The clustered Fcgamma receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner.
Phosphorylation of clustered Fcgamma receptor II (FcgammaRII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between FcgammaRII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross-linked FcgammaRII from non-ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol-anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross-linked FcgammaRII displayed a low buoyant density. The FcgammaRII present in the complexes underwent tyrosine phosphorylation. Cross-linked FcgammaRII and Lyn occupied common 100-200 nm detergent-resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with beta-cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent-resistant complexes. In parallel, the association of Lyn with cross-linked FcgammaRII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent-resistant membrane fraction and restored both Lyn association with cross-linked FcgammaRII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol-enriched membrane rafts can facilitate tyrosine phosphorylation of clustered FcgammaRII by Lyn kinase.
Kwiatkowska
K
K
Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland.
Sobota
A
A
D000818:Animals; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D046911:Macromolecular Substances; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011940:Receptor Aggregation; D017452:Receptors, IgG / Q000737:chemistry / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism; D020298:U937 Cells; D047392:beta-Cyclodextrins; D019061:src-Family Kinases / Q000378:metabolism*
D003505:Cyclodextrins; D046911:Macromolecular Substances; D017452:Receptors, IgG; D013107:Sphingolipids; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D019000:Phosphotyrosine; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D002784:Cholesterol; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C031215:betadex