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Laboratoire d’Immunologie Cellulaire et Clinique, Institut National de la Santé et de la Recherche Médicale U.255, Institut Curie, Paris, France; and
Centre d’Immunologie Institut National de la Santé et de la Recherche Médicale/Centre National de la Recherche Scientifique de Marseille Luminy, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) 6 . KIRs belong to a wider group of negative
receptors that possess immunoreceptor tyrosine-based inhibition motifs
(ITIMs) in their intracytoplasmic (IC) domain 7 . In
addition to KIRs 8, 9 , these receptors include type IIB receptors for
the Fc portion of IgG (Fc
RIIB), which inhibit BCR- 10, 11 , TCR-,
and FcR-dependent cell activation 12 ; Ly49 13 and NKG2A 14 , which
inhibit cell-mediated cytotoxicity; CD22 15 and CD72 16, 17 , which
inhibit B cell activation; paired Ig-like receptors of the B type
(PIR-B) 18 which inhibit the activation of B cells and
myeloid cells; gp49B1 19 and mast cell function-associated Ag
(MAFA) 20 , which inhibit IgE-induced mast cell activation; products
of Ig-like transcripts (ILTs) 21, 22 and leukocyte Ig-like
receptors (LIRs) 23 , which inhibit the activation of
monocytes and dendritic cells; leukocyte-associated inhibitory
receptors (LAIRs) 24 , which inhibit the activation of
lymphoid cells; and signal-regulatory proteins of the 
type
(SIRP) 25, 26 , which inhibit the proliferation of hemopoietic and
nonhemopoietic cells induced by hormones and growth factors via protein
tyrosine kinase receptors.
ITIMs were structurally defined as a consensus sequence made of a
tyrosine residue, an N-terminal isoleucine, valine, leucine, or serine
at position Y-2, and a C-terminal leucine or valine at position Y + 3
(I/V/L/SxYxxL/V) 27, 28 . ITIMs were functionally defined as
specifically binding Src homology 2 (SH2) domain-bearing cytoplasmic
phosphatases when tyrosyl-phosphorylated 7 . Phosphorylated peptides
corresponding to ITIMs of most ITIM-bearing receptors, including KIRs,
were found to bind in vitro to the two protein tyrosine phosphatases
SHP-1 and SHP-2 with variable affinities 27, 28, 29 . Phosphorylated
peptides corresponding to ITIMs of a few ITIM-bearing receptors,
including FcRIIB, bound to SHP-1 and SHP-2 and also to the inositol
5-phosphatase SHIP 30, 31 . It was demonstrated that the
binding of SHP-1 and SHP-2 to KIR and FcRIIB ITIM phosphopeptides
was dependent upon the Y-2 residue 27, 28 . The structural basis of
the binding of ITIM phosphopeptides to SHIP is not known.
Interestingly, ITIM-bearing receptors were found to differentially
recruit phosphatases in vivo among those that they bind in vitro. Thus,
FcRIIB was shown to selectively recruit SHIP 30, 31 , whereas most
other negative receptors recruit SHP-1. KIRs were reported to recruit
SHP-1 following pervanadate treatment of KIR-expressing cells 28, 29 ,
following KIR aggregation with specific Abs 32 , following
coaggregation of KIRs with BCR in KIR-transfected B cells 5 , and
during cell-cell interactions 33 . The molecular basis of the
selective in vivo recruitment of phosphatases by ITIM-bearing receptors
is not known.
