Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation.
IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.
Katsumata
O
O
Department of Physiology, Nihon University School of Dentistry, Matsudo, Japan.
Hara-Yokoyama
M
M
Sautès-Fridman
C
C
Nagatsuka
Y
Y
Katada
T
T
Hirabayashi
Y
Y
Shimizu
K
K
Fujita-Yoshigaki
J
J
Sugiya
H
H
Furuyama
S
S
D002478:Cells, Cultured; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism* / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D017452:Receptors, IgG / Q000378:metabolism* / Q000502:physiology*; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases; D047392:beta-Cyclodextrins; D019061:src-Family Kinases / Q000378:metabolism
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D003505:Cyclodextrins; D003902:Detergents; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D017452:Receptors, IgG; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D013481:Superoxides; D019000:Phosphotyrosine; D014212:Tretinoin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D016392:Proto-Oncogene Proteins pp60(c-src); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C067419:CBL protein, human