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J Biol Chem. Author manuscript; available in PMC 2007 October 2.
Published in final edited form as:
PMCID: PMC1997429
EMSID: UKMS517

The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signalling cascade

Abstract

The two lectin receptors, CLEC-2 and Dectin-1 have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In the present study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signalling by ITAM receptors including Src, Syk and Tec family kinases and on PLCγ. Strikingly, mutation of either SH2 domain of Syk blocks signalling by CLEC-2 despite the fact that it has only a single YXXL motif. Further, signalling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of ITAM receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same 4 amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2-domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signalling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif which requires the tandem SH2 domains of Syk but which is only partially dependent on the SLP-76/BLNK family of adapters.

INTRODUCTION

The C-type lectin superfamily of transmembrane proteins consists of at least members in the human genome (1). The superfamily can be divided into ‘classical’ C-type lectins which contain a carbohydrate recognition domain (CRD) and bind sugars in a calcium-dependent manner and the ‘non-classical’ C-type lectin-like proteins which contain a C-type lectin-like domain (CTLD), homologous to a CRD but which lacks the consensus sequence for binding sugars and calcium (2). Protein ligands for a number of classical and non-classical C-type lectin receptors have been described.

C-type lectin-like receptor 2 (CLEC-2) is a type II transmembrane protein and a non-classical C-type lectin (3). The CTLD domain in CLEC-2 is supported by a 41 amino acid neck region, a single transmembrane domain and 31 amino acid cytoplasmic domain (3). CLEC-2 mRNA has been identified in liver and in blood cells, mostly of myeloid origin, including monocytes, granulocytes and dendritic cells (3). Recently, we have identified expression of CLEC-2 in platelets and have shown that it functions as a receptor for the snake venom toxin rhodocytin (also known as aggretin), which elicits powerful platelet activation (4). Rhodocytin, however, also binds to several other platelet receptors (5, 6), making it unclear whether CLEC-2 is sufficient to mediate activation alone and thereby hampering analysis of the mechanism of activation.

The cytosolic domain of CLEC-2 contains a single tyrosine residue within a YXXL motif, a consensus sequence for phosphorylation by Src family kinases in immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ITAMs have the sequence Yxx(L/I)x6-12Yxx(L/I), and ITIMs, have the sequence (L/I/V)xYxx(L/I/V). Phosphorylation of the two tyrosine residues within an ITAM leads to recruitment of the tyrosine kinases Syk and Zap-70 via their tandem Src-homology 2 (SH2) domains, leading to cellular activation (7, 8). Phosphorylated ITIMs binds to the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, or the lipid phosphatases SHIP1 and SHIP2, leading, in most cases, to cellular inhibition (9).

Signalling by ITAM receptors, such as the platelet collagen receptor complex, GPVI/FcR γ-chain, or the B- and T-cell antigen receptors, is mediated via members of the Src, Syk, Tec, Vav, SLP-76/BLNK and PLCγ families of signalling proteins (reviewed in (10-12)). The specific members of each family which mediate ITAM signalling is cell dependent. For example, SLP-76 is used by the T-cell receptor (13) and the platelet collagen receptor GPVI (14), whereas B cells use the homologous protein BLNK (15).

We have shown that activation of platelets by rhodocytin is critically dependent on the tyrosine kinase Syk and many of the proteins which participate in ITAM signalling in platelets (4). This has led us to propose that the snake venom toxin signals through a similar pathway to that of ITAM receptors, with Syk being recruited via the phosphorylated YXXL sequence in the cytosolic tail of the lectin-like receptor. A similar coupling to Syk has been proposed for a second C-type lectin receptor, Dectin-1, which mediates activation of dendritic cells by zymosan (16). A third YXXL containing member of the C-type lectin superfamily, DC-SIGN, has also recently been reported to signal to PLCγ in dendritic cells, although the role of Syk in signalling by this receptor is not known (17).