Noticeably, most SHP-1-recruiting receptors possess two or more ITIMs, and one may wonder whether these play redundant roles. Supporting this possibility, the mutation of the ITIM C-terminal tyrosine in a human KIR-based chimeric molecule had no effect, and the mutation of the ITIM N-terminal tyrosine only partially suppressed inhibition of the NF of activated T cells (NFAT) promoter activity 34 . However, these chimeras, which comprised the extracellular and the transmembrane domains of CD8, were expressed as dimeric molecules, and one may argue that the mutated cis ITIM may have been replaced by an identical ITIM in trans. Against a redundant role of ITIMs, SHP-1 and SHP-2 have two tandem SH2 domains that have different affinities for ITIMs 28, 29 and which are thought to differentially mediate the interaction of these phosphatases with ITIM-phosphorylated receptors 35, 36 . The aim of our work was to determine the respective contributions of the two KIR ITIMs during the inhibition of cell activation. To address this issue, we analyzed the properties of chimeric molecules whose IC domain contained the N-terminal and/or the C-terminal KIR ITIMs in their polypeptidic environment. We found that the two KIR ITIMs play distinct roles that may synergize to inhibit cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RBL-2H3 cells 37 were cultured in DMEM supplemented with 10%
FCS, 100 international units/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine; only adherent
cells (recovered with trypsin-EDTA) were used. The FcRIIB-negative
variant 38 of the A20/2J lymphoma B cell line IIA1.6 39 and CTLL-2
cells were cultured in RPMI 1640 supplemented with 10% FCS,
0.5 µM 2-ME, 2 mM sodium pyruvate, 100 international units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine;
nine U/ml murine rIL-2 (R&D Systems, Minneapolis, MN) were
added to CTLL-2 cultures. All culture reagents were obtained
from Life Technologies (Paisley, U.K.).
Antibodies
The mouse IgE mAb 2682-I was used as culture supernatant of a
subclone of DNP-H1--26 hybridoma cells 40 . The rat anti-mouse
FcRIIB 2.4G2 mAb 41 was purified by affinity-chromatography on
protein G-Sepharose from ascitic fluid of nude mice inoculated i.p.
with 2.4G2 hybridoma cells. F(ab')2 fragments were obtained
by pepsin digestion for 48 h. The purity of IgG and
F(ab')2 fragments was assessed by SDS-PAGE.
F(ab')2 fragments and intact IgG of polyclonal mouse
anti-rat Ig (MAR) were obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA). MAR F(ab')2 was
trinitrophenylated by incubation for 1 h at room temperature with
trinitrobenzene sulfonic acid (Eastman Kodak, Rochester, NY) in
borate-buffered saline (pH 8). Trinitrophenyl
(TNP)4-MAR F(ab')2 was obtained after
purification on Sephadex G25 (Pharmacia, Uppsala, Sweden). Rabbit Abs
against recombinant extracellular domains of FcRIIB were kind gifts
of Dr. Catherine Sautès (Institut Curie, Paris, France).
Horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine (PY)
mouse mAbs PY-20 were purchased from Chemicon (Temecula, CA). Mouse
monoclonal anti-SHP-1 and anti-SHP-2 were purchased from
Transduction Laboratories (Lexington, KY). Rabbit anti-SHIP Abs
were purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal
goat anti-rabbit and goat anti-mouse Ig Abs were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit
anti-mouse Ig (RAM) IgG and F(ab')2 used for IL-2
release were purchased from Jackson ImmunoResearch Laboratories; those
used for biochemical analysis were purchased from Cappel Laboratories
(West Chester, PA).
Expression constructs
cDNA of mouse FcRIIB was modified by a point mutation of the
codon coding for V212 (GTT
GTA), which induced no
amino acid change but created a KpnI restriction site. This
cDNA encoding the entire extracellular and transmembrane domains and
the six first IC amino acids of FcRIIB encoded by the transmembrane
exon was inserted into an expression vector under the control of the
SR promoter in pBR322 42 and in which a resistance gene to
neomycin was introduced (NT-neo). The cDNAs encoding the entire
(N+C-KIR), the N-terminal half (Lys272-Cys309)
(N-KIR), or the C-terminal half (Val310-Pro341)
(C-KIR) of the IC domain of p58.183 KIR 43 were fused to FcRIIB
cDNA by cloning them into the created KpnI site and the
SacI site of the vector after PCR with the following
primers: sense primers, 5'-TCGCTGGTGGGTACCCAAAAAAAATGCTGTT-3' (for
N+C-KIR and N-KIR) and 5'-CAGTTGAAGGTACCCGTTTTCACACAGAGA-3' (for
C-KIR); antisense primers, 5'-CTGACTGTGGAGCTCATGGGCAGG-3' (for
N+C-KIR and C-KIR) and 5'-TCTGTGGAGCTCAGCAGTGATTCAACTGTGC-3' (for
N-KIR).
