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*
Hospital de la Princesa, Madrid, Spain; and
Departament de Ciències Experimentals i de la Salut (Inmunología), Universitat Pompeu Fabra, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R-induced serotonin release
in rat basophilic leukemia cells were studied. Both biochemical and
functional analyses revealed tyrosines 644 (SIYATL) and 614
(VTYAQL) as the SHP-1 docking sites required for ILT2 inhibitory
function. Substitution of tyrosine 562 (VTYAEV) did not alter receptor
function. By contrast, mutation of tyrosine 533 (NLYAAV) interfered
with ILT2 tyrosine phosphorylation and the subsequent SHP-1
recruitment, thus supporting a regulatory role for this
motif. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ITIMs are also displayed by other surface molecules such as Fc
RIIB,
CD22, and CD72 in B cells; MAFA in mast cells; CDw150 (SLAM), CD33,
CD31, and others (4, 5). Among them, a group of inhibitory
receptors named Ig-like transcripts (ILTs) or leukocyte Ig-like
receptors has been described in humans (6, 7). ILTs belong
to the Ig superfamily and are encoded by genes that cluster in
chromosome 19p13.4 together with KIRs. This region of chromosome 19 is
known as the leukocyte receptor complex (8). In contrast
to CD94/NKG2 heterodimers and KIRs, ILTs expression is not restricted
to NK and T cells, and these genes have a broader distribution in
different leukocyte lineages (9).
ILT2/leukocyte Ig-like receptor 1 is a 110-kDa surface glycoprotein
detected on the surface of NK and T cell subsets, B cells, dendritic
cells, and monocytes (10). According to a recently
proposed nomenclature, ILT2 is now termed CD85j (11). It
has been shown that ILT2 is expressed primarily by memory T cells
(12), although some authors propose that it is present at
variable levels in most T cells (13). ILT2 includes four
Ig-like C2 domains in the extracellular region and a cytoplasmic tail
containing four ITIM-like sequences (6). This receptor has
been found to interact through its amino-terminal Ig domain with
several classical and nonclassical class I molecules (10, 14, 15, 16) and with UL18, a HCMV protein homologous to HLA class I
(7). Upon ligand binding, ILT2 is able to inhibit
cytotoxicity in NK and T cells; moreover, ligation of ILT2 inhibited
BcR-dependent Ca2+ mobilization in B cells
(10) and signaling triggered through the FcRI (CD64) in
monocytes; in fact, cross-linking of ILT2 with FcR impaired tyrosine
phosphorylation of the associated -chain and of Syk kinase,
inhibiting Ca2+ mobilization (17).
Upon treatment with the phosphatase inhibitor, sodium pervanadate ILT2
becomes tyrosine phosphorylated and associates with the SH2
domain-containing protein tyrosine phosphatase 1 (SHP-1)
(10). SHP-1 contains two SH2 domains, and its activity is
negatively controlled through the interaction of the N-terminal SH2
domain with the catalytic domain of the phosphatase
(18).
Despite the fact that several reports have addressed the structural
basis of SHP-1 binding to ITIMs (19, 20), a number of
questions regarding the mechanisms underlying inhibitory signaling
still exist. In particular, little is known about the putative
relevance of the different ITIM-like sequences for ILT2 function. To
address this issue we generated different mutants of the ILT2
cytoplasmic tail and transfected them in the rat basophilic leukemia
(RBL) and COS-7 cell lines. The ability of the different mutants to
recruit SHP-1 and to inhibit FcR-induced serotonin secretion in RBL
cells was analyzed. Our data revealed that the ILT2 C-terminal
tyrosines 614 and 644 are central sites for binding to SHP-1. Mutation
of residue Y562 had no effect, whereas residue Y533 appears involved in
the regulation of receptor tyrosine phosphorylation
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The RBL cell line was grown in RPMI 1640 with 10% heat-inactivated FCS. Stable transfectants were generated as previously described (21). Clones were obtained by culture under limiting dilution, selected on the basis of flow cytometry analysis, and maintained in the presence of 1 mg/ml G418. Polyclonal T cells were obtained by coculturing nonadherent PBMC with irradiated lymphoblastoid 721.221 cells. Cell populations were used after 12–14 days of culture. Anti-ILT2 HP-F1 mAb has been previously described (10). Anti-phosphotyrosine mAb 4G10 and rabbit polyclonal anti-SHP-1 were purchased from Upstate Biotechnology (Lake Placid, NY). Peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Pierce (Rockford, IL). Sheep anti-mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO).
