From the Immunology Unit, Department of Cellular Biology and Pathology, University of Barcelona School of Medicine, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona E-08036, Spain
Received for publication, February 13, 2003, and in revised form, February 26, 2003
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ABSTRACT |
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CD229 (Ly9) is a cell surface
receptor selectively expressed on T and B lymphocytes, and it belongs
to the CD150 receptor family. Like other receptors of this family,
CD229 interacts with SAP/SH2D1a protein, mutation of which is
responsible for the fatal X-linked lymphoproliferative disease.
Receptors of the CD150 family function as costimulatory molecules,
regulating cytokine production and cytotoxicity. Thus, their signaling
and regulation in lymphocytes may be critical to an understanding of
the pathogenesis of the X-linked lymphoproliferative disease. Here we
show that CD229 interacts with the µ2 chain of the
AP-2 adaptor complex that links transmembrane proteins to
clathrin-coated pits. CD229 was the only member of the CD150 family
associated with AP-2. We also show that the µ2 chain
interacts with the Y470EKL motif of CD229. The
integrity of this site was necessary for CD229 internalization, but it
was not involved in SAP recruitment. Moreover, CD229 binds to the
AP-2 complex in T and B cell lines, and it is internalized rapidly from
the cell surface on T cells after antibody ligation. In contrast,
cross-linking of CD229 receptors with intact antibody inhibited CD229
internalization on B cells. However, when F(ab')2
antibodies were used, CD229 internalization was similar on T and B
cells, suggesting that Fc CD229 (also termed Ly9) is a cell surface glycoprotein of relative
mass 120 kDa found on T and B cells (1). It is a member of the
immunoglobulin superfamily, and it belongs to the CD150 family of
receptors (2, 3). Six members of the CD150 family (CD150, CD229, CD84,
CD244, NTB-A, and CS1) have one or more cytoplasmic tyrosine motifs
with the consensus sequence
Thr-(Ile/Val)-Tyr(P)-X-X-(Ile/Val). This motif
functions as a docking site for the
SAP1/SH2D1a protein (4-9).
Mutations on the SAP gene, sh2d1a, are the
responsible for the fatal X-linked lymphoproliferative disease after
Epstein-Barr virus infection, familial hemophagocytic
lymphohistiocytosis, and some cases of combined variable
immunodeficiency (2, 4, 10-12). These disease phenotypes point to
distinct roles for the six CD150 family/SAP signaling pathways in the
control of T, B, and natural killer cell activation and homeostasis.
Studies on SAP knock-out mice reveal that T cells have an impaired
ability to differentiate into T helper 2 cells, resulting in increased resistance to T helper 2-mediated disease such as infection with Leishmania major. In addition, SAP In this study, we have investigated the biochemical mechanism involved
in CD229 signaling and intracellular trafficking by identifying
proteins that interact with the CD229 cytoplasmic domain. Two clones
encoding human AP-2 µ2 chain were isolated using CD229 as
bait in a three-hybrid screen of a human B cell library.
µ2, also termed AP50, is the 50-kDa subunit of the AP-2 complex. AP-2 is a key component of the endocytic machinery that links
cargo membrane proteins to the clathrin lattice. It is a heterotetramer
consisting of two large (100-115 kDa) Cells and Reagents--
COS-7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Jurkat and Daudi
cells were maintained in RPMI 1640 medium supplemented with 10% fetal
calf serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Biotin, brefeldin A (BFA),
and anti-mouse IgG peroxidase were obtained from Sigma. Monoclonal
anti-
Anti-human CD229 (clone HLy-9.1.25) was generated at our laboratory
(1). Biotinylated anti-CD229 was generated using EZ-Link biotinylation
kit from Pierce. F(ab')2 anti-CD229 was prepared using
immobilized ficin from Pierce. The purity of the F(ab')2 preparation was confirmed by SDS-PAGE under nonreducing conditions and
reducing conditions followed by silver staining.
Yeast Three-hybrid Screen--
The cytoplasmic tail of human
CD229, cloned in pBridge and transformed in the yeast strain CG1945,
was used as bait to screen a human B cell cDNA library in pACT2
vector (Clontech, Palo Alto, CA). CD229 and mutated
Fyn were inserted in the bicistronic vector pBridge. In mutant
Fyn, the regulatory tyrosines 420 and 531 were replaced by
phenylalanine, and arginine was replaced by glutamine (FynY420F,Y531F,R176Q) as described elsewhere (7).