Transfectants
Fifty micrograms of cDNA were transfected by electroporation at
260 V and 960 µF into RBL-2H3 cells or into the FcRIIB-deficient
murine cell line, IIA1.6. RBL-2H3 and IIA1.6 transfectants were
selected by culture with 0.5 and 1 mg/ml neomycin, respectively (Cayla,
Toulouse, France). RBL transfectants recovered after selection were
cloned as described previously 44 . After selection, IIA1.6 cells were
cultured for 10 days in RPMI 1640 supplemented with 1 mg/ml neomycin
and 0.07% SeaPlaque agarose (FMC Corporation, Rockland, ME) in
the presence of feeder cells (rat embryonic cells). The clones, grown
up in soft agar, were picked up with a Pasteur pipette. The expression
of recombinant receptors by cloned cells was assessed by indirect
immunofluorescence. The expression of recombinant receptors on clones
remained stable over the duration of experiments. Several clones of
each transfectant were used for experiments and gave similar results.
Indirect immunofluorescence
Cells were incubated for 1 h at 0°C with 10 µg/ml 2.4G2 IgG in BSS containing 5% FCS. Cells were washed and stained by being incubated for 30 min at 0°C with 50 µg/ml FITC-labeled MAR F(ab')2. Fluorescence was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Mountain View, CA).
Serotonin release
Transfected RBL cells (resuspended in RPMI 1640 medium supplemented with 10% FCS (RPMI-FCS) at 1 x 106 cells/ml) were incubated at 37°C for 1 h with 2 µCi/ml [3H]serotonin (Amersham, Les Ulis, France), washed, resuspended in RPMI-FCS, incubated for another hour at 37°C to remove excess [3H]serotonin, washed again, resuspended in the same medium, distributed in 96-well microculture plates at 2 x 105 cells/well, and incubated for 1 h at 37°C in the presence or absence of IgE anti-DNP and/or 2.4G2 F(ab')2 in a final volume of 50 µl. Adherent cells were washed four times with 200 µl HBSS; next, 25 µl of culture medium was added to each well, and cells were warmed at 37°C for 15 min before challenge. Cells were challenged for 30 min at 37°C with 25 µl TNP-MAR F(ab')2, previously warmed at 37°C for 15 min. Reactions were stopped by the addition of 50 µl of ice-cold medium and by placing plates on ice. Fifty microliters of supernatants was mixed with 200 µl of Aqualuma-Plus scintillation fluid (Wallac, Turku, Finland) and counted in a beta-plate counter (Pharmacia). The percentage of [3H]serotonin released was calculated using as 100%, cpm in 50 µl harvested from wells containing the same number of cells that were lysed in 100 µl of 0.5% SDS and 0.5% Nonidet P-40.
IL-2 secretion
Aliquots of 5 x 105 IIA1.6 transfectants, resuspended in culture medium and distributed in 96-well microculture plates, were incubated with various concentrations of intact IgG or F(ab')2 fragments of RAM for 18 h at 37°C. Cell-free supernatants were harvested and assayed for IL-2 on CTLL-2 cells as described previously 45 .
