Biochemical analysis of the receptor for ubiquitin-like polypeptide.
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-beta, an isoform of the MNSF, has been isolated and characterized. MNSF-beta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to unstimulated D.10 cells. Detergent-extracted membrane proteins were applied to an immobilized Ubi-L column. SDS-polyacrylamide gel electrophoresis of eluted fraction revealed a band of Mr = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of Mr = 90,000 was visualized by immunoprecipitation of 125I-Ubi-L cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide) antibody, indicative of the association of this protein with the Ubi-L receptor complex. Amino acid sequence analysis of the 82-kDa polypeptide revealed that the Ubi-L receptor may be a member of a cytokine receptor family.
Nakamura
M
M
Department of Biochemistry, Shimane Medical University, Izumo 693, Japan.
Tanigawa
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D019921:Biotinylation; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018121:Receptors, Cytokine / Q000737:chemistry*; D012269:Ribosomal Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013491:Suppressor Factors, Immunologic / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D014357:Trypsin; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018121:Receptors, Cytokine; D012269:Ribosomal Proteins; D013491:Suppressor Factors, Immunologic; C119949:Ubi-L receptor; D014452:Ubiquitins; C072169:monoclonal nonspecific suppressor factor; C066109:monoclonal-nonspecific suppressor factor receptor; D014357:Trypsin