Flow cytometry
Flow cytometric analysis was performed on a FACScan (BD Biosciences, San Jose, CA). Indirect immunofluorescence staining was performed as previously described (21).
DNA reagents
Briefly, ILT2 cDNA subcloned into pCDNA3 (Invitrogen, Carlsbad,
CA) was subjected to site-directed mutagenesis using the Quick Change
Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) to generate
Y533F, Y614F, and Y644F mutants. Primers used to generate the Y533F
mutant were 5'-GAAGAAAACCTCTTTGCTGCCGTG-3' and reverse, those for Y614F
mutant were 5'-GGATGTGACCTTCGCCCAGCTGC-3' and reverse, and those for
Y644F mutant were 5'-GTGCCCAGCATCTTCGCCACTCTG-3' and reverse. ILT2
Y562F was generated by conventional PCR techniques using as forward
primer 5'-GGGTCGGATCCCCAGAGTGGTCTG-3' and as reverse primer
5'-CCTAGGTCTGGAGTGTTTCACCTCGGCAAA-3'; the 370-bp fragment was
subcloned into BamHI-StyI sites of ILT2 cDNA. The
sequences were confirmed by automatic dideoxy sequencing. Double
mutants were made by restriction subcloning and conventional molecular
biology techniques. The deletion mutant ILT2.
2Y was generated by
ILT2 cDNA digestion with EcoRI (at nt 1750) and
XbaI restriction enzymes and religation after filling in
with Klenow fragment of DNA polymerase. ILT2.Cyt was made by plasmid
digestion with StuI at nt 1548 and ligation with blunted
XbaI. Mutants were transfected into RBL cells using DOTAP
liposomal reagent (Roche, Indianapolis, IN) as previously described
(21). Stable transfectants were selected in 1 mg/ml G418.
Clones were obtained by culture under limiting dilution conditions and
were selected by flow cytometric analysis. PSVL-SHP-1 and SHP-1/D419A
mutants were provided by Dr. Z.-H. Xie (National Institutes of Health,
Bethesda, MD).
For expression in yeast, a cDNA sequence encoding human ILT2 cytoplasmic tail was amplified by PCR and cloned in the BamHI site in multiple cloning site I of the pBRIDGE/Fyn420Y-F,531Y-F,176R-Q cassette (22) (ILT2 5' sense primer, 5'-CGCGGATCCCCCGACATCGACGTCAGGGCAAACAC-3'; ILT2 3' antisense primer, 5'-CGCGGATCCCTAGTGGATGGCCAGAGTGGCGTA-3'). Wild-type and two mutated cDNA sequences encoding for the two SH2 domains of human SHP-1 were subcloned in the GAL4 DNA activation domain vector pGAD424 using the EcoRI/BglII sites. Wild-type SHP-1 cDNA was amplified by PCR using the primers SHP-1 5' sense (5'-CCGGAATTCATGCTGTCCCGTGGGTGGTTT-3') and SHP-1 3'-antisense (5'-GGAAGATCTTCGGACTCCTGCTTCTTGTTC-3'). For the construction of mutant SHP-1 R30Q, two SHP-1 cDNA fragments were amplified by PCR, then annealed at overlapping ends containing 30 R-Q substitutions, filled in, and further amplified to produce the mutant (the first fragment was generated using SHP-1 5' sense primer and primer SHP-130Q 3' antisense, 5'-CTTGCGACTGGGCTGAGCCAGGAAGCTACC-3', and the second cDNA fragment was generated using primers SHP-130Q 5' sense, 5'-GAGAACACCGTGTTTGCACAAGTGTTCAAC-3', and SHP-1 3' antisense primer). Mutant SHP-1 R136Q was generated in a similar way using the primers SHP-1136Q 5' sense (5'-ACGTTTCTTGTGCAAGAGAGCCTCAGCCAG-3') and SHP-1136Q 3' antisense (5'-GCT GAGGCTCTCTTGCACAAGAAACGTCCA-3').