Flow cytometric analysis of Ca2+ mobilization
The intracellular free calcium concentration was determined by preloading 1 x 106 IIA1.6 cells with 5 mM Fluo-3 AM (Molecular Probes, Eugene, OR) in the presence of 0.2% Pluronic F-127 (Sigma, St. Louis, MO) for 30 min at room temperature. Cells were washed three times in RPMI 1640 and resuspended at 1 x 106 cells/ml in complete medium; the intracellular free calcium concentration was monitored with a flow cytometer. The mean [Ca2+]i was evaluated with FCS assistant 1.2.9 ß software (Becton Dickinson). In these experiments, the intracellular Ca2+ mobilization and the extracellular Ca2+ influx were recorded separately, as described previously 46 . To detect intracellular Ca2+ mobilization, cells were suspended in medium in which calcium was buffered to 60 nM (equivalent to [Ca2+]i in B cells) using EGTA. Cells were immediately (within 1 min) stimulated with RAM IgG or F(ab')2, and [Ca2+]i was measured. To detect extracellular Ca2+ influx, the extracellular Ca2+ concentration ([Ca2+]o) was raised to a final concentration of 1.3 mM with CaCl2.
Immunoprecipitation and Western blot analysis
Transfected RBL cells, resuspended at 1 x
107/ml, were incubated for 1 h at 37°C with IgE
anti-DNP (culture supernatant diluted 1/10) and 2.4G2
F(ab')2 (3 µg/ml) in complete culture medium, washed
three times, resuspended in the same medium at 1 x
107 cells/ml, and challenged for various periods of time at
37°C with 10 µg/ml of TNP-MAR F(ab')2. Cells were
centrifuged, and pellets were lysed for 10 min at 0°C at 8 x
107 cells/ml in lysis buffer containing 10 mM Tris (pH
7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM
Na3VO4, 5 mM NaF, 5 mM sodium pyrophosphate,
0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, and 1 mM PMSF. Lysates were centrifuged at 12,000 rpm for 10
min at 4°C. Protein G-Sepharose (Pharmacia) (50 µl beads diluted
1/2) was used to precipitate 2.4G2-bound FcRIIB-KIR chimeras.
Transfected IIA1.6 cells, resuspended at 3 x 107/ml,
were stimulated at 37°C for 3 min in complete culture medium with 45
µg/ml IgG or 30 µg/ml RAM F(ab')2. The cells were
centrifuged, and pellets were lysed for 10 min at 0°C at 8 x
107 cells/ml in lysis buffer. We used 2.4G2-coated protein
G-Sepharose (5 µg of purified Ab per 50 µl beads diluted 1/2) to
precipitate FcRIIB-KIR chimeras.
Immunoadsorbents were washed three times in 1 ml of lysis buffer and
boiled for 3 min in reducing or nonreducing sample buffer. Eluted
material was fractionated by SDS-PAGE and transferred onto Immobilon-P
membranes (Millipore, Bedford, MA). Membranes were saturated with
either 5% BSA (Sigma) or 5% skimmed milk (Régilait,
Saint-Martin-Belle-Roche, France) diluted in 10 mM Tris buffer (pH 7.4)
containing 0.5% Tween 20 (Merck, Schuchardt, Germany) (Western
buffer) and blotted with appropriate dilutions of either HRP-conjugated
anti-PY Abs or anti-FcRIIB, anti-SHP-1, anti-SHP-2,
and anti-SHIP, followed by HRP-conjugated goat anti-rabbit or
goat anti-mouse IgG. Peroxidase-labeled Abs were detected using the
Amersham enhanced chemiluminescence kit (Amersham, Little
Chalfont, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIB and an IC
domain consisting of the N-terminal half, the C-terminal half, each
containing one ITIM, or the whole IC domain, containing both ITIMs of
human p58 KIRs (N-KIR chimera, C-KIR chimera, and N+C-KIR chimera,
respectively) (Fig. 1
A). cDNAs
encoding the three KIR chimeras were stably transfected in the RBL-2H3
rat mast cells, which constitutively express FcRI, and in the
FcRIIB-deficient IIA1.6 mouse lymphoma B cells 38 , which
constitutively express BCRs composed of IgG2a of an unknown specificity
39 . Clones expressing comparable levels of recombinant chimeric
molecules in each cell type were selected and used for the experiments
(Figs. 1B and 2A).
