Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.
A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.
Constantinescu
S N
SN
Department of Pathology (576 BMH), University of Tennessee Health Science Center, Memphis 38163.
Croze
E
E
Wang
C
C
Murti
A
A
Basu
L
L
Mullersman
J E
JE
Pfeffer
L M
LM
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism
D000911:Antibodies, Monoclonal; D016898:Interferon-alpha; D046911:Macromolecular Substances; D008565:Membrane Proteins; D017471:Receptors, Interferon; D053633:Receptor, Interferon alpha-beta; D019000:Phosphotyrosine; D014443:Tyrosine