Three-hybrid system assay
The three-hybrid assay was conducted by transforming
sequentially the yeast strain CG1945 with
pBRIDGE/Fyn420,531Y-F,176R-Q containing ILT2 and
then with the vector pGAD424 containing SHP-1, wild type or mutated.
Transformants were plated in synthetic dropout medium
supplemented with a -Trp, -Leu dropout. Clones were tested by a
-galactosidase liquid culture assay using O-nitrophenyl
D-galactopyranoside as a substrate as previously
described (22)
Serotonin release
RBL cells (106/ml) were pulsed for 3 h at 37°C in RPMI 1640/10% FCS containing 2 µCi/ml [3H]serotonin (DuPont-NEN, Boston, MA). Cells were washed, incubated at 37°C for an additional hour, washed again, and resuspended at 4 x 106 cells/ml. Cells were then plated in 50-µl aliquots (2 x 105 cells) in 96-well flat-bottom plates precoated with 20 µg/ml anti-trinitrophenyl (anti-TNP) mouse IgE (Sigma-Aldrich) alone or in combination with 20 µg/ml HP-F1 whole Ab or F(ab')2 or control HP-3B1 mAb whole Ab or F(ab')2 in triplicate. After 1 h at 37°C the reaction was stopped by adding 150 µl cold medium; subsequently, 100 µl supernatant was collected from each well, and radioactivity was measured. Serotonin release was calculated as % serotonin release = (cpm sample - cpm spontaneous release)/(cpm total - cpm spontaneous release).
Pervanadate treatment, receptor cross-linking, immunoprecipitation, and Western blotting
For pervanadate treatment cells were incubated in RPMI 1640 containing freshly prepared sodium pervanadate (0.1 mM sodium orthovanadate and 10 mM H2O2) at 37°C for 10 min. After stimulation, cells were lysed in buffer containing 1% (v/v) Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM Na3VO4, and 1 mM NaF. For receptor cross-linking, cells were resuspended at 108 cells/ml and preincubated for 15 min at 4°C with 2 µg HP-F1 F(ab')2. F(ab')2 sheep anti-mouse (5 µg) was added before incubating at 37°C. Cells were lysed, and immunocomplexes were isolated with protein G-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Lysates from pervanadate-treated cells were incubated at 4°C with HP-F1 mAb, followed by protein G-Sepharose. Precipitates were washed three times with lysis buffer, resolved by 8.5% SDS-PAGE under nonreducing conditions, and transferred onto nitrocellulose membranes.
Cell haptenization and conjugation
For cell haptenization .221 cells (5.106 cells/ml) were incubated for 15 min at 37°C in PBS containing 0.5 mM trinitrobenzene sulfonic acid (Sigma-Aldrich), washed twice in 50 ml PBS, and resuspended in RPMI 1640/10% FCS medium at 8 x 106cells/ml. Twenty-five microliters (2 x 106) of haptenized cells were coincubated with 25 µl (2 x 106) RBL cells for 1 min at 37°C, lysed, and immunoprecipitated as previously described. To measure serotonin release stimulation, 4 x 105 haptenized cells were cocultured with 2 x 105 [3H]serotonin-loaded RBL cells in the presence of anti-TNP IgE for 1 h in round-bottom 96-well plates in a final volume of 50 µl. The reaction was stopped by addition of 150 µl cold medium. One hundred microliters were collected from each well, and serotonin release was determined as described above. All experiments were performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Engagement of KIR and CD94/NKG2A receptors has been reported to be
sufficient for activating their tyrosine phosphorylation (21, 23, 24, 25). By contrast, it has been proposed that other inhibitory
receptors require coligation with activating receptors to recruit
tyrosine kinases involved in ITIM phosphorylation (26, 27). We reported that pervanadate stimulation induced tyrosine
phosphorylation of ILT2 and recruitment of SHP-1 phosphatase
(10). To determine whether receptor engagement is
sufficient to promote ILT2 tyrosine phosphorylation we cross-linked
F(ab')2 of the ILT2-specific mAb HP-F1 on
polyclonal activated T cell populations or ILT2 transfected RBL cells
(RBL.ILT2) (10). As shown in Fig. 1
, ILT2 engagement was sufficient to
trigger tyrosine phosphorylation, which was detectable within 1–2 min
of stimulation in both cell systems. To further confirm these data, we
assayed the effect of ILT2 interaction with its natural ligands.