The aim of the present study was to characterise the mechanism of CLEC-2 signalling in platelets and in two haematopoietic-derived cell line model systems, and to compare this to signalling by Dectin-1 and DC-SIGN. The results demonstrate that signalling by CLEC-2 is completely dependent on the cytoplasmic YXXL motif and requires both SH2 domains of Syk. The signalling pathway activated by CLEC-2 involves Src, Syk and Tec family kinases and PLCγ, but is distinct from that of ITAM signalling in that it has a partial, rather than absolute dependence on the SLP-76/BLNK family of adapter proteins. Dectin-1 signals in a similar way to CLEC-2 whereas the mechanism of signalling by DC-SIGN is distinct. The results demonstrate that some but not all lectin receptors signal through a single YXXL motif leading to activation of PLCγ.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents

Polyclonal goat α-human CLEC-2, α-mouse Dectin-1 and normal goat IgG were purchased from R & D systems Inc. (Minneapolis, MN, USA). Monoclonal α-CD209 (DC-SIGN) was purchased from BD Pharmingen (San Diego, CA). Rabbit polyclonal antibodies α-Syk, α-PLCγ2 and α-Btk have been described previously (18, 19). Anti-phosphotyrosine monoclonal antibody 4G10, α-SLP-76 polyclonal antibody and α-LAT polyclonal antibody were purchased from Upstate Biotechnology (TCS Biologicals Ltd., Bucks, UK). Anti-human Vav3 antibody was a kind gift from Dr Daniel Billadeau and was raised in rabbits as described previously (20). Anti-MYC antibody was purchased from Cell Signalling Technology (NEB UK Ltd., Herts, UK). F(ab’)2 fragments of the anti-human FcγRIIA antibody IV.3 were generated as described previously (21). FITC-conjugated donkey anti-goat IgG secondary antibody was from Jackson ImmunoResearch Laboratories Inc. (Stratech Scientific Ltd., Cambs, UK). HRP-conjugated sheep anti-mouse secondary antibody, HRP-conjugated donkey anti-rabbit secondary antibody and enhanced chemiluminescence reagents (ECL) were purchased from Amersham Biosciences (Bucks, UK). GST-fusion proteins corresponding to single or tandem SH2 domains of Syk were prepared as described previously (22). Rhodocytin was purified by Dr Johannes Eble as previously described (23). The GPIIbIIIa antagonist lotrafiban was a gift from GlaxoSmithKline (King of Prussia, PA, USA) and the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide was obtained from Peptide Institute (Osaka, Japan). The Src kinase inhibitor PD0173952 was a gift from Pfizer Global Research and Development (Ann Arbor, MI, USA). The Src kinase inhibitor PP2 was purchased from Calbiochem (Nottingham, UK). All other reagents were purchased from Sigma or from previously described sources (18, 24).

Platelet studies

Blood was drawn on the day of experiment from healthy, drug-free volunteers into 1:10 (v:v) sterile sodium citrate and 1:9 (v:v) acid citrate dextrose (ACD: 120 mM sodium citrate, 110 mM glucose, 80 mM citric acid). Washed platelets were prepared as described previously (18).

Platelet aggregation studies were carried out using washed platelets at a concentration of 2 x 108/ml in a Born aggregometer (ChronoLog, Havertown, PA, USA) at 37 °C with continuous stirring at 1200 rpm for five minutes. Aggregation of platelets in response to rhodocytin (300nM) or α-CLEC-2 antibody (10 μg/ml) was recorded by measuring change in optical density. Platelets were pre-incubated with IV.3 F(ab’)2 (12 μg/ml), PP2 (10 μM) or PD0173952 (25 μM) for 10 minutes prior to stimulation where indicated. Platelets were used at 1 x 109/ml for protein studies. Lotrafiban (10 μM) or GRGDS peptide (1 mM) was included in the resuspension buffer to block aggregation and signalling through GPIIbIIIa. Stimulations were carried out in a Born aggregometer for the times shown. Following stimulation platelets were lysed with an equal volume of 2 x lysis buffer (300 mM NaCl, 20 mM Tris, 2 mM EGTA, 2 mM EDTA, 2% NP-40 pH7.4 with 2.5 mM Na3VO4, 100 μg/ml AEBSF, 5 μg/ml leupeptin, 5 μg/ml aprotonin and 0.5 μg/ml pepstatin).

