Artificially lipid-anchored proteins can elicit clustering-induced intracellular signaling events in Jurkat T-lymphocytes independent of lipid raft association.
We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquid-ordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoyl- or dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaf-folding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that cross-linking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.
Wang
Tian-yun
TY
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Leventis
Rania
R
Silvius
John R
JR
D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D018957:CD59 Antigens / Q000096:biosynthesis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003720:Densitometry; D003902:Detergents / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal; D008954:Models, Biological; D010766:Phosphorylation; D015398:Signal Transduction; D019809:Streptavidin / Q000737:chemistry; D013601:T-Lymphocytes / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D018957:CD59 Antigens; D003432:Cross-Linking Reagents; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008055:Lipids; D005677:G(M1) Ganglioside; D014443:Tyrosine; D001710:Biotin; D019809:Streptavidin; D002118:Calcium