For the three-hybrid screen in the presence of
FynY420F,Y531F,R176Q, the yeast strain CG1945 was
cotransformed sequentially with the vector (pBridge), containing both
CD229 and the Fyn mutant, and these transformants were selected on SD
medium lacking tryptophan and transformed further with 1 mg of the
human B cell cDNA library. Double transformants were then plated in
SD medium lacking tryptophan, leucine, histidine, and methionine in the
presence of 3 mM 3-aminotriazole.
Yeast clones that grew in these restrictive conditions were then tested
by the
The Plasmid Constructions--
CD229 cloned in pCIneo was used as a
template (1) to generate two mutated DNA sequences of human CD229
(mutants CD229Y470F and CD229Y470A). The
primers used to generate all the mutations were:
5'-CTCCCAAGGATTTGAGAAGCTGG-3' (primer 1),
5'-GTGTCCAGCTTCTCAAATCCTTGGG-3' (primer 2),
5'-CTCCCAAGGAGCTGAGAAGCTGG-3' (primer 3), and
5'-CCAGCTTCTCAGCTCCTTGGGAG-3' (primer 4). Construct
CD229Y470F was generated using primers 1 and 2, and
CD229Y470A using primers 3 and 4. PCR products were cloned
using the TOPO subcloning kit (Invitrogen) and sequenced. For
expression in yeast, cytoplasmic tails of CD229WT and
CD229Y470F were generated from CD229WT and
CD229Y470F templates using sense primer
5'-GGGGAATTCAAGCGAAAAGGACGG-3' and antisense primer
5'-GGGGGATCCGGTGAAATTT TCATAGC-3' and subcloned in the pBridge binding
domain vector using the EcoRI/BamHI sites.
To generate µ2-EGFP fusion protein, µ2 was
amplified by PCR using a cDNA peripheral blood lymphocyte library
as a template with sense primer
5'-AAACTGCAGGCCGCCATGATTGGAGGCTTATTC-3' and antisense primer
5'-TTCCCCGGGGCGAGTTTCATAAATGCCACTGCGG-3' and cloned in-frame into
the pEGFP-N2 vector (Clontech) using the PstI/SmaI sites. The presence of desired
nucleotide changes was established by bidirectional nucleotide
sequencing using dye terminator chemistry (PE Biosystems, Foster City, CA).
Transfections--
COS-7 cells (1 × 106) were
seeded into 100-mm dishes and 24 h later transfected with 4 µg
of each plasmid DNA/dish using the LipofectAMINE Plus kit (Roche
Applied Science) following the manufacturer's instructions. Cells were
incubated for 48 h, and the experiments were performed.
Immunoprecipitation and Immunoblotting--
Transfected COS-7
cells were biotinylated and lysed with 1% CHAPS (Pierce), and
immunoprecipitation was carried out using antibodies against CD229.
Likewise, previously biotinylated Jurkat and Daudi cells (100 × 106) were lysed with 1% Triton X-100 as described
elsewhere (7). Briefly, cell lysates were precleared for 1 h with
50 µl of protein A-Sepharose beads (Amersham Biosciences).
Immunoprecipitations were performed using 2 µg of each indicated
antibody, and 40 µl of protein A-Sepharose beads was added for 2 h at 4 °C. Beads were then washed, and samples were boiled. All of
the precipitates were separated by SDS-PAGE. Proteins were transferred
onto nitrocellulose membranes (Millipore, Bedford, MA). Filters were
blocked for 1 h with 5% skim milk (or 3% bovine serum albumin)
and then probed with the indicated antibodies. Bound antibody was
detected using horseradish peroxide-conjugated secondary antibodies and
enhanced chemiluminescence (Pierce).
Internalization Assay--
Cells (1 × 106/sample) were incubated with anti-human CD229 mAb (1 µg/ml = 1 × 106) at 37 °C. At various time
points (0-90 min), cells were chilled rapidly and acid stripped to
remove cell surface-bound mAb as described elsewhere (28). Samples were
washed twice in cold complete culture medium, and they were incubated
for 25 min with biotinylated anti-CD229 followed by avidin-PE
incubation (1). For recycling experiments, cells were incubated at
37 °C with anti-CD229 in the presence or absence of BFA at 10 µg/ml in complete culture medium for the indicated time periods.
Samples were stripped and stained with biotin-conjugated anti-human
CD229 followed by streptavidin-PE. For stimulation, cells were treated
for 5 min with anti-CD3 or anti-IgM. Intact anti-CD229 or
F(ab')2 was added for an additional 30 min. Thereafter,
cells were harvested, chilled, and acid stripped as described above.