Constructs

Human CLEC-2 cloned into pcDNA3 has been previously described (25). For these experiments CLEC-2 was subcloned into pEF6 vector with a C-terminal MYC tag, pEF6/Myc-His A (Invitrogen). A point mutation of the cytoplasmic tyrosine residue of CLEC-2 to a phenylalanine (Y7F) was generated by a two-step PCR method (26). The mutating primers CLEC-2-Y7F-REV (5’-GTT TTA ATA TTT AAG GTG ATG AAT CCA TCT TCA TCC TG-3’) and CLEC-2-Y7F-FWD (5’-CAG GAT GAA GAT GGA TTC ATC ACC TTA AAT ATT AAA AC-3’) were used along with vector specific primers T7 and 4150 (5’-AGG CAC AGT CGA GGC TGA TC-3’). D3A, E4A, D5A and G6A CLEC-2 were generated by a single step PCR approach. The mutating primers were D3A-FWD (5’-TAG TAG GGA TCC ATG CAG GCT GAA GAT GGA TAC-3’), E4A-FWD (5’-TAG TAG GGA TCC ATG CAG GAT GCA GAT GGA TAC-3’), D5A-FWD (5’-TAG TAG GGA TCC ATG CAG GAT GAA GCT GGA TAC ATC-3’), G6A-FWD (5’-TAG TAG GGA TCC ATG CAG GAT GAA GAT GCA TAC ATC ACC-3’) and hCLEC-2-REV (5’-TAG TAG GCG GCC GCA GGT AGT TGG TCC ACC TTG GTC-3’). Porcine Syk cloned into pcDNA3 has been described previously (27). Inactivating point mutants of each SH2 domain of Syk were made by mutating Arg-37 or Arg-190 to Ala. In both cases T7 and BGH were used as outside primers. Specific primers for the point mutants were Syk-R37A-FWD (5’-GGG CTC TAC CTG CTT GCC CAG AGC CGC AAC TAC-3’), Syk-R37A-REV (5’-GTA GTT GCG GCT CTG GGC AAG CAG GTA GAG CCC-3’), Syk-R190A-FWD (5’-GGG AAG TTT TTG ATC GCG GCC AGG GAC AAC GGG-3’), Syk-190A-REV (5’-CCC GTT GTC CCT GGC CGC GAT CAA AAA CTT CCC-3’). Human GPVI cloned into pRC plasmid has been described previously (24). For these experiments hGPVI was subcloned into pcDNA3 with a C-terminal MYC tag (Invitrogen). Human FcR γ-chain DNA was amplified from HEL cell cDNA by PCR (hFcRγ-FWD (5’-TAG TAG GGA TCC CAG CCC AAG ATG ATT CCA GC-3’), hFcRγ-REV (5’-TAG TAG GCG GCC GCC TAC TGT GGT GGT TTC TCA TG-3’)) and cloned into pEF6 vector with no tag (Invitrogen). Murine Dectin-1 was amplified from cDNA prepared from murine spleen by PCR (mDectin-1-FWD (5’-TAG TAG GGA TCA TGA AAT ATC ACT CTC ATA TAG-3’), mDectin-1-REV (5’-TAG TAG GCG GCC AGT TCC TTC TCA CAG ATA C-3’) and cloned into pEF6 vector with a C-terminal MYC tag, pEF6/Myc-His. All sequences were verified by sequencing. Wild type DC-SIGN in pcDNA3 has been described previously (28). A point mutation of the YXXL tyrosine (Y31) to phenylalanine was generated by a two-step PCR method. The mutating primers were hDCSIGNY31F-FWD (5’-CGA CAG ACT CGA GGA TTC AAG AGC TTA GCA GGG-3’) and hDCSIGNY31F-REV (5’-CCC TGC TAA GCT CTT GAA TCC TCG AGT CTG TCG-3-). The NFAT luciferase reporter contains three copies of the distal NFAT site from the IL-2 promoter (29) and was kindly provided by Prof. A. Weiss. The pEF6-lacZ expression construct was obtained from Invitrogen.

