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Association of FcRII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation¹

Osamu Katsumata^*, Miki Hara-Yokoyama^2,, Catherine Sautès-Fridman, Yasuko Nagatsuka, Toshiaki Katada^¶, Yoshio Hirabayashi^||, Kazufumi Shimizu, Junko Fujita-Yoshigaki^*, Hiroshi Sugiya^* and Shunsuke Furuyama^*

^* Department of Physiology, Nihon University School of Dentistry, Matsudo, Japan; Department of Hard Tissue Engineering, Biochemistry, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; Centre de Recherches Biomédicals des Cordeliers, Paris, France; Department of Microbiology, Nihon University School of Medicine, Tokyo, Japan; ^¶ Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and ^|| Brain Science Institute, Institute of Physical and Chemical Research, Wako, Japan

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcRs resulted in a marked increase in the tyrosine phosphorylation of FcRIIa, p58^lyn, and p120^c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcRs 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 FcRs as well as p120^c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl--cyclodextrin, the cross-linking did not induce the association of FcRs as well as p120^c-cbl with DRMs. In addition, although the physical association between FcRIIa 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--cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcRII to lipid rafts is important for the activation of FcRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

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