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1
*
Section of Rheumatology, Departments of Medicine and
Pathology, University of Chicago, Chicago, IL 60637; and
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742
3 Abbreviations used in this paper: BCR, B cell Ag receptor; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; BLNK, B cell linker protein; GFP, green fluorescent protein; ITAM, immunoreceptor tyrosine-based activation motif; Lamp-1, lysosome-associated membrane protein-1; MIIC, MHC class II-enriched compartment; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PLC, phospholipase C; PVDF, polyvinylidene difluoride; SH2, Src homology 2; SNARE, soluble N-ethylmalemide-sensitive factor attachment protein receptor; TfR, transferrin receptor.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. We postulated
that the Ig nonimmunoreceptor tyrosine-based activation motif
tyrosines, Y176 and Y204, contributed to
receptor trafficking. Ig(Y
F176,204)/Ig
receptors
were targeted to late endosomes, but were excluded from the vesicle
lumen and could not facilitate the presentation of Ag to T cells.
Subsequent analysis demonstrated that phosphorylation of
Y176/Y204 recruited the B cell linker protein,
Vav, and Grb2. Reconstitution of Ig(YF176,204)/Ig
with the B cell linker protein rescued both receptor-facilitated Ag
presentation and entry into the MHC class II-enriched compartment.
Thus, aggregation accelerates receptor trafficking by recruiting two
separate signaling modules required for transit through sequential
checkpoints. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indeed, the endocytic compartments preferentially targeted by the BCR are specialized for Ag processing. The best described of these is the MHC class II-enriched compartment (MIIC), which in murine B cells is multivesicular (7), lysosome-associated membrane protein-1+ (Lamp-1+), MHC class II+ (8), H2-M+, transferrin receptor (TfR)-, and M6PR- (9, 10, 11, 12, 13). In this compartment, MHC class II molecules loaded with specific peptides are first detected following antigenic recognition by the BCR (8). Peptide loading has also been described in an early endocytic compartment bearing the TfR termed the CIIV (14). It is thought that the MIIC, CIIV, and potentially other compartments can be used within a particular cell line to process Ag (15). The MIIC, however, might be preferred because the low pH of this compartment enhances the activity of many hydrolases and may increase the activity of H2-M (16, 17, 18).
The MIIC is not static, but its biochemical and physical properties are responsive to BCR-initiated signals (19, 20). First, receptor activation induces an accumulation of newly synthesized MHC class II within the MIIC (12, 21, 22). This may be facilitated by protein kinase C-dependent phosphorylation of invariant chain that augments late endosomal targeting (19). Second, receptor activation increases the acidity of the MIIC (12). Finally, BCR aggregation can induce the coalescence and fusion of MIIC vesicles to form large central vesicles of >1 µm in diameter (12). These results suggest that signals transduced through the BCR induce changes in the environment of the MIIC that enhance Ag processing and peptide loading onto MHC class II.
In addition to regulating the MIIC, BCR cross-linking greatly accelerates the internalization and targeting of antigenic complexes to it. The endocytic route through which the receptor traffics is not changed (23). However, aggregation of the BCR decreases the time spent by the bulk of receptor complexes in early endosomes, while hastening the appearance of MHC class II-peptide complexes within the MIIC (5, 23, 24, 25). The accelerated delivery of multivalent Ags favors the presentation of both high- and low-affinity Ags captured from the membranes of cells such as follicular dendritic cells (24). Receptor aggregation enhances the presentation of high affinity Ags by as much as 50-fold (23) and may allow initiation of immune responses to low-affinity Ags (24).
Aggregation of the BCR induces the phosphorylation of several tyrosines
in the cytoplasmic tails of the receptor-associated chains Ig and
Ig (26). Receptor-associated kinases phosphorylate
these immunoreceptor tyrosine-based activation motif (ITAM) tyrosines
upon engagement with a polyvalent ligand (27, 28, 29).
Phosphorylation of the Ig ITAM serves both to recruit and activate
the tyrosine kinase Syk (30, 31). One of the most proximal
substrates of Syk, and possibly of other receptor-associated kinases,
is the adaptor molecule B cell linker protein (BLNK) (32),
which is required for the activation of phospholipase C
(PLC) and
c-Jun N-terminal kinase (33). Phosphorylation of BLNK
forms a scaffold for the assembly of Grb2, Vav, Nck, and PLC2 at the
plasma membrane (32).
