Expression of the human GM-CSF receptor alpha subunit in Saccharomyces cerevisiae.
The alpha subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 45 kDa membrane protein with a higher apparent molecular weight of 50-85 kDa due to glycosylation. Previously, we had demonstrated that N-glycosylation plays a critical role in the GM-CSF receptor-ligand interaction. To assess the activity of the alpha subunit of the human GM-CSF receptor (GMRalpha) in a lower eukaryote, we expressed GMRalpha in the yeast S. cerevisiae and found that the protein has a lower apparent molecular weight compared with that expressed in mammalian cells. Using indirect immunofluorescence microscopy, we showed that GMRalpha protein expressed in yeast localizes to the plasma membrane. Although the yeast-expressed GMRalpha is able to interact with anti-GMRalpha antibody, the heterologously expressed receptor does not bind GM-CSF. Our results indicate that specific sites and/or forms of glycosylation of the GM-CSF receptor are crucial for ligand binding.
Tu
J
J
Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Golde
D W
DW
Vera
J C
JC
Heaney
M L
ML
D015870:Gene Expression; D006801:Humans; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae
D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins