Molecular associations involving CD16, CD45 and zeta and gamma chains on human natural killer cells.
zeta (CD3-zeta) and gamma (Fc epsilon RI gamma) chains associate with CD16, the low affinity receptor for IgG (Fc gamma RIII) on human NK cells and are essential for the cell surface expression of CD16 and for CD16-mediated effector functions. This study has investigated whether, on NK cells, molecules other than CD16 associate with zeta and gamma chains, as a method of identifying other NK cell surface molecules important in NK cell function. Cell surface biotinylated NK cells were lysed in digitonin, and the lysates immunoprecipitated with mAb to CD16, zeta and gamma, and the immunoprecipitates analysed by SDS-PAGE. CD16 mAb co-precipitated zeta and gamma chains (16 and 12kD, respectively) and in addition molecules of 24, 32-35, 100, 150 and 180-200 kD. Also, zeta mAb co-precipitated gamma chain, and molecules of 24-26, 32-35, 48, 50-66, 100, 150 and 180-200 kD; and gamma co-precipitated zeta chain, and molecules of 24-26, 29, 32-35, 37, 45, 49, 50-66 and 100 kD. While significant amounts of zeta and gamma were co-precipitated with CD16, 10 to 12-fold more zeta and gamma were immunoprecipitated with their respective mAb. Furthermore, depletion of CD16 from the lysate resulted in only a partial (10-12%) depletion of zeta and gamma, indicating that only a relatively small proportion (10-12%) of these molecules are associated with CD16. Interestingly, substantial amounts of molecules with electrophoretic mobility similar to CD16 (50-66 kD) were co-precipitated with zeta and gamma chain mAb from lysates depleted of CD16. In contrast to NK cells where zeta associated with a number of different molecules, the majority of zeta in T cells was found to be associated only with the TCR:CD3 complex. NK cells showed a strong association between CD45, CD16 and a 33 kD molecule and often a strong association of zeta with CD16, CD45 and an unidentified molecule of approximately 150 kD. Our results show first, that CD16, zeta and gamma each can be efficiently labelled by cell surface biotinylation, and second, that CD16, zeta and gamma each can form a complex with each other, and with a number of additional molecules including a 33 kD molecule and CD45 potentially important in NK cell function.
Altin
J G
JG
Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
Pagler
E B
EB
Kinnear
B F
BF
Warren
H S
HS
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000276:immunology; D015415:Biomarkers; D017252:CD3 Complex / Q000276:immunology*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D017493:Leukocyte Common Antigens / Q000276:immunology*; D011233:Precipitin Tests; D017455:Receptors, IgE / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015415:Biomarkers; D017252:CD3 Complex; D017455:Receptors, IgE; D017452:Receptors, IgG; D017493:Leukocyte Common Antigens