Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor.
The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.
Cofano
F
F
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Moore
S K
SK
Tanaka
S
S
Yuhki
N
N
Landolfo
S
S
Appella
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011971:Receptors, Immunologic / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017471:Receptors, Interferon; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013945:Thymoma; D013953:Thymus Neoplasms; D014162:Transfection
D004273:DNA, Neoplasm; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D007371:Interferon-gamma