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KIR chimeras were coaggregated with FcRI in RBL transfectants
sensitized with mouse IgE anti-DNP, incubated with
F(ab')2 fragments of the rat anti-mouse FcRIIB mAb
2.4G2, and challenged with TNP-MAR F(ab')2 as described
previously 47 . Serotonin release induced under these conditions was
compared with serotonin release induced by aggregating FcRI with
TNP-MAR F(ab')2 in the same transfectants sensitized with
mouse IgE anti-DNP but not incubated with 2.4G2
F(ab')2. When coaggregated with FcRI, the N+C-KIR
chimera virtually abolished serotonin release, the N-KIR chimera
induced partial inhibition, and the C-KIR chimera induced no
inhibition (Fig. 1
B).
KIR chimeras were coaggregated with BCR in IIA1.6 transfectants
challenged with intact RAM IgG. IL-2 secretion induced under these
conditions was compared with IL-2 secretion induced by aggregating BCR
with RAM F(ab')2 fragments in the same transfectants. When
coaggregated with BCR, the N+C-KIR chimera and the N-KIR chimera
abolished IL-2 secretion, whereas the C-KIR chimera induced no
inhibition (Fig. 2A).
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B).
Finally, we examined the tyrosyl-phosphorylation of intracellular
proteins in IIA1.6 transfectants following the aggregation or
coaggregation of BCR with the three KIR chimeras. The N+C-KIR chimera
and, to a lower extent, the N-KIR chimera, but not the C-KIR chimera,
inhibited BCR-induced phosphorylation (Fig. 2C).
Taken together, these results indicate that to fully inhibit
FcRI-induced mast cell activation and BCR-induced B cell activation,
KIRs require the conservation of sequences containing the two tandem
ITIMs. Although both contain one ITIM, the N-terminal and C-terminal
halves of the KIR IC domain do not appear to exert redundant functions,
because the N-KIR chimera still inhibited cell activation, whereas the
C-KIR chimera did not.
The N+C-KIR chimera and the N-KIR chimera, but not the
C-KIR chimera, become tyrosyl-phosphorylated upon
coaggregation with FcRI or BCR
KIR-dependent inhibition of cell activation is correlated with the
tyrosyl-phosphorylation of KIR ITIMs, and the double mutation of the
two ITIM tyrosines abolished inhibition 34 . Therefore, we examined
the tyrosyl-phosphorylation of KIR chimeras immunoprecipitated from RBL
and from IIA1.6 transfectants, following their coaggregation with
FcRI or BCR, respectively (Fig. 3A), using the same ligands as
in Fig. 1. Phosphorylation of the N+C-KIR and, with a lower intensity,
of the N-KIR chimera, but not of the C-KIR chimera, was observed in RBL
cells incubated with 2.4G2 F(ab')2 only and in resting
IIA1.6 cells. Following coaggregation for 3 min with FcRI in RBL
cells or with BCR in IIA1.6 cells, phosphorylation of the N+C-KIR and
N-KIR chimeras increased. No phosphorylation of the C-KIR chimera was
induced (Fig. 3A). Phosphorylated proteins coprecipitated
with the three chimeras when coaggregated with the BCR, particularly a
77-kDa molecule that was heavily phosphorylated in cells expressing the
C-KIR chimera. To determine whether the differences in phosphorylation
seen in the N+C-KIR and N-KIR chimeras might be due to differences in
phosphorylation kinetics, the two chimeras were coaggregated with
FcRI for various periods of time before immunoprecipitation and
Western blot analysis with anti-PY Abs. The induced phosphorylation
of both the N+C-KIR and the N-KIR chimeras was detectable as early as
30 s and slightly increased until 15 min. The difference between
the phosphorylation of the two chimeras remained constant with time
(Fig. 3B).