RBL.ILT2 cells were incubated in the presence of 721.221 or 721.221
cells expressing either HLA-B*2705 (.221.B2705) or HLA-E (.221.AEH)
(16). Interaction between ILT2+
cells and HLA+ cells was sufficient to induce
receptor tyrosine phosphorylation and SHP-1 recruitment (Fig. 2B).
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R. RBL.ILT2 cells were loaded
with [3H]serotonin and incubated in the
presence of either the lymphoblastoid cell line 721.221 or 721.221
cells expressing HLA-B*2705 (.221.B2705). Both cell lines were
previously haptenized with TNP and incubated with anti-TNP IgE. As
shown in Fig. 2
A, FcR-induced serotonin release was
diminished when RBL.ILT2 cells were incubated with .221.B*2705 cells.
When this assay was conducted in the presence of an anti-ILT2 mAb
to block the interaction between ILT2 and HLA-B molecules, no
inhibitory effect was detected (Fig. 2A). Taken together,
these data support that ILT2-class I interaction is able to inhibit the
signal triggered through FcR in RBL cells, rendering this system
appropriate to analyze the structural basis for ILT2 receptor
function. Tyrosines 614 and 644 are necessary for ILT2 inhibitory function
We have previously shown that coligation of ILT2 and FcR
inhibits serotonin secretion in RBL cells challenged with IgE
(10). To elucidate the relative contributions of the
different ITIM-like sequences in the delivery of the negative signal we
first generated two ILT2 deletion mutants. Constructs encoding for a
truncated version of ILT2 in which tyrosines 614 and 644 had been
removed (ILT2.2Y) as well as a receptor lacking the cytoplasmic tail
(ILT2.Cyt; see Fig. 3D)
were transfected into RBL cells. Fig. 3A shows surface
expression of the different proteins in the RBL clones selected to
perform the functional studies. To assay the ability of the receptor to
inhibit serotonin secretion, ILT2 was coligated to FcR using mouse
IgE and F(ab')2 of HP-F1 mAb adsorbed onto
plastic. As expected, the mutant lacking the whole cytoplasmic region
(ILT2.Cyt) was unable to mediate inhibition of serotonin release
(data not shown) or SHP-1 recruitment (Fig. 3C). The
truncated protein in which the carboxyl-terminal tyrosines are missing
(ILT2.2Y) was phosphorylated in cells treated with pervanadate (Fig. 3B, upper panel), but it did not coprecipitate
SHP-1 (Fig. 3B, lower panel) or inhibit serotonin
secretion when ILT2.2Y was cross-linked to FcR (Fig. 3C). It is noteworthy that the lower molecular band detected
in transfectants by Western blotting, which predominates in the
wild-type ILT2 (ILT2wt) immunoprecipitates, was not phosphorylated
after pervanadate treatment (Fig. 3B).
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2Y could account for the
lack of interaction with SHP-1, it was still possible that residue Y562
was also involved in the recruitment of SHP-1. To test this hypothesis
we mutated Y562 to phenylalanine and expressed it in RBL cells (Fig. 4A). ILT2.Y562F mutant was
able to inhibit serotonin release in response to IgE almost as
efficiently as the wild-type receptor (Fig. 4B) and to
recruit SHP-1 phosphatase upon receptor phosphorylation (Fig. 4C). According to these data, Y562 does not appear to be
essential to deliver the inhibitory signal. Thereafter we generated
single mutants in which tyrosine 614, 644, or both were mutated to
phenylalanine and individually transfected them into RBL cells. During
the selection process of stable transfectants we did not find any clone
whose ILT2 surface expression was comparable to that of RBL.ILT2wt.