Many of the molecules involved in signal initiation have been
implicated in trafficking of the aggregated receptor complex. The
cytoplasmic tails of Ig and Ig are both necessary and sufficient
to recapitulate the trafficking of the BCR (34). Mutation
of the Ig ITAM tyrosines (Y182 and
Y193) to phenylalanines inhibits both Ag
presentation and sorting to the MIIC (34). Syk also plays
a crucial role in sorting receptors to late endosomes (35, 36). The mechanisms by which Syk, or any other molecules that
may bind the phospho-Ig ITAM, mediates receptor trafficking are
unknown. In addition, Ig contains two non-ITAM tyrosines at residues
Y176 and Y204. One study
indicated that BLNK might bind to phosphorylated
Y204; however, the contribution of this
association to signaling or receptor trafficking was not investigated
(37).
In this study, we report that phosphorylation of the non-ITAM Ig
tyrosines is necessary for efficient delivery of aggregated receptor
complexes into late endosomes. Both mutant receptor complexes
(Ig(YF176,204)/Ig) and wild-type
Ig/Ig sort efficiently to late endosomes. However, the transport
vesicles carrying mutant complexes did not enter the MIIC endosomes.
These excluded receptors were compromised in their ability to
facilitate the presentation of pulsed Ag to T cells. Subsequent
experiments demonstrated that phosphorylation of
Y204 served to recruit BLNK, Vav, and Grb2 to the
receptor. Reconstituting
Ig(YF176,204)/Ig with BLNK rescued the
ability of the receptor both to enter late endosomes and to facilitate
the presentation of Ag. These data reveal an unexpected mechanism by
which the direct recruitment of BLNK to the BCR mediates the rapid
delivery of antigenic complexes into late endocytic processing
compartments.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine B cell lymphoma A20IIA1.6 (IgG2a+, FcR-, I-Ad+, I-Ed+) (2) was cultured in IMDM (Life Technologies, Grand Island, NY) containing 10% FCS (HyClone, Logan, UT), 2 mM of glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 7.5% CO2. The myoglobin-specific T cell clone (Ach 8.2; A. Sant, University of Chicago, Chicago, IL) was maintained in DMEM (Life Technologies) supplemented as above for IMDM.
Platelet-derived growth factor receptor (PDGFR) chimera
The basic construction, expression, and activation of the PDGFR
chimeras have been previously described (38, 39). Tyrosine
to phenylalanine mutations were engineered using complementary primers
and PCR. To fuse BLNK to the C terminus of
Ig(YF176,204), BLNK was first PCR-amplified
from murine cDNA using primers complementary to the entire open reading
frame. The 5' primers contained an EcoRI site, and the 3'
primer contained a XhoI site. Using these sites, the cDNA
was cloned to the 3' end of a cDNA encoding
PDGFR/Ig(YF176,204) and then inserted
into the retroviral vector MIGR1. Virus was packaged in the GP-293
packaging cell line (Clontech Laboratories, Palo Alto, CA), and
supernatants were used to infect A20IIA1.6 cells expressing
PDGFR/Ig. Cells were sorted by FACS for green fluorescent protein
(GFP) expression. All other cDNAs were cloned into pMuTKneo that
contains the thymidine kinase promoter and the µ enhancer. Plasmids
were cotransfected with plasmids encoding PDGFR/Ig into A20IIA1.6
cells and clonal transfectants selected with G418 (Life Technologies).
To determine surface expression of each chimeric molecule, or the BCR,
cells were first stained with mouse anti-PDGFR (Genzyme,
Cambridge, MA; catalog number 1264-00) and anti-PDGFR Abs (R&D
Systems, Minneapolis, MN; catalog number MAB1263), followed by
FITC-conjugated anti-IgG1 (Zymed Laboratories, San Francisco, CA;
catalog number 61-0100), or with goat anti-mouse IgG2a (Southern
Biotechnology Associates, Birmingham, AL; catalog number 1080-01),
followed by donkey anti-goat IgG FITC (Jackson ImmunoResearch
Laboratories, West Grove, PA; catalog number 705-095-147). Samples were
then examined by flow cytometry (FACScan; BD Biosciences, Bedford,
MA).