Cell culture and transfection

DT40 chicken B cells were grown in RPMI supplemented with 10% foetal bovine serum, 1% chicken serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μM mercaptoethanol and 20mM GlutaMAX. DT40 cells rendered deficient for syk (30), lyn (30), lyn/syk (31), BLNK (32), btk (31), and plcγ2 (33) were described previously and kindly provided by Dr. T. Kurosaki (Kansai Medical University, Moriguchi, Japan). Jurkat T cells were grown in RPMI supplemented with 10% foetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 20 mM GlutaMAX. Jurkat derivatives JCaM1 and JCaM1/Lck and J14 and J14/SLP-76 (J14-76) were kindly provided by Dr. A. Weiss (UCSF, CA, USA) and have been described previously (34, 35). Cells were transfected in a volume of 400 μl non-supplemented RPMI by electroporation using a GenePulser II (Biorad) set at 350V, 500 μF for DT40 and 250V and 950 μF for Jurkats. 293T cells were grown in DMEM supplemented with 10% foetal bovine serum, 100 units/ml penicillin, 100μg/ml streptomycin and 20mM Glutamax. 293T cells were transfected with 5μg DNA of each DNA construct by calcium phosphate precipitation.

Cell line protein studies

Cells were transfected as described above with 10 μg of CLEC-2 or 10 μg of Y7F CLEC-2. Twenty hours following transfection cells were washed and resuspended in non-supplemented RPMI. Cells were stimulated with 500 nM rhodocytin at room temperature for ten minutes. Where indicated inhibitors were preincubated with the cells for 10 minutes prior to stimulation. Following stimulation cells were lysed with an equal volume of 2 x lysis buffer. 293T cells were harvested 48 hours after transfection, washed once in PBS and lysed in 500μl 1 x lysis buffer.

Luciferase assay

Cells were transfected as described above with either 10 μg of CLEC-2, 10 μg of Y7F CLEC-2, 10μg Dectin-1 or 2 μg GPVI and 2 μg FcR γ-chain constructs, in addition to 15 μg of the luciferase reporter construct and 2 μg of pEF6-lacZ to control for transfection efficiency. Where indicated the receptor of interest was contransfected along with 5 μg wild-type Syk, R37ASyk or R190ASyk into Syk deficient DT40 cells. Twenty hours after transfection, live cells were counted by Trypan blue exclusion, and samples were divided for luciferase assay, β-Galactosidase assay and flow cytometry. Luciferase assays were as described previously (36). Rhodocytin was used at 50 nM, α-CLEC-2 antibody at 40 μg/ml, α-DC-SIGN 10 μg/ml crosslinked with sheep α-mouse F(ab’)2 fragments at 30 μg/ml, Zymosan at 250μg/ml and convulxin at 10 μg/ml. Luciferase activity was measured with a Centro LB 960 microplate luminometer (Berthold Technologies, Germany). Data is expressed either as luminescence units normalised to β-galactosidase activity or as fold increase in luminescence units over basal as indicated. All luciferase data is averaged from 3 readings. Data is represented as one experiment representative of three +/- s.e.m for the three readings of the experiment. β-galactosidase assays were performed with half a million cells using the Galacto-Light chemiluminescent reporter assay, according to the manufacturer’s instructions (Applied Biosystems, Bedford, Mass, USA). β-Galactosidase activity was measured in triplicate using a microplate luminometer. All luciferase assay data were normalized to β-galactosidase values.

