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

Abstract

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.

Altered lipid raft–associated proximal signaling and translocation of CD45 tyrosine phosphatase in B lymphocytes from patients with systemic lupus erythematosus

“…Similar observations were reported in an unstimulated mouse cell line in which LR aggregation induced signaling analogous to TCR stimulation (27). Interestingly, our study showed that the intensity of staining for LRs decreased upon in vitro resting.…”

“…To trace LRs, B lymphocytes were stained with Alexa Fluor 647-conjugated CTB, which binds to the LR-associated GM1 gangliosides (27,28). Confocal microscopy revealed intense staining and an irregular morphologic appearance of LRs in B cells from patients with SLE.…”

Abnormal CTLA‐4 function in T cells from patients with systemic lupus erythematosus

“…After 72 h, a significant increase in membrane lipid GM1 (measured by binding to cholera toxin subunit B (CTB) [35] was noted in healthy T-responder cells stimulated via CD3/CD28 (p 5 0.005), which was downregulated by cross-linking CTLA-4 (p 5 0.004) as reported previously [6] (Fig. 5A upper panels).…”

CD40 translocation to lipid rafts: Signaling requirements and downstream biological events

“…The crucial role of lipid rafts in the signal transduction of several receptors, such as BCR, TCR and other members of the multichain immune recognition receptor family, has been well documented [29][30][31]. In each case, the lipid rafts concentrate the Src-family kinase (Lyn, Fyn and Lck among others) that initiates signaling and the immune receptors are excluded from the lipid rafts in resting or inactivated cells [32,33].…”

“…Such translocation was even shown not to necessitate the CD40 cytoplasmic tail, confirming therefore its independence of intracellular signaling. On the other hand, we show herein that CD40 localization into lipid rafts is essential for some CD40-mediated events such as PI-3K-induced Akt phosphorylation and subsequent CD80 upregulation, but not for p38, ERK and JNK activation.The crucial role of lipid rafts in the signal transduction of several receptors, such as BCR, TCR and other members of the multichain immune recognition receptor family, has been well documented [29][30][31]. In each case, the lipid rafts concentrate the Src-family kinase (Lyn, Fyn and Lck among others) that initiates signaling and the immune receptors are excluded from the lipid rafts in resting or inactivated cells [32,33].…”