Ag presentation
Preparation of the myoglobin/goat anti-rabbit IgG conjugate
(final concentration of 
20 ng/ml) has been previously described
(34). Myoglobin was targeted to the chimeric complexes on
each transfectant as a pulse by incubating the transfected cells on ice
consecutively with PDGF-BB (100 ng/ml; Sigma-Aldrich, St. Louis,
MO) for 5 min, mouse anti-human PDGFR (5 µg/ml) for 3 min, and
rabbit anti-mouse IgG1 (5 µg/ml) for 10 min. The cells were
washed and then incubated with the noted dilutions of
myoglobin-conjugated goat anti-rabbit IgG for 15 min on ice. Cells
were then incubated at 37°C for 30 min and washed, and 1 x
105 cells/sample were incubated with 1 x
105 myoglobin-specific T cell clones (Ach 8.2)
for 24 h at 37°C. Supernatants were then harvested for IL-2
analysis by ELISA. Briefly, rat anti-mouse IL-2 (1 µg/ml; BD
PharMingen, San Diego, CA; catalog number 18161D) was coated in binding
buffer (0.1 M of Na2HPO4
(pH 9)) onto an Immunolon microtiter plate (Dynatech Laboratories,
Chantilly, VA) at 4°C overnight. Plates were washed with PBS/Tween,
blocked with 1% BSA/PBS for 30 min at room temperature, and then
washed. Supernatants (100 µl) were incubated on coated wells for
2 h at room temperature, then washed with PBS/Tween. The IL-2
standard was purchased from R&D Systems. Biotin rat anti-mouse IL-2
(1 µg/ml; BD PharMingen; catalog number 554426) in blocking buffer
was added to wells for 1 h at room temperature and then washed.
Peroxidase-conjugated streptavidin (1 µg/ml; Jackson ImmunoResearch
Laboratories; catalog number 016-030-084) was added for 30 min, and the
plates were then washed. DAKO (Carpinteria, CA) served as the
substrate, and the plates were read on an ELISA reader (Corixa,
Hialeah, FL) at 650 nm. The data reported are representative of that
obtained in three separate experiments.
Confocal microscopy
For BCR or chimera/Lamp-1 costaining, transfectants were
incubated with either goat anti-mouse IgG2a (Southern Biotechnology
Associates), followed by donkey anti-goat IgG FITC (Jackson
ImmunoResearch Laboratories) or PDGF-BB ligand, mouse anti-PDGFR
Ab as described above, and then with FITC-labeled (Zymed Laboratories)
rabbit anti-mouse IgG1 (Jackson ImmunoResearch Laboratories) or
rabbit anti-mouse IgG1, followed by
7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labeled donkey
anti-rabbit IgG (Jackson ImmunoResearch Laboratories; catalog
number 711-155-152). Cells were placed at 37°C for 30 min and then
fixed with 3% paraformaldehyde/3% sucrose in PBS, or in the case of
GFP-expressing cells, fixed with PBS containing 2% formaldehyde and
0.05% glutaraldehyde (both from Sigma-Aldrich). Following fixation,
cells were permeabilized with 0.05% saponin and stained for Lamp-1, as
described previously (34). Confocal sections (0.75–1
µm) were acquired using a Zeiss (Oberkochen, Germany) 410 confocal
microscope. Images are displayed by pseudocoloring using LSM
software.
Immunoelectron microscopy
Cells were incubated with PDGF-BB ligand, mouse anti-human
PDGFR, and rabbit anti-mouse IgG1, followed by 5-nm gold
particle-conjugated goat anti-rabbit IgG (British Biocell
International, Cardiff, U.K.; catalog number 15725) on ice, stimulated
for 30 min at 37°C, then fixed in 8% paraformaldehyde/250 mM of
HEPES (pH 7.2). Cells were washed in PBS, infiltrated with 2.3 M of
sucrose, frozen in liquid nitrogen, and sectioned at -110°C. To
immunostain sections for the late endosomal marker Lamp-1, samples were
incubated in 10% FCS/0.12% glycine/PBS at room temperature for 30
min, then incubated with ID4B (Developmental Studies Hybridoma Bank;
University of Iowa, Iowa City, IA) for 30 min at room temperature. The
grids were washed in PBS/0.12% glycine and then stained with 10-nm
gold particle-conjugated goat anti-rat IgG (catalogue 15771). Grids
were then washed with PBS/glycine and then distilled water, followed by
incubation with 1.8% methyl cellulose/0.3% uranyl acetate. A 100 CX
transmission electron microscope (JEOL, Peabody, MA) was used at an
accelerating voltage of 60 kV to view the sample (40).
Quantitation of the micrographs was analyzed by blind scoring.
Internalization
Internalization was quantitated as previously described (34). Briefly, cells were stimulated via the chimera receptor, as described above, except the anti-mouse IgG1 Ab was HRP-labeled (Zymed Laboratories; catalog number 61-0120). After washing, 2.5 x 107 cells were placed at 37°C for 5, 10, or 30 min, and then put on ice to prevent further endocytosis and then washed again. The concentration of noninternalized chimera labeled with HRP-conjugated Ab was identified colorimetrically by the addition of 5 mM of O-phenylenediamine HCl in 0.15 M of phosphate buffer (pH 6) with 0.15% H2O2 for 15 min at room temperature (41). The total HRP activity for the individual clones was determined by solubilizing a duplicate sample with PBS/1% Nonidet P-40 for 10 min at room temperature and then adding substrate, as outlined above for 15 min. Peroxidase reactions were terminated by the addition of 6 N of HCl and centrifugation at 20,000 x g for 10 min at 4°C. For a given experiment, total HRP activity varied <20% between clones.