Flow cytometry

Expression of each receptor was confirmed by flow cytometry. For CLEC-2 or Dectin-1 detection, 5 x 105 cells were stained in 50 μl volume for 20 minutes with either 10 μg/ml goat α-CLEC-2 or 10 μg/ml goat α-Dectin-1 antibody alongside goat IgG as a negative control. Cells were then washed and incubated for 20 minutes with 15 μg/ml FITC-conjugated α-goat IgG secondary antibody. Stained cells were analysed using a FACScalibur (BD Biosciences). Data was collected and analyzed using CellQuest software.

Immunoprecipitation, pull downs and Western blotting

Cell lysates were precleared and detergent-insoluble debris was removed as described (37). Following pre-clearing 50 μl aliquots of the stimulation were added to an equal volume of 2 x Laemlli sample buffer for whole cell phosphorylation studies. For immunoprecipitation and pull down studies lysates were incubated with the indicated antibodies and a mixture of PAS and PGS or GST-fusion proteins corresponding to single or tandem SH2-domains of Syk associated with glutathione sepharose. Following immunoprecipitation the sepharose beads were washed and resulting protein complexes eluted with 2 x Lamelli sample buffer.

The resulting whole cell lysates and immunoprecipitates were resolved by SDS-PAGE, and western blotting was carried out as described previously (18).

Analysis of data

Experiments were performed on at least three occasions and are shown as representative data from one experiment. Where experiments were carried out in triplicate, results are presented as the mean of this data.

RESULTS

Anti-CLEC-2 antibody and rhodocytin stimulate similar patterns of tyrosine phosphorylation in platelets.

The snake toxin rhodocytin binds to multiple receptors on the platelet surface therefore making it unclear which signalling events are mediated through CLEC-2 or by other receptors for the snake toxin. To address this, we used an α-CLEC-2 antibody which we have previously shown is able to induce platelet aggregation and phosphorylation of CLEC-2 in platelets independent of the low affinity immune receptor, FcγRIIA (4). Experiments were carried out in the presence of F(ab’)2 fragments of the antibody IV.3, to block the FcγRIIA receptor on platelets. The antibody to CLEC-2 (10 μg/ml) stimulated platelet shape-change and aggregation whereas a non-specific goat IgG control antibody had no effect (Fig 1Ai). The onset of aggregation in response to the CLEC-2 antibody occurs after a lag time that is characteristic of platelet aggregation to rhodocytin (Fig 1Ai). Aggregation to the CLEC-2 antibody is completely inhibited by the Src family kinase inhibitor, PP2 (Fig 1Aii), as is also the case for rhodocytin (4). The same result was observed with the structurally distinct Src kinase inhibitor PD0173952 (data not shown). Tyrosine phosphorylation of platelet lysates induced by rhodocytin and the CLEC-2 antibody was compared by western blotting with the antiphosphotyrosine antibody, 4G10. The two agonists stimulated a similar pattern of tyrosine phosphorylation suggesting that the increase in tyrosine phosphorylation induced by rhodocytin is mediated through CLEC-2 (Fig 1B). No increase in whole cell tyrosine phosphorylation was observed in platelets incubated with non-specific goat IgG antibody (Fig 1B). In addition, immunoprecipitation studies confirmed that the CLEC-2 antibody induced tyrosine phosphorylation of the same set of proteins that are regulated by rhodocytin in platelets, namely Syk, PLCγ2, Vav3, LAT, SLP-76 and Btk (Fig 1C). The greater level of tyrosine phosphorylation of Syk, LAT and PLCγ2 induced by the CLEC-2 antibody may reflect slight differences in the kinetics of activation or differences in the level of stimulation. Importantly, none of these proteins became phosphorylated following stimulation with control goat IgG. These results demonstrate that CLEC-2 is sufficient to cause platelet activation and suggest that the major mechanism of platelet activation by rhodocytin is through the lectin receptor.

