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© The Rockefeller University Press, 0021-9525/1999//447 $5.00
The Journal of Cell Biology, Volume 147, Number 2, , 1999 447-461

Original Article

Aggregation of Lipid Rafts Accompanies Signaling via the T Cell Antigen Receptor



Peter W. Janesa,b, Steven C. Leyb, and Anthony I. Mageea

a Division of Membrane Biology, National Institute for Medical Research, London NW7 1AA, United Kingdom
b Division of Cellular Immunology, National Institute for Medical Research, London NW7 1AA, United Kingdom
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.44-181-906-447744-181-959-3666

t-magee{at}nimr.mrc.ac.uk

s-ley{at}nimr.mrc.ac.uk

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)2–green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B–induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.

Key Words: lipid rafts • T cell antigen receptor • LCK • cholera toxin-B • signal transduction

© 1999 The Rockefeller University Press

1.used in this paper: APC, antigen-presenting cell; CT-B, cholera toxin B subunit; DAF, decay accelerating factor; ERK, extracellular-regulated kinase; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; ITAM, immunoreceptor tyrosine-based activation motif; MβCD, methyl-β-cyclodextrin; NFAT, nuclear factor of activated T cells; PTK, protein tyrosine kinase; PTyr, phosphotyrosine; SH2, Src-homology 2; TCR, T cell antigen receptor; TfR, transferrin receptor

S. Ley, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel.: 44-181-959-3666. Fax: 44-181-906-4477. E-mail: t-magee@nimr.mrc.ac.uk or s-ley@nimr.mrc.ac.uk


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