Peptide precipitations
Peptides were synthesized at the University of Chicago peptide
facility. The sequence of the peptide encompassing
Y176 was MPDDYEDENLY, and that encompassing
Y204 was LQGTYQDVGNL (using the single letter
amino acid code). The peptides were synthesized with or without a
phosphate group on the fifth tyrosine of the peptide. The peptides were
covalently coupled to normal human serum-activated Sepharose (Amersham
Biosciences, Piscataway, NJ). For precipitations, 2 x
106 cells were washed and resuspended in 400-µl
serum-free medium. Cells were either left unstimulated or stimulated
with 25 µg/ml rabbit anti-mouse IgG (Jackson ImmunoResearch
Laboratories; catalog number 315-005-045) for 2 min at 37°C. Cells
were lysed by the addition of 2x lysis buffer (1% Nonidet P-40, 150
mM of NaCl, 10 mM of Tris, 0.4 mM of
Na4P2O7,
0.4 mM of EDTA, 10 mM of PMSF, and 1 µg/ml aprotinin, leupeptin, and
-1-antitrypsin final). Lysates were clarified by centrifugation and
rotated overnight at 4°C with Sepharose-coupled peptide beads.
Precipitations were then washed, resolved by SDS-PAGE, transferred to
polyvinylidene difluoride (PVDF) membrane, and immunoblotted with 4G10
and anti-BLNK Abs.
Immunoprecipitations
Anti-Syk (sc-929), anti-Grb2 (sc-255), and anti-Vav
(sc-132) Abs were all purchased from Santa Cruz Biotechnology (Santa
Cruz, CA), 4G10 anti-phosphotyrosine (catalog number 05-321) was
purchased from Upstate Biotechnology (Lake Placid, NY), and polyclonal
rabbit antisera were made to a GST fusion of the BLNK Src homology 2
(SH2) domain, while the anti-Ig rabbit antisera has been
described previously (39). For each immunoprecipitation,
1 x 107 cells were either left untreated or
stimulated by incubating on ice with either rabbit anti-mouse IgG
(Jackson ImmunoResearch Laboratories) at 25 µg/ml or PDGF-BB,
mouse anti-human PDGFR, and rabbit anti-mouse IgG1, as
described above, and then incubated at 37°C for 3 min. Cells were
then washed and lysed in modified RIPA buffer (1% Nonidet P-40, 150 mM
of NaCl, 10 mM of Tris, 0.1% SDS, 0.5% deoxycholate, 0.4 mM of
Na4P2O7,
0.4 mM of EDTA, 10 mM of NaF, 1 mM of PMSF, and 1 µg/ml aprotinin,
leupeptin, and -1-antitrypsin). Lysates were clarified by
centrifugation and precleared by incubating with 150 µl of a mixture
of protein A- and protein G-Sepharose (Amersham Biosciences). The
resulting supernatants were immunoprecipitated by incubation overnight
at 4°C with the indicated Abs prebound to protein A-Sepharose.
Immunoprecipitations were washed extensively, boiled in loading buffer,
loaded onto SDS-PAGE gels, transferred to PVDF membranes (Millipore,
Bedford, MA), and Western blotted with the indicated Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y176 and Y204
Within the complex structure of the BCR, the cytoplasmic tails of
the Ig/Ig heterodimer determine the primary trafficking and
signaling capacities of the receptor (34, 36, 42). To
define the specific functional domains within the tails necessary for
receptor trafficking, we used an approach that allowed different
receptor complexes containing the cytoplasmic tails of Ig and Ig
to be assembled on the cell surface. Chimeric molecules were
constructed and expressed, in the FcR-deficient B cell lymphoma
A20IIA1.6, in which the extracellular and transmembrane domains of the
human PDGFR or were fused to the cytoplasmic tails of Ig or
Ig (Fig. 1
) (39).
PDGFR and have equal affinity for PDGF-BB, therefore adding this
ligand to cells expressing two different chimeras (PDGFR/Ig and
PDGFR/Ig) forms Ig/Ig heterodimers that can then be
aggregated first with mouse mAbs to the extracellular domain of
PDGFR and then by rabbit anti-mouse IgG1.
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(Y182 and
Y193) (34). These phosphotyrosines
function to recruit and activate Syk (43, 44), which has
also been implicated in receptor targeting (35, 36). Other
conserved tyrosines (Y176 and
Y204) exist within the cytoplasmic tail of Ig,
but their role in receptor trafficking has not been investigated.