Figure 1
Platelet activation by rhodocytin and a specific antibody to CLEC-2. (A)(i) Washed platelet aggregation was measured following addition of 300 nM rhodocytin, 10 μg/ml goat α-CLEC-2 or 10μg/ml goat IgG. Addition of agonist is indicated ...

CLEC-2 expressing DT40 cells and Jurkat cells are activated by stimulation of the receptor.

To further investigate the mechanism of CLEC-2 signalling, CLEC-2 was cloned into an expression vector and transiently transfected into cell lines. DT40 B cells and Jurkat T cells were used as model systems for studying CLEC-2 signalling since B-cells and T-cells express many of the same signalling proteins as platelets and mutants of both cell lines are available with deficiencies in key signalling proteins. Transfection of CLEC-2 into DT40 cells and Jurkat cells led to expression at the cell surface as measured by flow cytometry, whereas it was absent from mock-transfected cells (Fig 2A).

Figure 2
CLEC-2 expressing DT40 and Jurkat cells are activated by rhodocytin and anti-CLEC-2 antibody. Wild-type DT40 chicken B cells and Jurkat T cells were transiently transfected with NFAT-luciferase reporter construct, β-galactosidase construct and ...

The ability of CLEC-2 to activate PLCγ in DT40 and Jurkat cells was investigated by cotransfection with a luciferase reporter construct encoding the luciferase enzyme under control of NFAT/AP1 sites from the IL-2 promoter. This reporter construct is activated by PKC and Ca2+ and the amount of luciferase activity is therefore a measure of PLCγ activity. Transfection of CLEC-2 conferred activation of luciferase in response to the CLEC-2 antibody and to rhodocytin (Fig 2B and 2C). Cells co-transfected with the reporter construct and empty pEF6 vector showed no response to rhodocytin or the CLEC-2 antibody, thereby confirming that activation is mediated by expression of CLEC-2 (Fig 2B and C). All cells exhibited a marked increase in expression of luciferase in response to the PKC activating phorbol ester PMA and the calcium ionophore ionomycin which served as a positive control (Fig 2B). These results demonstrate that neither DT40 nor Jurkat cells express functional, endogenous CLEC-2, and that expression of CLEC-2 is sufficient to confer responses to rhodocytin and the CLEC-2 antibody.

Tyrosine phosphorylation of CLEC-2 is critical for activation

We have previously shown that CLEC-2 becomes tyrosine phosphorylated in platelets in a Src kinase-dependent manner following stimulation with rhodocytin (4). To investigate the functional significance of CLEC-2 phosphorylation, we have expressed wild type CLEC-2 and a mutant of CLEC-2, Y7F, in which the cytosolic tyrosine has been replaced by phenylalanine, in DT40 cells. Rhodocytin stimulated an increase in whole cell tyrosine phosphorylation in DT40 cells expressing the wild type receptor through a pathway that was blocked by the Src kinase inhibitor, PP2 (Fig 3A). In contrast, no increase in tyrosine phosphorylation was seen in mock-transfected DT40 cells or in DT40 cells expressing the Y7F mutant of CLEC-2 in response to rhodocytin. Further, rhodocytin was unable to support activation of PLCγ2 in cells expressing the Y7F mutant, as measured in the luciferase assay (Fig 3B). Importantly, in all of these studies, wild type and Y7F-CLEC-2 were expressed on the cell surface at a similar level (Fig 3C). These results demonstrate that CLEC-2 requires the cytoplasmic tyrosine to mediate activation of PLCγ.

Figure 3
Role of CLEC-2 cytoplasmic tyrosine residue in signalling by the receptor. (A) Wild-type DT40 cells were transfected with pEF6-MYC-CLEC-2 or pEF6-MYC-Y7FCLEC-2 and were stimulated with 50 nM rhodocytin for 10 minutes. Where indicated cells were preincubated ...