The samples were analyzed in a flow cytometer (FACS Caliber; BD
Biosciences, Mountain View, CA) to detect PE (FL2) fluorescence (mean
fluorescence intensity; MFI). The percentage of endocytosis was
calculated using MFI values of PE as follows: 100 Immunofluorescence Microscopy--
COS-7 cells were
cultured on coverslips, transfected with µ2-EGFP
cDNA, fixed, and permeabilized with CD229 Interacts with µ2 in a Yeast Three-hybrid
Screen--
The cytoplasmic tail of human CD229 was used as bait to
screen a human B cell library. The CD229 cytoplasmic tail was cloned in
the pBridge vector together with Fyn (7). The screening of ~2 × 106 clones yielded two clones that encoded portions of
human µ2, the medium chain of clathrin-associated coated
pit adaptor protein complex AP-2 (Fig.
1A). Because the
µ2 interaction with proteins is independent of
phosphorylation, we assayed the binding by depleting the catalytic
activity of Fyn. CD229 binding to µ2 was similar in the
presence and absence of kinase activity (Fig. 1B).
CD229 Is the Only Member of the CD150 Family of Receptors That
Interacts with µ2--
We assayed the interaction of
µ2 with all of the CD150 members using a semiquantitative
CD229 Binds to µ2-EGFP Chimera and Integrates in AP-2
Endogenous Complex in COS-7 Cells--
To test the interaction
CD229-µ2 in a mammal system, we made the
µ2-EGFP construct and transiently transfected COS-7 cells alone or together with CD229. To determine whether CD229 and the µ2-EGFP chimera integrated in the endogenous AP-2 complex
in COS-7 cells, we performed an immunoprecipitation with anti- Tyrosine 470 Is Required for CD229 Binding to
µ2--
Analysis of the CD229 sequence revealed several
putative motifs for µ2 binding
(Y-X-X- Tyrosine 470 Is Essential for CD229 Endocytosis--
To determine
the relevance of CD229-µ2 interaction for receptor
internalization, we generated CD229 mutant constructs in which the
Tyr470 residue was replaced by phenylalanine or alanine. We
transiently transfected CD229 wild-type and mutants encoding constructs
into COS-7 and measured their internalization after ligation with
anti-CD229 mAb at 37 °C. Thereafter, cells were stripped and stained
with CD229 biotinylated antibody plus avidin-PE labeling, and samples were measured by flow cytometry (Fig.
5A). Within 30 min, 30-40% of wild-type CD229 surface receptors had been internalized. In contrast, internalization was completely abrogated, and CD229 surface
expression even increased slightly, in CD229 mutants, which
Tyr470 had been replaced by phenylalanine or alanine. We
also transfected COS cells with the CD229WT and
CD229Y470 mutants plus µ2-EGFP. After 24 h, cells were stained with biotinylated antibody at 4 °C followed by
avidin-Cy3 at 37 °C (Fig. 5B). In cells transfected with CD229WT, the receptor was internalized and colocalized
with the µ2-EGFP, whereas CD229Y470F and
CD229Y470A internalization was impaired, and no
colocalization with µ2 was found. Both results indicate
that Tyr470 is needed for CD229 binding to µ2
and further endocytosis.
The Cytoplasmic Tail of CD229 Is Associated with the AP-2 Adaptor
Complex in T and B Lymphocytes--
Because CD229 is expressed only in
cells of the immune system, we next determined whether the association
of the AP-2 adaptor complex with CD229 occurred in lymphocytes. To this
end, we performed coimmunoprecipitation experiments in two lymphocytic
cell lines: Jurkat (T cell line) and Daudi (B cell line). Both cell
lines were immunoprecipitated using anti-CD229 mAb and an anti-CD84 as
a control mAb. Western blot was probed with anti-AP-2 Anti-CD229 mAbs Trigger CD229 Internalization--
Anti-CD229 mAbs
induce the internalization of CD229 in COS cells after 30 min of mAb
coligation. Because CD229 is expressed on lymphocytes and associates
with the AP-2 complex, we assayed CD229 internalization in these cells.
Endocytosis was assayed in the Jurkat T cell line and in the Daudi B
cell line. CD229 was endocytosed rapidly in Jurkat cells, reaching a
plateau within 15 min. The internalization of CD229 after incubation
with intact anti-CD229 was comparable with that obtained with
F(ab')2 in Jurkat cells (Fig.
7A). In contrast,
internalization of CD229 with intact mAb was impaired in B cells.