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Once they have been tyrosyl-phosphorylated, KIRs recruit the protein tyrosine phosphatase SHP-1 32 , which was shown to be necessary and sufficient to account for a KIR-mediated inhibition of B cell activation 5 . SHP-1 is thought to dephosphorylate proteins that become tyrosyl-phosphorylated upon aggregation of ITAM-bearing receptors 48 .
To identify the protein tyrosine phosphatase(s) recruited by the KIR
chimeras, these chimeras were coaggregated with FcRI for 5
min in RBL transfectants. Phosphatases coprecipitated with the chimeras
were examined by Western blotting with anti-SHP-1 and
anti-SHP-2 Abs. Detectable amounts of SHP-1 and SHP-2 were
coprecipitated with the N+C-KIR chimera but not with the N-KIR or the
C-KIR chimeras in unstimulated cells. Upon coaggregation with FcRI,
higher amounts of both SHP-1 and SHP-2 were coprecipitated with the
N+C-KIR chimera. SHP-2, but not SHP-1, was coprecipitated with the
N-KIR chimera. Comparable amounts of SHP-2 were recruited by the
N+C-KIR and N-KIR chimeras. Under the same conditions, no phosphatase
was coprecipitated with the C-KIR chimera (Fig. 5A). To determine whether the
differential recruitment of SHP-1 by the N+C-KIR and N-KIR chimeras
might be explained by differences in the kinetics of recruitment,
phosphatases coprecipitated with the two chimeras were examined
following coaggregation with FcRI for various periods of time. Both
SHP-1 and SHP-2 were coprecipitated with the N+C-KIR chimera as early
as 15 s after coaggregation, and the amount of coprecipitated
phosphatases remained constant over a 30-min period. SHP-2 was
coprecipitated with the N-KIR chimera with the same kinetics, but no
SHP-1 was coprecipitated at any timepoint (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI in RBL cells or with BCR in IIA1.6 cells. As
a consequence, the N+C-KIR and N-KIR chimeras, but not the C-KIR
chimera, recruited SH2-domain-bearing phosphatases and
inhibited cell activation. Surprisingly, the N+C-KIR chimera recruited
both SHP-1 and SHP-2, whereas the N-KIR chimera recruited SHP-2 only.
Therefore, the N-terminal half of the KIR IC domain is sufficient for
the in vivo recruitment of SHP-2, whereas the recruitment of SHP-1
requires the whole KIR IC domain.
One unexpected finding of our work was that the three KIR chimeras were
differentially phosphorylated under several conditions. The N+C-KIR and
N-KIR chimeras, but not the C-KIR chimera, were constitutively
phosphorylated in unstimulated cells. The N+C-KIR chimera was more
phosphorylated than the N-KIR chimera, probably because they possess
two and one tyrosine residues, respectively. Phosphorylation of the
N+C-KIR chimera dramatically increased following pervanadate treatment.
The phosphorylation of the N-KIR chimera also increased, and the C-KIR
chimera became phosphorylated. This finding indicates that the three
KIR chimeras are constitutively associated with protein tyrosine
kinases. Supporting this conclusion, KIRs were reported to be
constitutively associated with lck in NK cells 48, 49 , and the
aggregation of KIRs by anti-KIR Abs, in NK cells 32 , or in RBL
cells 28 induced their phosphorylation. lck is not expressed in RBL
or in IIA1.6 cells, but it could possibly be replaced by another src
family protein tyrosine kinase such as lyn. Phosphorylation of the
N+C-KIR and N-KIR chimeras, but not of the C-KIR chimera, was enhanced
following coaggregation with BCR in IIA1.6 cells or with FcRI in RBL
cells. The phosphorylation induced under these conditions is most
likely due to kinases associated with BCR and FcRI, as was shown for
FcRIIB 47 . The reason why these kinases failed to phosphorylate
the C-KIR chimera is unclear.