Therefore, we mutated these tyrosines singly and in combination. The
resulting mutant or wild-type PDGFR/Ig chimeras were expressed
with wild-type PDGFR/Ig because trafficking to late endosomes and
the presentation of pulsed Ags require both cytoplasmic tails
(34).
We first examined whether Ig/Ig required Ig
Y176 and/or Y204 to
facilitate the presentation of Ag to specific T cell clones. Chimeric
receptors on each transfectant were aggregated by sequential incubation
on ice with PDGF-BB, by mouse anti-PDGFR Abs, and then by rabbit
anti-mouse IgG1. These aggregated receptor complexes were pulsed
with Ag by incubation with myoglobin conjugated to goat anti-rabbit
IgG at 37°C for 30 min. In separate samples, Ag was targeted to the
endogenous BCR on each transfectant, first by ligating the receptor
with rabbit anti-mouse IgG and then by pulsing with myoglobin
conjugated to goat anti-rabbit IgG. In both cases, cells were then
washed and used as APCs in assays with the T cell clone Ach 8.2
(34). IL-2 production was assayed by ELISA after 24
h. Ag targeted either to the chimeric complexes containing wild-type
Ig/Ig cytoplasmic tails or to the BCR was presented efficiently
(Fig. 2). As expected, the magnitude of
IL-2 produced correlated with the amount of available Ag. Although the
A20IIA1.6 cell line constitutively secretes low levels of IL-2, no
increase in IL-2 was detected in the absence of T cells nor in the
absence of Ag (data not shown). In contrast, Ig(YF176,204)/Ig
was incapable of effectively facilitating the presentation of Ag.
Concurrent cross-linking of the endogenous BCR with anti-IgG2a Abs
did not rescue
Ig(YF176,204)/Ig-facilitated Ag
presentation, suggesting that the defect is intrinsic to
Ig(YF176,204)/Ig (data not shown).
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(YF176,204)/Ig,
mutation of Y204 completely abrogated the ability
of the chimeric receptor to facilitate the presentation of pulsed Ag
(Fig. 2
). Ig(YF176)/Ig was partially
defective in facilitating presentation. These data indicate that the
efficient presentation of peptides derived from aggregating ligands
requires both tyrosines.
Entry into late endosomes requires Ig Y176 and
Y204
To begin to examine the receptor-mediated processes dependent upon
Y176 and Y204, we first
determined whether efficient chimeric complex internalization depends
upon either tyrosine. For these assays, HRP-coupled goat
anti-rabbit IgG was used to label the aggregated receptor
complexes, and internalization was allowed to proceed 5, 10 (data not
shown), or 30 min (Fig. 3A).
The percentage of total HRP activity still remaining on the cell
surface was assayed colorimetrically. There was no significant
difference in the degree of internalization between the wild-type and
mutant chimeric complexes at any of the time points examined (Fig. 3A) (34) (data not shown). Accordingly, we next
examined whether any of the chimeric complexes were aberrant in their
ability to target late endosomes.
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Each clone was stimulated through either the BCR or the chimera with
FITC-coupled Abs for 30 min and then fixed, permeabilized, and stained
for Lamp-1 (ID4B, red). Upon stimulation of the clones through the BCR
or the Ig/Ig chimera for 30 min (Fig. 3B), receptor
complexes colocalized with a large perinuclear aggregate of
Lamp-1+ vesicles (large arrows). In contrast,
stimulation of Ig(YF176,204)/Ig did not
induce the perinuclear coalescence of Lamp-1-bearing late endosomal
vesicles. The majority of endocytosed
Ig(YF176,204)/Ig mutant chimeric
complexes did colocalize with peripherally distributed endosomes
(arrowheads), but some receptor complexes appeared to be adjacent to
Lamp-1+ vesicles rather than actually occupying
the same region (small arrow), suggesting that
Ig(YF176,204)/Ig might be excluded from
their lumen. Analysis of cells expressing
Ig(YF204)/Ig revealed a similar defect
in receptor trafficking, while in those expressing
Ig(YF176)/Ig, receptor entry into
Lamp-1+ vesicles appeared to be partially
inhibited (Fig. 3C). To further characterize the defect in
mutant receptor trafficking, we used immunoelectron microscopy.
The Ig/Ig or mutant chimeric complexes were stimulated with Abs
conjugated with 5-nm gold particles for 30 min at 37°C. Cells were
then fixed, and frozen sections were prepared and stained with ID4B and
10-nm gold-conjugated secondary Abs. Fig. 4 shows representative immunoelectron
micrographs of Ig/Ig and Ig(YF176,204)/Ig. As observed
when the BCR was similarly stimulated and analyzed (12),
>80% of internalized Ig/Ig chimeric complexes were
detected within Lamp-1+ vesicles (arrows), often
within the intraluminal multivesicular bodies thought to be derived
from sorted transport vesicles (45) (Fig. 4A).