However, when F(ab')2 was used to coligate, the effect on
CD229 internalization resembled that observed in T cells, indicating
that CD229 endocytosis may be regulated through Fc Endocytosed CD229 Mainly Follows the Lysosomal Degradation
Pathway--
We next analyzed the events following CD229 ligation in
lymphocytes. CD229, once internalized, may traffic to lysosomal
vesicles for degradation or be recycled back to the cell surface. To
distinguish these two events we assessed CD229 endocytosis in the
presence or absence of BFA. If most CD229 were recycled back to the
cell surface, the loss of CD229 after mAb ligation would be much
greater in the presence of BFA. On the other hand, if most endocytosed CD229 were transferred to lysosomes, we would obtain identical results
in the presence or absence of BFA. CD229 surface levels decreased
slightly on T cells induced by F(ab')2 anti-CD229 in the
presence of BFA, suggesting that CD229 was partially recycled back to
the cell surface (20-25%) but mostly trafficked to lysosomal degradation (Fig. 8A). In B
cells, CD229 surface levels were similar in both cases. Thus,
endocytosed CD229 follows mainly the lysosomal degradation pathway in
these cells (Fig. 8B). Taken together, these results suggest
that most CD229 is transported to the lysosomal compartment rather than
recycled back to the cell surface.
CD229 Internalization Is Regulated after TCR or BCR
Cross-linking--
We next investigated whether antigen receptor
cross-linking modulates CD229 internalization in T and B lymphocytes.
When we assayed CD229 expression after TCR ligation at 30-60 min, the expression of CD229 did not vary significantly. In contrast, CD229 ligation did not affect CD3 expression in this period (data not shown).
Unexpectedly, the coligation of CD229 and TCR enhanced CD229
internalization (Fig. 9A).
Daudi cells were incubated with anti-IgM F(ab')2 and
anti-CD229 for 30 min, and CD229 endocytosis was studied. CD229
internalization after IgM coligation was enhanced (Fig.
9B).
Taken together, these data suggest that signals delivered by the TCR
and BCR regulate CD229 cell surface expression.
In this study, we have shown that CD229 binds to the
µ2 chain of the AP-2 complex. It is well established that
the µ2 subunit binds to the consensus sorting signal
NPXY or YPP We have also demonstrated that the cytoplasmic tail of CD229 but not
that of any of the other members of CD150 family can interact with the
AP-2 complex via its µ2 chain. This intriguing difference
suggests that the expression and internalization of CD229 are tightly
and differentially regulated. The structure of CD229 differs from that
of the rest of the CD150 family of receptors. It is the only member
that has four extracellular Ig-like domains, and it has the longest
cytoplasmic tail, comprising 180 amino acids, 16 threonines, 17 serines, and 8 tyrosines. Two unique tyrosine-based motifs (with the
consensus amino acid sequence TV/I YXXV/I) are critical for
the binding to the SAP/SH2D1a protein and the Eat-2 SH2 domain protein
homolog to SAP in B cells (41).
Furthermore, CD229 can bind to endogenous AP-2 in COS cells,
µ2-EGFP chimera can efficiently integrate in the
endogenous AP-2 complex, and CD229 can coprecipitate with
µ2-EGFP and the AP-2 endogenous complex. The complex is
functional because CD229 internalization and
CD229·µ2-EGFP colocalization were detected in COS cells
after antibody-induced endocytosis. In agreement with our mapping data, CD229Y470F and CD229Y470A endocytosis was
severely impaired. Although in our model a phenylalanine substitution
abrogates the binding to µ2, the FXX Because CD229 is expressed in T and B lymphocytes, we have defined the
mechanism of CD229 internalization and demonstrated that it can be
regulated differentially in T and B cell lines. CD229 was endocytosed
rapidly in response to antibody ligation in Jurkat T cells, whereas no
internalization was observed in B cells. The same results were obtained
in lymphocytes from peripheral blood. CD229 in B cells was inhibited by
the Fc Moreover, TCR and anti-IgM coligation enhances CD229 endocytosis,
suggesting that the antigen receptor signaling regulates the
availability of CD229 at the plasma membrane. Thus, endocytosis may
represent a critical mechanism that modulates CD229 cell surface expression rapidly. After TCR activation, phosphatidylinositol 3-kinase may become activated and catalyze the conversion of
phosphatidylinositol 4,5-biphosphate to phosphatidylinositol
3,4,5-trisphosphate. The pleckstrin homology domain in the
Because CD229 endocytosis is impaired when Fc receptors control CD229 cell surface
expression. Furthermore, CD229 was regulated by T cell receptor and B
cell receptor signaling because coligation with antibodies
against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis.