Therefore, we selected a new RBL.ILT2wt clone whose expression levels
were similar to those of RBL.ILT2Y614F, RBL.ILT2Y644F, and
RBL.ILT2.Y614, 644F cell lines. This clone, referred to as RBL.ILT2wtL,
was used in the functional studies for comparative purposes (Fig. 4A). Ligation of the ILT2.Y614F single mutant on RBL cells
was able to partially inhibit serotonin release in response to IgE
(Fig. 4B). Accordingly, SHP-1 was recruited to the receptor
upon tyrosine phosphorylation (Fig. 4C, middle
panels). In contrast, the ILT2.Y644F mutant had markedly reduced
its ability to inhibit degranulation (Fig. 4B), although its
ability to recruit the phosphatase was not lost (Fig. 4C).
The double mutant ILT2.Y614, 644F appeared inactive (Fig. 4B), further supporting that the loss of function of the
ILT2.2Y truncated protein is due to the lack of residues Y614
and Y644.
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To circumvent the variability of ILT2 expression in different RBL
clones and to estimate the relative contribution of each tyrosine to
the recruitment of the phosphatase, we performed transient transfection
experiments in COS-7 cells. The Src kinase Lck has been reported to
catalyze tyrosine phosphorylation of KIR ITIMs (24),
suggesting that members of this family of kinases would be able to
phosphorylate ILT2 cytoplasmic tyrosine residues. Thus, we tested in
transient transfection experiments in COS-7 cells whether either Lck or
Fyn was able to phosphorylate the receptor, promoting SHP-1
recruitment. To that purpose we used a trapping mutant of SHP-1 in
which D419 has been changed to A (SHP-1/D419A). This mutation within
the catalytic domain still allows the phosphatase to bind to its
substrate, but impairs the reaction, thus stabilizing phosphatase
binding to the substrate (28). Cotransfection experiments
in COS-7 cells showed that both Src kinases were able to phosphorylate
the receptor and promote SHP-1 recruitment (Fig. 5), yet Fyn appeared to be slightly more
efficient in several experiments performed (not shown).
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, A and B). The double mutant ILT2.Y533, 562F also
bound SHP-1 upon tyrosine phosphorylation. By contrast, the double
mutant ILT2.Y614, 644F did not coprecipitate SHP-1 when cotransfected
with Fyn (Fig. 6C) even when the remaining tyrosines were
phosphorylated (not shown). These results support the data obtained in
RBL cells indicating that tyrosines 614 and 644 play a major role in
SHP-1 recruitment and activation.
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To determine whether both SH2 domains of SHP-1 are involved
in the interaction with the ILT2 cytoplasmic tail, mutations that
eliminate phosphotyrosine binding (R30Q and R136Q) (29)
were introduced into the individual SH2 domains. Wild-type SHP-1 SH2
domains or single mutants were assayed in a yeast three-hybrid system
for binding to the ILT2wt cytoplasmic tail. Fyn kinase was introduced
into the yeast system to induce phosphorylation of ILT2
(22). As a result of bait and prey protein interaction,
-galactosidase gene transcription was activated, and the enzyme
activity was detected. As shown in Fig. 7, wild-type SH2 domains were able to
bind to ILT2, but this interaction was completely abrogated when single
mutations were introduced into SHP-1 SH2 domains. Therefore, at least
in the absence of the catalytic and C-terminal domains, both SH2
domains of SHP-1 appear to be required for interaction with the ILT2
cytoplasmic tail.
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A single Y533F mutation was introduced in the ILT2
cytoplasmic tail, and the cDNA was transfected into RBL cells. Single
clones expressing ILT2.Y533F were tested in a serotonin release assay
for the ability of ILT2 to inhibit the signal mediated through FcR
(Fig. 8, A and B).
In none of the tested clones was the ILT2.Y533F mutant able to inhibit
serotonin release stimulated by IgE (Fig. 8B shows results
obtained with a representative clone). Moreover, in cells treated with
sodium pervanadate, no phosphorylation of the receptor was detected by
Western blotting (data not shown). In accordance with these data, the
double mutant ILT2.Y533, 562F (Fig. 8A) was not able to
inhibit degranulation of RBL cells (Fig. 8B). Recently, it
has been shown that a specific sequence of events is required for
efficient phosphorylation of some receptors (30, 31).