We provide here the first evidence that KIRs can recruit SHP-2 in
addition to SHP-1 in vivo. SHP-2 is a ubiquitous protein tyrosine
phosphatase that is expressed by human NK cells 29 . Interestingly,
the N-KIR chimera was sufficient to recruit SHP-2, indicating that a
single ITIM is sufficient. Comparable amounts of SHP-2 were recruited
by the N-KIR and N+C-KIR chimeras, suggesting that the C-terminal ITIM
did not contribute to SHP-2 recruitment. Thus, SHP-2 might bind to
molecules bearing a single ITIM, possibly through an interaction of its
two tandem SH2 with ITIMs in trans borne by adjacent
phosphorylated molecules. This hypothesis is supported by a mutational
analysis showing that SHP-2 binds to a single tyrosine-containing
sequence of the IC domain of the platelet-derived growth
factor receptor (PDGF-R) in vivo 50 and by the loss of
catalytic activity of SHP-2 when binding to two different tandem
tyrosine-based motifs on a doubly phosphorylated PDGF-R-derived peptide
in vitro 35 . Surprisingly, the N-KIR chimera selectively recruited
SHP-2 in vivo. This finding is in contrast to the fact that
phosphopeptides corresponding to the N-terminal KIR ITIM bind SHP-1
with higher affinity than SHP-2 in vitro 28, 29 . The selective
recruitment of SHP-2 by the N-KIR chimera is reminiscent of the
selective recruitment of SHIP by single-ITIM FcRIIB 31, 47 , when
corresponding phosphopeptides bind SHP-1 and SHP-2 as well as SHIP in
vitro 30, 31, 47 . The structural basis for such a
selectivity is unknown.
Like the N-KIR chimera, the N+C-KIR chimera recruited SHP-2 when phosphorylated; comparable amounts of SHP-2 were coprecipitated with the two chimeras. Unlike the N-KIR chimera, the N+C-KIR chimera also recruited SHP-1. Whether SHP-1 was recruited by the C-terminal ITIM in the N+C-KIR chimera could be neither ascertained nor excluded in the absence of detectable phosphorylation of the C-KIR chimera. Alternatively, two tandem ITIMs may be required for the recruitment of SHP-1. That 13-aa long phosphopeptides containing one ITIM can bind SHP-1 apparently does not favor this hypothesis. However, the in vitro binding of phosphopeptides heavily coupled to beads may permit a binding that cannot occur in vivo. Moreover, the phosphorylation of both tyrosines, in a 46-aa long peptide encompassing the two ITIMs of a KIR molecule, was found to be mandatory for the binding of SHP-1 34 . The binding of phosphopeptides with two KIR ITIMs was also shown to increase the phosphatase activity of SHP-1 27 . Finally, with the exception of Ly49, other molecules that recruit SHP-1 in vivo (i.e., PIR-B, CD22, ILT-3, LAIRs, and NKG2A) have several ITIMs, and one can notice a C-terminal tyrosine in Ly49, distant of 26 residues from the ITIM tyrosine. This finding suggests the existence of cooperative rather than additive interactions between the N-terminal and the C-terminal ITIMs in KIRs and implies that the C-terminal ITIM is phosphorylated in the N+C-KIR chimera.
The differential phosphorylation and the differential recruitment of
phosphatases by the three chimeras can be correlated with their
biological properties. The heavily phosphorylated N+C-KIR chimera,
which recruited both SHP-1 and SHP-2, induced more profound inhibitions
in both RBL and IIA1.6 cells than the more lightly phosphorylated N-KIR
chimera, which recruited SHP-2 only. SHP-1 is thought to
dephosphorylate the phosphorylated substrates of protein tyrosine
kinases, which are associated and/or recruited by ITAM-bearing
receptors, and to abort early phosphorylation signals 48 . SHP-1 was
shown to be sufficient to inhibit B cell activation by coaggregating
BCR with a chimeric molecule made of the extracellular and
transmembrane domains of FcRIIB and the catalytic domain of SHP-1 as
an IC domain in IIA1.6 cells 5 . The role of SHP-2 is less well known.