These data suggest that CD229 cell surface expression on lymphocytes
surface is strongly and differentially regulated within the CD150
family members.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice
fail to resolve the lymphocytic choriomeningitis virus, showing an
increased number of interferon-
-producing cells in the spleen and
liver (13) and a nearly complete absence of virus-specific long lived
plasma cells and memory B cells (14). Altogether, these data indicate
that SAP controls several distinct key T cell signal transduction
pathways, which may be controlled differentially by the various CD150
receptors. It is crucial to elucidate the contribution of each member
to these immune functions. Unlike other CD150 family members, CD229 has
four rather than two extracellular Ig domains. Domains 1 and 3 are
similar, as are domains 2 and 4, suggesting that CD229 arose from a
progenitor with two domains (15). Although the function of CD229 is
unknown, other members of the subfamily such as CD84, CD244, and CD150
are known to activate lymphocytes (16-22), suggesting that the CD229
molecule is also involved in leukocyte activation. Recently,
NTB-A and CS1 have also been shown to regulate cytotoxicity in
natural killer cells (23, 24).
and
subunits or
adaptins, one medium µ2 (50 kDa), and one small (17 kDa)
2 subunit. The
-adaptin appears to contain the major
membrane binding interface. The hinge domain of the
subunit binds
to the clathrin heavy chain, providing a mechanism for the formation of
polyhedral lattices. In addition to its structural role in coat
assembly, AP-2 is also involved in the transport of proteins from the
plasma membrane to the endosomal and lysosomal compartments. For
effective cargo into coated pits, receptors should contain specific
internalization signals, namely tyrosine-based motifs such as
NPXY or YXX
(where X stands for any
amino acid and
for a bulky hydrophobic residue), dileucine motifs,
and acidic clusters (25, 26). The structural analysis of the AP-2 core
reveals that the binding site for YXX
endocytic motifs is
buried, indicating that a conformational change, probably triggered by
phosphorylation in the disordered µ2 linker, is necessary
to allow YXX
motif binding (27). Here, we demonstrate that the only member of the CD150 family which specifically binds µ2 is CD229. We mapped the CD229-µ2
interaction in Tyr470 of the CD229 cytoplasmic domain.
Moreover, we show that TCR and BCR signaling regulates CD229 expression.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adaptin AP-2 (clone 100/2), also from Sigma, was used for
immunoblotting. Rabbit anti-rat AP-2
-C, from Upstate Biotechnology
(Lake Placid, NY), was used for immunoprecipitation and microscopy.
Phosphotyrosine monoclonal antibody mixture horseradish
peroxidase-conjugated was from Zymed Laboratories Inc.
(San Francisco). Anti-GFP monoclonal was from Roche Applied Science.
Anti-CD84 (clone CD84 1.1), generated at our laboratory (16), was used
as a control IgG. Anti-human CD3 (clone T3b) was a gift from Dr.
Lopez-Botet (UPF, Barcelona). Anti-human IgM F(ab')2 was
from Caltac Laboratories (Burlingame, CA). Anti-CD71 was a gift from
Dr. Ramon Vilella (Hospital Clínic, Barcelona).
Streptavidin-PE was obtained from BD Pharmingen (San Diego).
Steptavidin-Cy3 and anti-rabbit peroxidase were from Dako (Carpinteria,
CA). Avidin-peroxidase was purchased from Roche Applied Science.
-galactosidase assay. DNA was extracted from the
-galactosidase-positive clones and then expanded after
transformation of HB101 bacteria and selection in M9 medium, lacking
leucine, to isolate the GAL4 activation domain plasmid pACT2. Purified plasmids were sequenced.
-galactosidase colony-lift filter assay and liquid
culture assay using o-nitrophenyl
-D-galactopyranoside as a substrate were carried out as
described in the Clontech yeast protocols handbook.
The vector pBridge containing CD229, in the absence of catalytic
activity of mutated Fyn, was generated after BglII digestion, ligation, and purification.
[(MFI at
37 °C/MFI at 4 °C) × 100].
20 °C methanol
for 15 min. After two washes, they were incubated for 30 min at room temperature with blocking buffer (phosphate-buffered saline containing 0.2% skim milk, 2% fetal bovine serum, 1% bovine serum albumin, 0.1 mM glycine). Cells were then washed and incubated with 2 µg/ml rabbit anti-rat AP-2
-C for 1 h at 4 °C. After three
washes, they were incubated with a biotinylated anti-rabbit (Biogenex,
San Ramon, CA) at 4 °C for 30 min. After several additional washes, cells were incubated with streptavidin-Cy3 (Jackson Immunoresearch, West Grove, PA) at 4 °C for 20 min. Samples were washed and mounted in Fluoromount-G (Southern Biotechnology, Birmingham, AL). For endocytosis experiments, COS-7 cells were cultured on coverslips and
cotransfected with human CD229 (WT or Y470F or Y470A mutants) with
µ2-EGFP cDNAs in duplicate. After 48 h they were
labeled with 1 µg/ml biotinylated mAb CD229 at 4 °C for 30 min.
