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Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

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Signaling proteins are concentrated in α-CD3–isolated PM fragments. α-CD3 and α-TfR immunoisolates were analyzed by Western blot using antibodies against the indicated proteins. Recovery of biotinylated cell surface was monitored by Western blot with HRP-avidin. 1:100 (A) and 1:50 (B) dilutions of pelleted homogenate were loaded. The positions of molecular mass markers (in kilodaltons) are shown. Closed circles mark the position of the Ab light chain.
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Figure 2: Signaling proteins are concentrated in α-CD3–isolated PM fragments. α-CD3 and α-TfR immunoisolates were analyzed by Western blot using antibodies against the indicated proteins. Recovery of biotinylated cell surface was monitored by Western blot with HRP-avidin. 1:100 (A) and 1:50 (B) dilutions of pelleted homogenate were loaded. The positions of molecular mass markers (in kilodaltons) are shown. Closed circles mark the position of the Ab light chain.

Mentions: LAT and TCR are enriched ∼50-fold over total pelletable protein in the α-CD3 immunoisolated PM fragments (Table ). Next, we tested whether they specifically accumulate in the α-CD3 isolated PM fragments. As a measure for PM recovery, we used biotinylated cell surface probed on a Western blot with avidin-HRP (Fig. 2). α-Transferrin receptor (TfR) antibody–coated beads were employed to isolate control PM fragments. The α-TfR beads did not elicit Ca2+ fluxes, whereas α-CD3 beads efficiently triggered a sustained rise in intracellular Ca2+ (data not shown). The amounts of PM retrieved by α-CD3 and α-TfR beads after different times of incubation at 37°C were similar, corresponding to 1–2% of the total biotinylated cell surface (Fig. 2, Table ). As expected, TfR was enriched in α-TfR bead isolates. The presence of TfR in the α-CD3 isolates indicates a background that corresponded to the amount of isolated PM. Likewise, the α-TfR beads contained background amounts of LAT. However, the amounts of LAT, PLC-γ, and Grb2 in α-CD3 immunoisolates was much higher, demonstrating that these proteins specifically accumulate in α-CD3–immunoisolated PM fragments together with the TCR ζ-chain. In contrast, α-CD3, α-TfR immunoisolates, and dilutions of cell homogenate, normalized to comparable amounts of PM, contained similar amounts of Lck and of Fyn (Fig. 2). Thus, Lck and Fyn were not detectably concentrated in the α-CD3 immunoisolated PM fragments. Next, we assayed recovery of raft glycolipid GM1 and cholesterol in the immunoisolates using an overlay with the choleratoxin B subunit or [3H]cholesterol-labeled cells, respectively. Neither GM1 nor cholesterol significantly accumulated in PM fragments immunoisolated with α-CD3 beads (Fig. 2 and Table ). Moreover, we did not detect an enrichment of GPI-anchored proteins using an overlay with the GPI probe bacterial toxin proaerolysin (data not shown; Abrami et al. 1998).


Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

Signaling proteins are concentrated in α-CD3–isolated PM fragments. α-CD3 and α-TfR immunoisolates were analyzed by Western blot using antibodies against the indicated proteins. Recovery of biotinylated cell surface was monitored by Western blot with HRP-avidin. 1:100 (A) and 1:50 (B) dilutions of pelleted homogenate were loaded. The positions of molecular mass markers (in kilodaltons) are shown. Closed circles mark the position of the Ab light chain.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192654&req=5

Figure 2: Signaling proteins are concentrated in α-CD3–isolated PM fragments. α-CD3 and α-TfR immunoisolates were analyzed by Western blot using antibodies against the indicated proteins. Recovery of biotinylated cell surface was monitored by Western blot with HRP-avidin. 1:100 (A) and 1:50 (B) dilutions of pelleted homogenate were loaded. The positions of molecular mass markers (in kilodaltons) are shown. Closed circles mark the position of the Ab light chain.
Mentions: LAT and TCR are enriched ∼50-fold over total pelletable protein in the α-CD3 immunoisolated PM fragments (Table ). Next, we tested whether they specifically accumulate in the α-CD3 isolated PM fragments. As a measure for PM recovery, we used biotinylated cell surface probed on a Western blot with avidin-HRP (Fig. 2). α-Transferrin receptor (TfR) antibody–coated beads were employed to isolate control PM fragments. The α-TfR beads did not elicit Ca2+ fluxes, whereas α-CD3 beads efficiently triggered a sustained rise in intracellular Ca2+ (data not shown). The amounts of PM retrieved by α-CD3 and α-TfR beads after different times of incubation at 37°C were similar, corresponding to 1–2% of the total biotinylated cell surface (Fig. 2, Table ). As expected, TfR was enriched in α-TfR bead isolates. The presence of TfR in the α-CD3 isolates indicates a background that corresponded to the amount of isolated PM. Likewise, the α-TfR beads contained background amounts of LAT. However, the amounts of LAT, PLC-γ, and Grb2 in α-CD3 immunoisolates was much higher, demonstrating that these proteins specifically accumulate in α-CD3–immunoisolated PM fragments together with the TCR ζ-chain. In contrast, α-CD3, α-TfR immunoisolates, and dilutions of cell homogenate, normalized to comparable amounts of PM, contained similar amounts of Lck and of Fyn (Fig. 2). Thus, Lck and Fyn were not detectably concentrated in the α-CD3 immunoisolated PM fragments. Next, we assayed recovery of raft glycolipid GM1 and cholesterol in the immunoisolates using an overlay with the choleratoxin B subunit or [3H]cholesterol-labeled cells, respectively. Neither GM1 nor cholesterol significantly accumulated in PM fragments immunoisolated with α-CD3 beads (Fig. 2 and Table ). Moreover, we did not detect an enrichment of GPI-anchored proteins using an overlay with the GPI probe bacterial toxin proaerolysin (data not shown; Abrami et al. 1998).

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

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