Many of the targeted Lamp-1+ vesicles were large,
often >1 µm in diameter (Fig. 4B). In contrast, <5% of
internalized Ig(YF176,204)/Ig chimeric complexes were found
within Lamp-1+ vesicles. Approximately 70% of
these complexes were located within proximity to
Lamp-1+ vesicles (Fig. 4, C–E). All of the observed
Lamp-1+ vesicles were small (<0.5 µm),
indicating that translocation and homotypic fusion between late
endosomal vesicles did not occur. Analysis of cells expressing
Ig(YF204)/Ig revealed a similar defect
in entry, while in cells expressing
Ig(YF176)/Ig, labeled complexes were
found both outside and inside of the Lamp-1+
vesicles (Fig. 5). These results indicate
that mutations in Y204, and to a lesser extent
Y176, inhibit the ability of Ig/Ig to enter
late endosomes.
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Y204
Examination of the amino acid sequences flanking
Y176 and Y204 revealed that
Y204 was part of a motif
(Y-hydrophilic-philic-phobic) capable of binding group I SH2 domains
(46). In contrast, Y176 was
not predicted to bind SH2 or any other phosphotyrosine binding domains.
To determine which molecules bound to Y176 or
Y204, we synthesized phosphorylated and
nonphosphorylated peptides corresponding to the regions encompassing
each tyrosine and coupled them to Sepharose beads. Precipitations from
the lysates of unstimulated or stimulated wild-type A20IIA1.6 cells
were resolved by SDS-PAGE, transferred to PVDF membrane, and
immunoblotted with anti-phosphotyrosine Abs (4G10). As seen in Fig. 6A, a single
tyrosine-phosphorylated band of 65 kDa was detected in
phospho-Y204 precipitations from the lysates of
stimulated cells. Of the molecules known to be tyrosine phosphorylated
following BCR aggregation, the linker protein BLNK has a molecular mass
of 65 kDa. Stripping and reprobing with anti-BLNK sera identified
the band as BLNK. BLNK was also efficiently precipitated by the
phospho-Y204 peptide from the lysates of
unstimulated cells.
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/Ig coprecipitated with BLNK. Cells
expressing chimeric complexes containing either wild-type or mutant
Ig/Ig were stimulated, lysed, and immunoprecipitated with Abs to
BLNK. Immunoprecipitations were subjected to SDS-PAGE, transferred to
PVDF membrane, and immunoblotted with 4G10 (Fig. 6B,
upper panels). A phosphoprotein of 140 kDa (arrow)
coprecipitated with BLNK from the lysates of Ig/Ig-stimulated
cells. This phosphoprotein was not detected following stimulation of
chimeric complexes bearing F204, and was
diminished in those containing F176. Subsequent
immunoblotting of the top portion of the membrane with anti-Ig
Abs identified the 140-kDa band as PDGFR/Ig (lower
panel). Immunoblotting the lower portion of the membrane revealed
that BLNK was phosphorylated following stimulation of either the BCR on
each clone or the Ig/Ig chimera. Mutation of
Y176 and Y204 ablated
inductive BLNK phosphorylation. As expected, chimeric complexes
containing mutations of the Ig ITAM tyrosines Y182 and Y193 did not
associate with, nor induce the phosphorylation of, BLNK (39, 43). These data indicate that Y204, and to
a lesser extent Y176, physically and functionally
link the BCR to BLNK.
Following BCR aggregation, Syk rapidly phosphorylates BLNK to form a
scaffold at the plasma membrane for the assembly and activation of a
variety of molecules including Vav and Grb2 (32, 47, 48).
Therefore, we next examined whether some of these molecules associated
with Ig/Ig. Cells were stimulated as before, and lysates were
immunoprecipitated with Abs to Vav. After SDS-PAGE, the membrane was
first immunoblotted with 4G10 and then stripped, divided, and
immunoblotted with either anti-Vav or anti-Ig Abs (Fig. 6B, middle panels). As was observed in the BLNK
immunoprecipitations, the Ig/Ig chimera coprecipitated with Vav,
and this coassociation depended upon receptor aggregation and
Y204 and/or Y176. Although
mutation of either tyrosine was sufficient to disrupt the detectable
association of Vav with Ig/Ig, mutation of both tyrosines was
required to uncouple chimera aggregation from Vav phosphorylation. The
apparent discordance between association and function is probably due
to the stringent conditions under which our immunoprecipitations were
performed. As with both BLNK and Vav, Grb2 associated with Ig/Ig
in an aggregation- and
Y176/Y204-dependent manner
(Fig. 6B, lower panels).