After two washes with ice-cold phosphate-buffered saline, samples were incubated with streptavidin-Cy3 at 37 °C (to induce endocytosis) for
30 min. Cells were then washed and fixed in
20 °C methanol for 15 min. After washing them twice, cells were mounted and visualized as
described above. Fluorescence images were obtained using a confocal
microscope (TCS NT; Leica, Heidelberg, Germany).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
CD229 interacts with
µ2 in a three-hybrid screen in
yeast. A, the sequence obtained in the two positive
clones overlaps (in bold) with the amino acidic sequence of
the µ2 (AP50) subunit of the human AP-2 complex.
B, -galactosidase semiquantitative assay after
cotransfection of pBridge CD229 (+Fyn) or (
Fyn) and clone 1.
-galactosidase assay. All bound to SAP/SH2D1a when Fyn was active,
with the exception of CD150, which bound to SAP regardless of its
phosphorylation status, as reported elsewhere (29) (Fig.
2A). Surprisingly, only CD229
was able to bind to µ2 in conditions where Fyn was
present or absent, whereas the rest of members failed to do so (Fig.
2B).
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Fig. 2.
CD229 is the only member of the CD150 family
which interacts with µ2. Yeast
cotransfected with pBridge cytoplamic tails of CD150 family members
(CD150, CD244, CD229, CD84, NTB-A, and CS1) +/ Fyn and pGAD-SAP
(A) or pACT2-µ2 (B) were analyzed
for
-galactosidase activity. These data are the mean of duplicates
and representative of three experiments.
chain
of AP-2 (Fig. 3A).
µ2-EGFP and CD229 coprecipitated with AP-2 endogenous complex (second and third lanes,
respectively). Moreover, CD229·µ2-EGFP and AP-2 were in
a complex (fourth lane). To test whether most µ2-EGFP was free or complexed with the endogenous AP-2,
we performed µ2-EGFP transfection in COS-7 cells and
immunochemical staining with anti-
chain AP-2. The majority of the
µ2-EGFP protein colocalized with the endogenous AP-2
(Fig. 3B).
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Fig. 3.
CD229 binds to
µ2 and the endogenous AP-2 complex in
COS cells. A, cells transfected with
µ2-EGFP, CD229, or both were biotinylated and
immunoprecipitated with anti-AP-2 . The blot was probed with
avidin-POD, anti-GFP, and anti-AP-2
. B, cells transfected
with µ2-EGFP were fixed and permeabilized, and
anti-AP-2
staining was carried out. Arrows indicate
colocalization of the µ2-EGFP·AP-2
endogenous
complex.
) (where X stands for any
amino acid and
for a bulky hydrophobic residue) (Fig.
4A). A more restricted binding
motif has been proposed for µ2: YPP
, where
P represents a polar amino acid (25). Following these
criteria we found a suitable motif for the binding (Fig.
4A). The Y470F mutation abolished the interaction between
CD229 and µ2 in a yeast system (Fig. 4B). COS
cells were transfected with CD229 and µ2-EGFP +/
Fyn.
Samples were biotinylated and divided. One set was immunoprecipitated with anti-CD229 and the second with anti-GFP. CD229 was found to
interact with µ2 in the absence of phosphorylation,
although some binding was found in the triple transfection, maybe
because of residual nonphosphorylated CD229 in this sample (Fig.
4C). Moreover, only nonphosphorylated CD229 was found to
bind µ2 (anti-GFP immunoprecipitation) in the samples
where Fyn was present. Altogether, these data indicate that CD229
interacts with µ2 when the receptor is not
phosphorylated, and CD229 phosphorylation by Fyn abrogates the binding
of the CD229 to the µ2 subunit.
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Fig. 4.
CD229Y470 is critical for
µ2 binding. A, CD229
cytoplasmic tail showing binding sites for the µ2 subunit
of the AP-2 (underlined). B, -galactosidase
assay measuring the interaction between pBridge CD229WT or
pBridge CD229Y470F with pACT2-µ2 in yeast
cells in the presence or absence of Fyn activity. The binding of
CD229WT and CD229Y470F to SAP was also
performed. These data are the mean of duplicates and are representative
of three experiments. C, CD229 was cotransfected with
µ2 +/
Fyn in COS cells. Samples were divided and
immunoprecipitated (Ip) with anti-GFP or anti-CD229
antibody. Avidin-peroxidase, anti-Tyr(P), and anti-GFP blots were
performed.
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Fig. 5.
CD229 internalization in COS cells
transfected with CD229Y470 mutants is impaired.
