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PDBsum entry 2k4f
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Immune system, signaling protein
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PDB id
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2k4f
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References listed in PDB file
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Key reference
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Title
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Regulation of t cell receptor activation by dynamic membrane binding of the cd3epsilon cytoplasmic tyrosine-Based motif.
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Authors
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C.Xu,
E.Gagnon,
M.E.Call,
J.R.Schnell,
C.D.Schwieters,
C.V.Carman,
J.J.Chou,
K.W.Wucherpfennig.
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Ref.
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Cell, 2008,
135,
702-713.
[DOI no: ]
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PubMed id
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Abstract
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Many immune system receptors signal through cytoplasmic tyrosine-based motifs
(ITAMs), but how receptor ligation results in ITAM phosphorylation remains
unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon
cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through
fluorescence resonance energy transfer between a C-terminal fluorescent protein
and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon
residues and acidic phospholipids enriched in the inner leaflet of the plasma
membrane were required for binding. The nuclear magnetic resonance structure of
the lipid-bound state of this cytoplasmic domain revealed deep insertion of the
two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor
ligation thus needs to result in unbinding of the CD3epsilon ITAM from the
membrane to render these tyrosines accessible to Src kinases. Sequestration of
key tyrosines into the lipid bilayer represents a previously unrecognized
mechanism for control of receptor activation.
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Figure 3.
Figure 3. The CD3  epsilon
Cytoplasmic Domain Is Bound to the Inner Leaflet of the Plasma
Membrane
(A and C) The FRET efficiency was determined for
transfectants expressing the CD3 epsilon
WT or a CD3 epsilon
mutant cytoplasmic domain (Emut1+2) (10 cells/construct, error
bars indicate SEM) using the quenching approach, as in Figures
2C and 2E. R18-PB: R18 photobleached.
(B and D) The donor
dequenching method was used to measure the FRET efficiency for
the same transfectants (20 cells shown here from one
experiment), as in Figures 2D and 2F.
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Figure 7.
Figure 7. Membrane Binding Renders the Tyrosines of CD3 epsilon
Inaccessible to Lck
(A) Lipid binding prevents tyrosine
phosphorylation of CD3 epsilon
[CD]. The tyrosine residues of CD3 epsilon
[CD] were efficiently phosphorylated by Lck in an in vitro
kinase assay. Addition of POPG/DHPC bicelles but not control
POPC/DHPC bicelles prevented tyrosine phosphorylation. Western
blots were probed with a phospho-tyrosine antibody (biotinylated
P-Tyr-100 and streptavidin-horseradish peroxidase, SA-HRP) and a
CD3 epsilon
[CD] antibody (CD3 epsilon
m20 and anti-goat IgG-HRP).
(B) Neither endogenous CD3 epsilon
nor the HA-KIR-CD3 epsilon
[CD]-TFP reporter protein are basally phosphorylated. HA-KIR-CD3
epsilon
[CD]-TFP transfected Jurkat cells were solubilized using 1% NP40
and proteins were immunoprecipitated with CD3ζ (6B10.2), CD3
epsilon
(UCHT1), HA (3F10), or FLAG (M2, isotype control) antibodies.
Phosphorylated and total proteins were detected with a
phospho-tyrosine antibody (biotinylated P-Tyr-100 and SA-HRP,
upper panel), a CD3 epsilon
[CD] antibody (CD3 epsilon
m20 and anti-goat IgG-HRP, which detects CD3 epsilon
and the reporter protein), or an antibody to the ζ chain
(anti-CD3ζ 6B10.2-HRP, lower panel). SDS-PAGE was performed
under nonreducing conditions.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Cell
(2008,
135,
702-713)
copyright 2008.
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