CLEC-2 signals via Src, Syk and Tec family kinases and PLCγ

The signalling pathway used by CLEC-2 was further investigated using mutant DT40 cells lacking key signalling proteins (30-33). In each case CLEC-2 was transfected into mutant DT40 cells along with the luciferase reporter construct described above. CLEC-2 was expressed on the surface at a similar level in all of the mutant cells as confirmed by flow cytometry (data not shown). The ability of each transfectant to respond to PMA/ionomycin within the expected range was also confirmed in each experiment (data not shown). The results are shown as the fold increase over basal luciferase levels in each cell line.

CLEC-2-expressing wild-type DT40 cells exhibited a robust increase in luciferase activity in response to rhodocytin whereas cells deficient in Syk, Btk or PLCγ2 failed to respond (Fig 4A), demonstrating a critical role for these proteins in mediating CLEC-2 signalling. In contrast, CLEC-2-expressing DT40 cells deficient in the major Src family kinase that is expressed in these cells, Lyn, exhibited a potentiated response to rhodocytin (Fig 4Bi). This activation is also dependent on Syk since DT40 cells deficient in both Lyn and Syk do not respond to rhodocytin (Fig 4Bi). This result may be due to a negative feedback role of Lyn, as previously described for the regulation of B-cell receptor signalling (30, 38). Interestingly, a similar negative feedback role for Lyn has also been proposed in platelets stimulated by the ITAM receptor, GPVI (39).

Figure 4
Role of Src, Syk and Btk family kinases downstream of CLEC-2. (A) Wild-type DT40 cells (WT) or DT40 cells engineered to lack indicated signalling proteins (Syk, Btk or PLCγ2) were transiently transfected with pEF6-MYC-CLEC-2. Signalling to PLCγ ...

In view of the potentiation observed in the absence of the Src family kinase Lyn in DT40 cells, we extended these studies to the Jurkat-derived cell line JCaM1, which is deficient in the T cell Src family kinase, Lck (34). Importantly, Lck is believed to be the only member of the Src family of tyrosine kinases in these cells and does not mediate feedback inhibition of PLCγ regulation. As a control for Lck deficiency, we have used JCaM1 cells stably transfected with wild type Lck (JCaM1/Lck(+)) (34). It was necessary to use Lck-transfected JCaM1 cells as a control in view of the possibility that these cells, which were originally made by chemical mutagenesis (34), have additional, unidentified defects. JCaM1/Lck(+) cells transfected with CLEC-2 exhibited an increase in luciferase activation in response to rhodocytin, whereas JCam1 cells transfected with CLEC-2 failed to respond (Fig 4Bii). Together these results demonstrate that CLEC-2 signalling is critically dependent on Src, Syk and Tec family kinases, and on PLCγ.

Both SH2 domains of Syk are required for binding and signalling downstream of CLEC-2