The recruitment and activation of Syk depend on the ITAMs in the
Ig/Ig cytoplasmic tails (43, 44). To ensure that
mutation of Y176/Y204 did
not perturb this function, immunoprecipitations of Syk from the lysates
of cells stimulated through either chimeras of Ig/Ig,
Ig(YF176,204)/Ig, or
Ig(YF182,193)/Ig were resolved by
SDS-PAGE and transferred to PVDF membrane. These membranes were
immunoblotted with 4G10, followed by stripping and reimmunoblotting
with either anti-Syk or anti-Ig Abs. As seen in Fig. 6C, aggregation of both Ig/Ig and
Ig(YF176,204)/Ig equally induced the
recruitment of Syk. As expected, mutation of the Ig ITAM tyrosines
prevented this interaction.
As illustrated in Fig. 6B, the inductive tyrosine
phosphorylation of BLNK and Vav is dependent upon
Y176 and Y204. To examine
whether mutation of these tyrosines globally compromised
receptor-induced tyrosine phosphorylation, we stimulated each receptor
complex, and total cell lysates from each sample were Western blotted
with 4G10 (Fig. 6C, lower panel). Total inductive
tyrosine phosphorylation was similar whether cells were stimulated
through either the chimeras or through the endogenous BCR expressed on
each cell line. From these data, we conclude that mutation of
Y176 and Y204 results in a
selective defect in Ig/Ig-dependent BLNK phosphorylation and
recruitment.
BLNK mediates receptor entry into late endosomes
BLNK serves as the primary scaffold on which Vav and Grb2 assemble
(32, 48). Furthermore, the BLNK SH2 domain can bind
directly to phosphorylated Y204 (Fig. 6A) (37, 68). Therefore, we examined
whether adding BLNK back to
Ig(YF176,204)/Ig restored normal
receptor trafficking. A cDNA was constructed encoding a chimera in
which the 3' sequence of
PDFGR/Ig(YF176,204) was linked in frame
to a cDNA-encoding murine BLNK. This cDNA was cloned into the
bicistronic GFP-expressing retroviral vector MIGR1. A20IIA1.6 cells
expressing PDGFR/Ig were infected with chimera-encoding
retrovirus, and GFP-expressing cells were isolated by FACS. Expression
of PDGFR/Ig(YF176,204)/BLNK was
confirmed both by flow cytometry and by immunoblotting with
anti-BLNK and anti-PDGFR Abs. Surface expression of the
chimera, as measured by flow cytometry, was 2-fold less than that of
PDFGR/Ig(YF176,204) (data not shown).
The PDGFR chimeras or the BCR on cells expressing either
Ig(YF176,204)/Ig or
Ig(YF176,204)BLNK/Ig were pulsed with myoglobin,
and their ability to present to the T cell clone Ach 8.2 was
determined. The presence of BLNK restored the ability of
Ig(YF176,204)/Ig to facilitate the presentation of Ag (Fig. 7A). We next determined
whether BLNK allowed entry into the MIIC. The chimeras on cells
expressing PDGFR/Ig with either
Ig(YF176,204) or
Ig(YF176,204)BLNK were aggregated, as
described previously, and labeled with AMCA-coupled Abs for 30 min.
Cells were then fixed and stained with rat anti-mouse Lamp-1
(ID4B), followed with Texas Red-conjugated anti-rat IgG. In
contrast to Ig(YF176,204)/Ig,
Ig(YF176,204)BLNK/Ig colocalized with,
and induced the coalescence of, Lamp-1+ late
endosomes (Fig. 7B). These data demonstrate that the
recruitment of BLNK by Ig is required for the delivery of
receptor-bound antigenic complexes to late endosomal processing
compartments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is required for the recruitment and activation of Syk
(43, 44, 49). Activated Syk both initiates downstream
signaling cascades and determines sorting of endocytosed complexes
through early endosomes (34, 35, 36). We now demonstrate that
a substrate of Syk, BLNK, is recruited to Ig
Y204 following receptor activation. Targeted
receptor complexes require BLNK to enter late endosomes, where
efficient peptide processing and MHC class II loading can occur.
In addition to being required for receptor trafficking, the non-ITAM
tyrosines were necessary for receptor-mediated BLNK and Vav
phosphorylation. Phosphorylation was associated with recruitment to
Ig/Ig, indicating that these two processes were interrelated. It
is known that BLNK is rapidly phosphorylated following receptor
ligation, and that Syk may be the responsible kinase (32).