Accordingly, it is possible that P-Y533 may be required to activate Src
kinases to further phosphorylate the cytoplasmic tail of ILT2. To test
this hypothesis, we expressed either ILT2wt or the Y533F mutant in
COS-7 cells in the presence of decreasing amounts of Fyn. As shown in
Fig. 8C, the wild-type receptor appears more efficiently
phosphorylated than the Y533F mutant. In the presence of comparable
levels of Fyn, a more efficient phosphorylation of ILT2wt was observed.
After densitometry and correction for the total amount of ILT2 in the
immunoprecipitates, phosphorylation of wild-type protein was 6-fold
that of the 533F mutant at the highest concentration of Fyn and about
12-fold at the lowest concentration (data not shown). These data
support the hypothesis that tyrosine residue 533 may be involved in
further activation of Src kinases (Fyn) and subsequent tyrosine
phosphorylation of ILT2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
strongly supporting the idea that coligation with activating receptors
is not necessarily required for ILT2 function. Certainly, the
possibility that tyrosine kinase activation via ITAM-bearing molecules
may also promote ILT2 function is not excluded. Our data are in line
with previous observations on inhibitory signaling delivery by
CD94/NKG2 and KIR (21, 23, 24, 25). In contrast, some
ITIM-containing receptors, such as FcRIIB and CD72, were shown to
need coligation to ITAM-containing receptors (i.e., BCR) to become
tyrosine phosphorylated (26, 27). Recently, it has been
reported that in ILT2-transfected Jurkat cells, cross-linking of the
TCR is needed for ILT2 tyrosine phosphorylation (32). This
was in contrast to our results with peripheral blood T lymphocytes,
which also show that coligation with TCR is not required for ILT2
activation in T cells. The basis for the discrepancy is uncertain and
probably reflects the different experimental cell systems used. In
Jurkat cells Fyn activity is diminished (33). The fact
that Fyn phosphorylated ILT2 more efficiently than Lck in transfected
COS-7 cells (not shown) might underlie the undetectable ILT2
phosphorylation upon receptor engagement in Jurkat cells.
The cytoplasmic region of ILT2 contains four putative ITIMs, of which
only two (those containing residues Y562 and Y614) fulfill the proposed
ITIM consensus sequence V/I/LxYxxV/L. Pei and colleagues
(29) have proposed a model for SHP-1 activation in which
the amino-terminal SH2 domain of SHP-1 serves both as a regulatory
domain and as a recruiting unit, whereas the C-terminal SH2 domain acts
merely as a recruiting unit. According to this model, we expected that
both SH2 domains would bind to residues Y562 and Y614, and we intended
to determine which was the docking site for the C-terminal SHP-1 SH2
domain. Unexpectedly, the results from our mapping experiments in RBL
and COS-7 cells indicated that SHP-1 docking sites were residues Y614
and Y644 (Figs. 3, 4, and 6). Functional data were in accordance with
biochemical information, further supporting that residues Y614 and Y644
were the SHP-1 docking sites required for ILT2 inhibition of
FcR-induced serotonin release (Figs. 3 and 4). The single mutant
ILT2.Y614F had partially lost its ability to inhibit degranulation,
while Y644F mutation markedly impaired the ability of the receptor to
inhibit serotonin release mediated by IgE, although in both cases a
residual binding to SHP-1 upon receptor phosphorylation was still
observed. According to the model proposed by Pei et al.
(29), binding to the C-terminal SH2 domain of SHP-1 would
not be able to fully activate the phosphatase. Apparently, the mutant
lacking Y644 but keeping Y614 could bind to SHP-1 without fully
activating it, whereas the Y614F mutant retained some inhibitory
activity. It is hypothesized that residue Y644 might act as the docking
site for the amino-terminal SH2 domain of SHP-1, while the C-terminal
SH2 domain would be recruited by residue Y614. We attempted to confirm
our hypothesis in a three-hybrid yeast system. Constructs including
either the wild-type SHP-1 SH2 domains or single mutants were assayed
for binding to ILT2wt cytoplasmic tail in the presence of Fyn.
Unfortunately, single mutations of SHP-1 SH2 domains abolished binding
to the ILT2 cytoplasmic tail. A similar result has been described for
the association between the CD22 cytoplasmic tail and SHP-1. Although
mutants with a single functional SH2 domain were still able to interact
with CD22, binding was dramatically decreased (31). In our
experiments no binding was detected, probably due to the fact that the
BD-SHP-1 constructs lack the catalytic domain, which is known to
stabilize the interaction with the receptor (34).
