GM-CSF receptor function. GMRbeta 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 GMRbeta 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 GMRbeta in the extracellular domain are not required for cell surface targeting but are essential for high-affinity GM-CSF binding.
Niu
L
L
Program in Molecular Pharmacology and Therapeutics and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Heaney
M L
ML
Vera
J C
JC
Golde
D W
DW
D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000409:Alanine