In vivo, the main determinant of BLNK binding,
Y204, is phosphorylated following receptor
ligation. Furthermore, the SH2 domain of BLNK can bind directly to this
phosphotyrosine (37, 68). These data indicate that
BLNK is recruited directly to Ig, the same chain to which Syk is
recruited and activated, providing a mechanism to ensure rapid
phosphorylation. Ig has been demonstrated to be capable of
activating signaling pathways downstream of BLNK, including PLC2
(43, 50, 51, 52). However, the cytoplasmic domain of Ig
contains only the two tyrosines of a single ITAM, and therefore does
not have a potential recruitment site for the SH2 domain of BLNK.
Furthermore, BLNK does not coprecipitate with Ig alone following
receptor ligation (data not shown). Therefore, it is likely that Ig
uses different proximal mechanisms to initiate signaling.
The incorporation of BLNK into the activated receptor complex raises
the possibility that it, or a molecule that it binds, such as Grb2 or
Vav, acts to chaperone the receptor into late endosomes rather than as
a linker to activate a permissive signaling pathway. In support of this
possibility, inhibition of many of the signaling pathways distal
to BLNK, including intracellular calcium mobilization
(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate
acid and EGTA), protein kinase C (Calphostin C), and Rac
(expression of N17-Rac), had no effect on
trafficking of the BCR (data not shown). Furthermore, providing a
concurrent signal in trans through the BCR fails to rescue
the presentation of Ag bound to
Ig(YF176,204)/Ig. However, BCR ligation
does allow entry of this receptor complex into late endosomes (K.
Siemasko, unpublished results). These data indicate that direct
binding of BLNK to Ig serves as both a signaling complex and a
chaperone to facilitate Ag presentation. To determine the mechanisms
underlying these complex functions will require identification of the
specific molecules and domains responsible for receptor trafficking. As
deletion of BLNK impairs B cell development (53, 54, 55),
studies in deficient mice are likely to be unrevealing.
Our data provide a model in which trafficking to late endosomes
proceeds through a series of sequential checkpoints, each regulated by
one or more signaling molecules bound to the Ig cytoplasmic tail
(Fig. 8). The demonstration of sequential
sorting and entry steps in the delivery of BCR complexes to late
endosomes is reminiscent of endocytic trafficking mechanisms defined in
yeast (56, 57, 58). Endocytosed yeast membrane proteins are
sorted in multivesicular bodies for transport to specific subcellular
compartments (59). Sorting is determined by specific
motifs present in the cytoplasmic tails of endocytosed proteins
(60, 61). To enter a compartment, transport vesicles must
first tether, then fuse to the limiting membranes of the targeted
vesicles. Rab proteins probably mediate tethering
(62, 63, 64), while fusion requires the interaction of vesicle
soluble N-ethylmalemide-sensitive factor attachment protein
receptors (SNAREs) on the donor compartment with target SNAREs on the
target or acceptor membranes (65). Mammalian structural
and functional homologues of both the Rab and SNARE yeast proteins have
been characterized in several cell types, including B lymphocytes
(62, 56), indicating that the general mechanisms of
endocytic trafficking are widely conserved.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marcus R. Clark, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC0930, Chicago, IL 60637. E-mail address: mclark{at}medicine.bsd.uchicago.edu
Received for publication August 21, 2001. Accepted for publication December 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dimers and facilitates peptide loading. Cell 82:155.[Medline]
/Ig heterodimer with membrane Ig and bound antigen to the major histocompatibility complex class II peptide-loading compartment. J. Biochem. 274:11439.
and Ig- with distinct cytoplasmic effectors. Science 258:123.2 and Rac1-JNK in B cells. Immunity 10:117.[Medline]
and Ig are required for efficient trafficking to late endosomes and to enhance antigen presentation. J. Immunol. 162:6518.-chain-mediated transport to lysosomes. EMBO J. 17:4606.[Medline]
subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation. J. Exp. Med. 188:819.. Eur. J. Immunol. 31:2126.[Medline]
and Ig-. Blood 89:1513./ heterodimer of the B cell antigen receptor complex. J. Biol. Chem. 271:5158./Ig immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590. cytoplasmic truncation renders immature B cells more sensitive to antigen contact. Immunity 11:537.[Medline]
couples the B cell antigen receptor
to distal signaling pathways. Mol. Cell. Biol. In press.This article has been cited by other articles:
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) or chimeric
receptor complexes (
) were first formed on ice, and then myoglobin
conjugated to goat anti-rabbit IgG was added. Cells pulsed with
different dilutions of myoglobin-conjugated Ab were placed at 37°C
for 30 min, washed, and then cocultured with the T cell clone Ach 8.2
for 24 h. IL-2 production was measured by ELISA. Each graph shows
the mean values of samples done in triplicate from a representative
single experiment. The experiment shown is representative of three
separate experiments. Additional independently derived clones gave
similar results (data not shown).
). In
contrast, a chimeric complex in which BLNK was fused to the C terminus
of Ig