Our results suggest that SHP-1 can amplify the inhibitory effects of
SHP-2. The fact that the N-KIR chimera could inhibit serotonin release
in mast cells and IL-2 secretion and Ca2+ mobilization in B
cells suggests that SHP-2 may be sufficient for inhibition. However,
one cannot exclude the possibility that not yet known phosphatases may
be recruited by the N-KIR chimera which would account for inhibition.
Unidentified phosphorylated proteins were indeed coprecipitated with
phosphorylated chimeras, the most prominent of which was a 77-kDa
molecule (Fig. 3A). Noticeably, however, the intensity of
phosphorylation of these proteins was correlated with that of the total
proteins seen in whole cell lysates, including lysates from cells
stimulated with RAM F(ab')2 (Fig. 2C),
suggesting that these molecules were not directly involved in
inhibition. In apparent contradiction to our data, SHP-1 was reportedly
necessary to inhibit B cell activation. Indeed, the same N+C-KIR
chimera as the one used in this study had markedly reduced inhibitory
properties in SHP-1-deficient chicken DT40 cells 5 . However, one may
notice that inhibition of Ca2+ responses and inhibition of
NFAT activity were not completely abolished in these cells. Direct
evidence that SHP-2 can inhibit is still lacking, and the inhibitory
role of SHP-2 51, 52 is controversial, as SHP-2 has been reported to
positively regulate cell activation 53 .
In conclusion, our results provide evidence that the two tandem ITIMs in KIRs may differentially contribute to the inhibition of cell activation. Our study suggests that two types of cooperative events may occur during KIR-mediated inhibition. Cooperative effects may be required for the phosphorylation of the C-terminal ITIM, and a cooperation of SHP-1 and SHP-2 may be required for KIRs to fully inhibit cell activation.
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Acknowledgments |
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RIIB Abs and Janine Moncuit (Institut Curie,
Paris, France) for rat embryonic cells. We are grateful to Dr. John C.
Cambier (Natl. Jewish Center, Denver, CO) for having taught us
Ca2+ measurement techniques. |
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2 Address correspondence and reprint requests to Dr. Marc Daëron, Laboratoire d’Immunologie Cellulaire et Clinique, Institut National de la Santé et de la Recherche Médicale U.255, Institut Curie, 26, rue d’Ulm, 75005 Paris, France. E-mail address:
3 Abbreviations used in this paper: KIR, killer cell inhibitory receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; FcRIIB, type IIB receptors for the Fc portion of IgG; SH2, Src homology 2; SHP, SH2 domain-bearing protein tyrosine phosphatase; FcRI, high-affinity IgE receptor; BCR, B cell receptor; MAR, mouse anti-rat Ig; PY, phosphotyrosine; RAM, rabbit anti-mouse Ig; SHIP, SH2 domain-bearing inositolphosphate phosphatase; TNP, trinitrophenyl; IC, intracytoplasmic; HRP, horseradish peroxidase.
Received for publication October 7, 1998. Accepted for publication December 9, 1998.
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T. Yokozeki, K. Adler, D. Lankar, and C. Bonnerot B Cell Receptor-Mediated Syk-Independent Activation of Phosphatidylinositol 3-Kinase |
) with 2.4G2
F(ab')2 before they were challenged for serotonin release
with 10 µg/ml TNP-MAR F(ab')2. The figure
represents the percentage of serotonin released as a function of the
concentration of IgE.
1 x 103 cells/s, as a function of
time. C, Inhibition of tyrosine phosphorylation of
intracellular proteins. IIA1.6 transfectants (5 x
105) expressing KIR chimeras were challenged or not with
RAM F(ab')2 or RAM IgG for 3 min and lysed. Whole cell
lysates were fractionated by SDS-PAGE, transferred onto Immobilon-P
membranes, and Western blotted with anti-PY Abs.