Our results showing Y644 (SIYATL) together with Y614 (VTYAQL) as the main sites responsible for SHP-1 recruitment differ from those of Dietrich et al. (32), who proposed Y562 (VTYAEV) as the main site for docking of SHP-1 to ILT2. However, these authors tested binding of SHP-1 to matrix-coupled phosphopeptides containing a single phosphotyrosine, and this could account for the discrepancy. By contrast, our data are in agreement with those obtained by Maeda et al. (35). These authors showed that mutation of Y771 (VTYAQL) and Y801 (SVYATL) within the C-terminal ITIMs of PIR-B (mouse ILT2 orthologue) abolished the ability of the receptor to inhibit Ca2+ mobilization.
Despite the finding that residue Y533 (NLYAAV) was not directly
involved in SHP-1 recruitment to the receptor (Figs. 3 and 6), we
observed that mutation of this residue completely abrogated the ability
of the receptor to inhibit serotonin release in RBL cells challenged
with IgE (Fig. 8). In agreement with these data, the double-mutant
ILT2.Y533, 562F behaved similarly (Fig. 8). Attempts to detect tyrosine
phosphorylation of these mutated receptors in RBL cells were
unsuccessful, yet phosphorylation was detected in COS-7 cells. It is
known that the expression of Src kinases in COS-7 leads to spontaneous
activation of these enzymes (22); thus, we hypothesized
that residue Y533 could play a role in the activation and optimal
phosphorylation of ILT2 tyrosine residues. Remarkably, in cell lines
transfected with CD19 cDNAs encoding for mutations of residues Y482 and
Y513 no phosphorylation of the remaining seven cytoplasmic tyrosine
residues could be detected (36). An interpretation for
this phenomenon has been recently proposed. Upon ligand engagement,
CD19 becomes phosphorylated in residue Y513 by the Src kinase Lyn.
Subsequently, Lyn binds phosphorylated Y513 through its SH2 domain and
phosphorylates residue Y482. This residue binds a second Lyn molecule,
which leads to Lyn transphosphorylation and autophosphorylation
(30). According to this model, upon ILT2-ligand
interaction Src kinases would phosphorylate Y533, rendering the kinase
active to catalyze further phosphorylation of ILT2. To approach this
hypothesis, we coexpressed ILT2wt and ILT2.Y533F mutant with decreasing
amounts of Fyn in COS-7 cells. We reasoned that if residue Y533 plays
any role in amplifying Src kinase activity, ILT2wt would be more
efficiently phosphorylated by low concentrations of Fyn than ILT2.Y533F
mutant. Indeed, ILT2wt became phosphorylated, whereas no
phosphorylation of ILT2.Y533F mutant was detected when the constructs
were cotransfected with 2 µg of a plasmid encoding for Fyn (Fig. 8C).
Interestingly, Y562 was not involved in SHP-1 recruitment and
subsequent negative signaling, but still became phosphorylated after
receptor engagement. We cannot rule out that other signaling mediators
may interact with this docking site. In fact, CD22, a
well-characterized inhibitory receptor that binds SHP-1, is also able
to recruit other effector molecules (i.e., Syk, phospholipase C-1,
and phosphatidylinositol 3-kinase) (37, 38). Further
studies will be necessary to fully understand the molecular basis of
ILT2 function.
In conclusion, we have explored the structural basis of ILT2 function, showing that ILT2 negative signaling takes place trough SHP-1 recruitment to phosphotyrosine residues Y614 and Y644. Additionally, we found that residue Y533 may play a pivotal role in ILT2 function through a mechanism that promotes the phosphorylation of SHP-1 docking sites.
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Footnotes |
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2 Current address: Servicio de Alergia, Hospital Universitario la Paz, Madrid, Spain.
3 Address correspondence and reprint requests to Dr. Miguel López-Botet, Departament de Ciències Experimentals i de la Salut (Inmunología), Universitat Pompeu Fabra, 08003 Barcelona, Spain. E-mail address: miguel.lopez-botet{at}cexs.upf.es
4 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; ILT, Ig-like transcript; ILT2wt, wild-type ILT2; ITIM, immunoreceptor tyrosine-based inhibition motif; RBL, rat basophilic leukemia; SH2, Src homology 2; SHP-1, SH2 domain-containing protein tyrosine phosphatase 1; TNP, trinitrophenyl.
