Binding and cross-linking of recombinant mouse interferon-. gamma. to receptors in mouse leukemic L1210 cells; interferon-. gamma. internalization and receptor down-regulation
Recombinant E. coli-derived murine IFN-..gamma.. (Mu-rIFN-..gamma..; 5 x 10/sup 7/ U/mg) was radiolabeled with /sup 125/I by the chloramine-T method without loss of its antiviral activity. The /sup 125/I-Mu-rIFN-..gamma.. showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 x 10/sup -10/ M. Binding of /sup 125/I-Mu-rIFn-..gamma.. to cells inhibited by both natural (glycosylated) and rIFN-..gamma.., but not by IFN-..gamma../..beta... Receptor-bound /sup 125/I-Mu-rIFN-..gamma.. was rapidly internalized when incubation temperature was raised from 4/sup 0/C to 37/sup 0/C. On internalization, almost no IFN-..gamma.. degradation was observed during 16 hr incubation. /sup 125/I-Mu-rIFN-..gamma.. binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-..gamma.., but not with IFN-..cap alpha../..beta... This receptor down-regulation was dose-dependent: 90% reduction of /sup 125/I-Mu-rIFN-..gamma.. binding was observed after preincubation with 100 U/ml. After removal of IFN-..gamma.. from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of /sup 125/I-Mu-rIFN-..gamma.. to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000.
- Research Organization:
- INSERM, Paris, France
- OSTI ID:
- 6936040
- Journal Information:
- J. Immunol.; (United States), Vol. 136:7
- Country of Publication:
- United States
- Language:
- English
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GLUCOPROTEINS
BIOSYNTHESIS
MOLECULAR STRUCTURE
INTERFERON
RECEPTORS
BIOCHEMICAL REACTION KINETICS
IODINE 125
LABELLED COMPOUNDS
LEUKEMIA
LYMPHOCYTES
LYMPHOKINES
MICE
RADIORECEPTOR ASSAY
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOHYDRATES
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DISEASES
ELECTRON CAPTURE RADIOISOTOPES
GROWTH FACTORS
HEMIC DISEASES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LEUKOCYTES
MAMMALS
MATERIALS
MEMBRANE PROTEINS
MITOGENS
NEOPLASMS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SACCHARIDES
SOMATIC CELLS
SYNTHESIS
TRACER TECHNIQUES
VERTEBRATES
550301* - Cytology- Tracer Techniques