Characterization of the alpha subunit of the IFN-alpha receptor. Evidence of N- and O-linked glycosylation and association with other surface proteins.
We studied the association of the alpha subunit of the (IFN-alpha-receptor) to other receptor components in the human H-929 and U-266 myeloma cell lines. Immunoprecipitation performed with the IFNaR3 mAb showed that two proteins with molecular masses of 205 and 145 kDa are co-precipitated with the alpha subunit. These complexes may not bind IFN-alpha as shown by studies using the heterobifunctional cross-linking reagent Denny-Jaffe and by partial cleavage of the homobifunctional cross-linker dithio succinimidyl propionate. These studies also provided evidence that at least two subunits with molecular mass of 130 kDa (alpha subunit) and 110 kDa (including 20 kDa corresponding to IFN-alpha) contribute to the formation of the IFN-alpha-receptor complex. To further characterize the alpha subunit of the IFN-alpha-receptor, immunoprecipitates using the mAb IFNaR3 were sequentially treated with N-glycanase, neuraminidase and O-glycanase. These studies showed that the alpha subunit is heavily glycosylated and has a protein precursor with a molecular mass of 68 kDa. Binding studies provided evidence for high and low affinity binding sites for IFN-alpha 2. Affinity cross-linking experiments under low and high affinity conditions suggest that the high affinity binding site of the IFN-alpha-receptor is formed by a complex containing the alpha subunit, whereas the 110-kDa subunit may bind IFN-alpha 2 under low affinity conditions.
Platanias
L C
LC
Department of Medicine University of Chicago, Pritzker School of Medicine, IL 68637.
Pfeffer
L M
LM
Cruciani
R
R
Colamonici
O R
OR
D001665:Binding Sites; D006031:Glycosylation; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D011233:Precipitin Tests; D017471:Receptors, Interferon / Q000737:chemistry* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016898:Interferon-alpha; D008565:Membrane Proteins; D017471:Receptors, Interferon