Received for publication August 6, 2001. Accepted for publication January 22, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor I-associated Lyn-dependent phosphorylation of Fc receptor IIb during negative regulation of mast cell activation. J. Immunol. 160:1647.
production but nor histamine release by SHP-1 in RBL-2H3 mast cells. J. Immunol. 164:1521.1 upon B cell activation. J. Exp. Med. 183:547.This article has been cited by other articles:
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  C. Li, B. L. Houser, M. L. Nicotra, and J. L. Strominger HLA-G homodimer-induced cytokine secretion through HLA-G receptors on human decidual macrophages and natural killer cells PNAS, April 7, 2009; 106(14): 5767 - 5772. [Abstract] [Full Text] [PDF]
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 M. Maffei, F. Ghiotto, M. Occhino, M. Bono, A. De Santanna, L. Battini, G. L. Gusella, F. Fais, S. Bruno, and E. Ciccone Human Cytomegalovirus Regulates Surface Expression of the Viral Protein UL18 by Means of Two Motifs Present in the Cytoplasmic Tail J. Immunol., January 15, 2008; 180(2): 969 - 979. [Abstract] [Full Text] [PDF]
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 C. S. Wagner, L. Walther-Jallow, E. Buentke, H.-G. Ljunggren, A. Achour, and B. J. Chambers Human cytomegalovirus-derived protein UL18 alters the phenotype and function of monocyte-derived dendritic cells J. Leukoc. Biol., January 1, 2008; 83(1): 56 - 63. [Abstract] [Full Text] [PDF]
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D. Alvarez-Errico, J. Sayos, and M. Lopez-Botet The IREM-1 (CD300f) Inhibitory Receptor Associates with the p85{alpha} Subunit of Phosphoinositide 3-Kinase J. Immunol., January 15, 2007; 178(2): 808 - 816. [Abstract] [Full Text] [PDF]
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 D. Imhof, A.-S. Wavreille, A. May, M. Zacharias, S. Tridandapani, and D. Pei Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains: CRITICAL ROLES OF RESIDUES BEYOND THE pY+3 POSITION J. Biol. Chem., July 21, 2006; 281(29): 20271 - 20282. [Abstract] [Full Text] [PDF]
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S. E. Kirwan and D. N. Burshtyn Killer Cell Ig-Like Receptor-Dependent Signaling by Ig-Like Transcript 2 (ILT2/CD85j/LILRB1/LIR-1) J. Immunol., October 15, 2005; 175(8): 5006 - 5015. [Abstract] [Full Text] [PDF]
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 K. Kuroki, N. Tsuchiya, M. Shiroishi, L. Rasubala, Y. Yamashita, K. Matsuta, T. Fukazawa, M. Kusaoi, Y. Murakami, M. Takiguchi, et al. Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis Hum. Mol. Genet., August 15, 2005; 14(16): 2469 - 2480. [Abstract] [Full Text] [PDF]
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H. Aguilar, D. Alvarez-Errico, A. C. Garcia-Montero, A. Orfao, J. Sayos, and M. Lopez-Botet Molecular Characterization of a Novel Immune Receptor Restricted to the Monocytic Lineage J. Immunol., December 1, 2004; 173(11): 6703 - 6711. [Abstract] [Full Text] [PDF]
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T. Gonen-Gross, H. Achdout, R. Gazit, J. Hanna, S.'a. Mizrahi, G. Markel, D. Goldman-Wohl, S. Yagel, V. Horejsi, O. Levy, et al. Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function J. Immunol., August 1, 2003; 171(3): 1343 - 1351. [Abstract] [Full Text] [PDF]
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M. Faure, D. F. Barber, S. M. Takahashi, T. Jin, and E. O. Long Spontaneous Clustering and Tyrosine Phosphorylation of NK Cell Inhibitory Receptor Induced by Ligand Binding J. Immunol., June 15, 2003; 170(12): 6107 - 6114. [Abstract] [Full Text] [PDF]
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