A, internalization was assessed 30 min after incubation with
anti-CD229 at 37 °C and was measured by flow cytometry. These data
are the mean of three experiments. B, endocytosis was
carried out in CD229WT and CD229 mutants and
µ2-EGFP-transfected COS cells. Cells were stained with
biotinylated anti-CD229, and avidin-Cy3 was added at 37 °C for 30 min, inducing endocytosis of the receptor. Cells were washed and
mounted.
mAb (Fig.
6). A band representing AP-2
was found
only in the lane in which CD229 was immunoprecipitated. Taken together,
these data demonstrate that the AP-2 adaptor complex specifically
interacts with the cytoplasmic tail of CD229 in T and B
lymphocytes.
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Fig. 6.
CD229 interacts with the AP-2 complex in T
and B lymphocytes. Jurkat and Daudi cell lines were biotinylated
and lysed. Anti-CD229 or anti-CD84 as a control Ig was used for
immunoprecipitation (ip). The blot was probed with
avidin-peroxidase and anti-AP-2 .
receptors (Fig.
7B). In agreement with this observation, a reduction of
intact antibody concentration used for ligation induced some CD229
endocytosis in B cells, indicating that a limiting mAb concentration
elicits coligation with CD229 with minimal Fc
receptor binding (data
not shown). The internalization of CD229 after antibody coligation may
represent a constitutive turnover rate of CD229 from the cell surface.
To distinguish these two events (endocytosis versus
turnover), we used cycloheximide and BFA, which block the expression of
newly synthesized protein. BFA blockage inhibits the transport of
proteins from the endoplasmic reticulum to the Golgi apparatus and also
recycling of endocytosed proteins (30-32). Treatment with
cycloheximide did not alter CD229 surface expression on T or B cells
within the time period assayed for endocytosis (data not shown).
Treatment with BFA for 2 h slightly reduced CD229 expression in T
cells after 60 min (20-25%) but did not significantly reduce the
surface expression of CD229 in B cells (Fig. 7C). The
transferrin receptor (CD71), which is rapidly recycled (33), was used
as a control. After 15 min, CD71 surface expression was reduced more
than 50% in BFA-treated cells (Fig. 7D). Altogether, these
results indicate that CD229 expression on the cell surface is more
stable on B than on T cells, and in both cases, the rate of turnover is
lower than that for CD71.
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Fig. 7.
Ab-dependent internalization of
CD229. Kinetics of CD229 internalization using intact
(squares) or F(ab')2 (triangles)
anti-CD229 mAb is shown in Jurkat T cells (A) and Daudi B
cells (B). The percentage of internalization was calculated
using MFI values of PE as follows: 100 (MFI at 37 °C/MFI at
4 °C) × 100. The turnover of cell surface CD229 was studied.
Changes in surface expression of CD229 (C) and CD71
(transferrin receptor) as a positive control (D) were
assessed after treatment with 10 µg/ml BFA. The percentage of surface
expression was calculated by determining the ratio of MFI of treated
cells compared with MFI of untreated cells at each time point and
extrapolated as a percentage. The data represent the mean of duplicates
and are representative of three experiments.
View larger version (17K):
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Fig. 8.
Effect of BFA on Ab-dependent
internalization of CD229. Internalization of CD229 in Jurkat T
cells (A) and Daudi B cells (B) is shown.
Internalization was determined at various times after incubation with
F(ab')2 anti-CD229 at 37 °C in the absence (white
columns) or presence (black columns) of 10 µg/ml BFA.
These data are the mean of duplicates and representative of three
experiments.
View larger version (22K):
[in a new window]
Fig. 9.
Effect of TCR and BCR coligation in CD229
endocytosis. A, Jurkat cells were incubated with medium
with anti-CD229 for 30 min (a), preincubated with 1 µg/ml
anti-CD3 for 5 min (b), and anti-CD229 was added for 30 min
(c). B, Daudi cells were incubated with medium
with anti-CD229 for 30 min (d), preincubated with 20 µg/ml
anti-IgM F(ab')2 (e), and cells were incubated
with anti-CD229 for 30 min (f). Cells were stripped, and
CD229 staining was performed (solid lines). Control Ig
staining was also performed (dotted lines). CD229
internalization (%) was measured after various stimuli for 30 min in
Jurkat (C) and Daudi cells (D). These data are
the mean of duplicates and representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(25). Although it has been
suggested that µ2 interacts with dileucine motifs (34),
peptides containing dileucine motifs bind only to the
subunit of
AP1 and AP-2 complexes and not to the other subunits (35, 36). Analysis
of CD229 revealed only one sequence that follows the YPP
consensus (Y470EKL), which is conserved in mouse (37).
