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J. Biol. Chem., Vol. 280, Issue 24, 22839-22846, June 17, 2005

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Artificially Lipid-anchored Proteins Can Elicit Clustering-induced Intracellular Signaling Events in Jurkat T-Lymphocytes Independent of Lipid Raft Association^*

Tian-yun Wang, Rania Leventis, and John R. Silvius

From the Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada

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.

Received for publication, March 16, 2005 , and in revised form, April 1, 2005.

^* This research was funded in part by an Operating Grant from the Canadian Institutes of Health Research (to J. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a studentship award from the Canadian Institutes of Health Research. Current address: Dept. of Chemistry, John Abbott College, Ste.-Anne-de-Bellevue, Québec H9X 3L9, Canada.

To whom correspondence should be addressed. Fax: 514-398-7384; E-mail: john.silvius{at}mcgill.ca.

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