Using site-directed mutagenesis, we mapped the µ2
interaction to CD229 in this motif. The single replacement of tyrosine
by phenylalanine abrogates µ2 binding to CD229 in yeast.
It has been proposed that the hydroxyl group of tyrosine contributes to
a network of interactions with µ2 residues that form the
hydrophobic pocket, explaining why phenylalanine does not efficiently
replace tyrosine in YPP
signals (38, 39). As expected,
the recruitment of SAP in the mutant is not altered because both
molecules do not compete for the same motif (7). CD229 binds to
µ2 when Fyn is active, or kinase activity of Fyn is
completely abrogated by depletion of its catalytic region in the yeast
system. This suggests various possibilities: CD229-µ2 interaction occurs when Tyr470 is phosphorylated, or
phosphorylation of the CD229 tail is partial, allowing the screening of
interactions dependent and independent of phosphorylation at the same
time. Our results point to this last possibility because the
CD229-µ2 interaction only occurred when the receptor was
not phosphorylated. This agrees with literature data, which show that
this type of interactions does not depend on phosphorylation. The
crystal structure of µ2 indicates that phosphotyrosine
does not fit into its hydrophobic pocket (40).
motif
can fit into the hydrophobic pocket of µ2 (42) but leads
to a weaker interaction and slower endocytosis. Thus,
Tyr470 is essential for CD229 endocytosis and may inhibit
the internalization of this receptor after its phosphorylation in
vivo. CD229 can be phosphorylated by Src-kinase Fyn in COS cells,
and its possible interaction with a SH2 domain protein may further
regulate the signaling and trafficking of the molecule, as proposed for
CTLA-4 (43).
receptor coligation because F(ab')2 antibodies
induce CD229 endocytosis in B cells. The CD229 uptake rate was similar
using the intact and F(ab')2 antibodies in Jurkat cells
lacking Fc
receptors. In agreement with our results, the coligation
of Fc
receptors and BCR has been suggested to inhibit endocytosis by
blocking the processing and presentation of the BCR-bound antigen (44).
Using BFA and cycloheximide, we have determined that CD229, unlike
transferrin receptor (CD71), exhibits a low constitutive rate of
turnover, and therefore, the rapid internalization of CD229 represents
ligand-induced endocytosis. After endocytosis, CD229 was segregated
into two distinguishable pools, one that is shuttled to a nonrecycling degradation pathway and another that is recycled to the cell surface. The results suggest that ~75-80% of CD229 in T cells become
degraded via lysosomal compartments, and the remaining 20-25% of
complexes are recycled to the cell surface. In contrast, most CD229 is
trafficked to the lysosomal degradation pathway in B cells.
2 subunit of the AP-2 adaptor complex binds with high
affinity to phosphatidylinositol 3,4,5-trisphosphate. This binding
mediates two effects: localization of AP-2 in the membrane and increase
in the affinity of µ2 binding to endocytosis sorting
signals (45, 46).
receptors are
triggered and enhanced when the BCR is activated, this molecule may
have a differential role in these different events. The ability to
regulate CD229 internalization in T and B cells differentially may
allow for different net effects of CD229 signaling which may be
cell-specific. Recent studies indicate that CD229, unlike CD150 and
CD84, inhibits interferon-
secretion after TCR activation in T
cells.2 TCR and BCR may
increase the rate of CD229 endocytosis to prevent CD229 from delivering
negative signals to the cell and Fc
receptor ligation retains the
CD229 on the cell surface, thus favoring signaling through this
receptor and the maintenance of an inhibitory response. The development
of CD229 knock-out mice may be a key step to define the physiological
role of CD229.
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ACKNOWLEDGEMENT |
---|
We thank Joan Sayós for a critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants SAF00-037 and FISS 02/3056 from the Comisión Interministerial de Ciencia y Tecnología.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a MAE/AECI fellowship from the
Ministerio de Asuntos Exteriores, Spain.
§ To whom correspondence should be addressed: Unidad de Inmunología, Departamento Biología Celular, Facultad de Medicina. C/Casanova 143, Barcelona E-08036, Spain. Tel.: 34-93-227-5400 (ext. 2338); Fax: 34-93-451-5272; E-mail: mmartin@medicina.ub.es.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301569200
2 J. M. Del Valle, P. Engel, and M. Martín, unpublished result.
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ABBREVIATIONS |
---|
The abbreviations used are: SAP, SLAM-associated protein; PE, R-phycoerythrin; TCR, T cell receptor(s); BCR, B cell receptor(s); BFA, brefeldin A; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; mAb, monoclonal antibody; MFI, mean fluorescence intensity; WT, wild-type.
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