We have previously reported that fusion proteins corresponding to the tandem SH2 domains of Syk associate with phosphorylated CLEC-2 in platelet lysates (4). Similarly, a phospho-peptide corresponding to the CLEC-2 cytoplasmic tail is able to pull-down Syk from platelet lysates (4). To investigate whether this interaction requires either or both of the SH2 domains of Syk, we have used recombinant N-terminal, C-terminal and tandem SH2 domains of Syk to precipitate CLEC-2 from platelet lysates. Strikingly, both SH2 domains were required for precipitation of CLEC-2 as shown in Figure 5A, thereby suggesting that both SH2 domains of Syk are required for the interaction with the lectin receptor. To confirm this conclusion, we introduced inactivating point mutations into the SH2 domains of Syk by mutating either Arg-37 or Arg-190 of porcine Syk to alanine residues. The residues were identified by alignment with rat, murine and human Syk sequences (Fig 5B). In addition, mutation of these residues, in combination with neighbouring amino acids have previously been demonstrated to inactivate the SH2 domains of porcine Syk and block the ability of porcine Syk to reconstitute signalling in the chicken cell line DT40 (30). Mutation of the corresponding residues in rat Syk have previously been reported to be required for Syk binding to phosphorylated tyrosine residues in ITAMs. In both cases these mutations have been demonstrated to render the SH2 domains inactive without affecting the autoactivated kinase activity of the enzyme (30, 40-42). In agreement with this, constructs for R37A and R190A porcine Syk translate a full-length Syk (Fig 5C) that induces a degree of basal signalling when overexpressed in DT40 cells (data not shown). Syk-deficient DT40 cells were co-transfected with wild-type or mutant Syk, and CLEC-2. In each case, expression of CLEC-2 was confirmed by flow cytometry and was of an equivalent level in each cell line (data not shown). Syk expression was confirmed by western blotting (Fig 5C). Signalling by CLEC-2 was measured by luciferase assay following stimulation with rhodocytin. Wild-type Syk was able to reconstitute signalling responses in Syk deficient cells following CLEC-2 stimulation (Fig 5C). However, inactivation of either SH2 domain of Syk was sufficient to block signalling responses following rhodocytin stimulation. Therefore, signalling by CLEC-2 is critically dependant on both SH2 domains of Syk.

Figure 5
Syk is recruited by CLEC-2 and signals via both its SH2 domains. (A) Washed human platelets were stimulated with or without 50 nM rhodocytin for five minutes in the presence of GRGDS peptide, lysed and incubated with Glutathione sepharose associated with ...

Differential regulation of BLNK/SLP-76 downstream of GPVI and CLEC-2

Signalling downstream of ITAM receptors is critically dependent on the SLP-76/BLNK family of adapter proteins (43-46). A notable difference between the signalling cascade used by the platelet ITAM receptor GPVI and by the CLEC-2 ligand rhodocytin is the ability of high concentrations of rhodocytin to overcome the blockade caused by the absence of SLP-76 (4). This difference could be due to the ability of rhodocytin to activate other receptors in platelets, such as integrin α2β1 or GPIbα (47), or because the adapter plays a partial role in signalling by the lectin receptor. Thus there may be a fundamental difference between the contribution of the SLP-76/BLNK family of adapter proteins to signalling downstream of CLEC-2 relative to ITAM receptors.

To compare the role of BLNK and SLP-76 in signalling by GPVI and CLEC-2, we have transfected CLEC-2 or GPVI and FcRγ-chain into mutant DT40 and Jurkat cells lacking BLNK and SLP-76, respectively (32, 35). Stimulation of wild-type DT40 cells expressing GPVI/FcR γ-chain complex with the snake venom toxin convulxin resulted in a robust increase in luciferase activity, which was totally abrogated in the absence of BLNK (Fig 6A). In comparison, rhodocytin generated a significant, but diminished (~ 30 % of wild type) response in the absence of BLNK even though it stimulated a similar increase to that induced by convulxin in wild type cells (Fig 6A). A similar set of observations were made in the Jurkat-derived cell line J14 which lacks SLP-76. J14 cells stably transfected with SLP-76 were used as a control (J14-76) for these studies as these cells were also made by chemical mutagenesis, which could have introduced additional genetic modifications. SLP-76-expressing cells transfected with CLEC-2 or GPVI/FcR γ-chain give similar robust responses to rhodocytin and convulxin, respectively (Fig 6B). The response to convulxin was abolished in the absence of SLP-76, whereas the response to rhodocytin was reduced by 60%. In order to further confirm that the partial dependency on BLNK/SLP-76 is not due to rhodocytin binding to another receptor on the cell lines, we have investigated whether CLEC-2 signalling induced by a CLEC-2 specific antibody is able to bypass SLP-76 in Jurkat cells. J14 and J14-76 cells were transfected with CLEC-2 and stimulated with the anti-CLEC-2 antibody. In response to CLEC-2 antibody, CLEC-2 signalling is significantly reduced in the absence of SLP-76 but the receptor is still able to signal without this adapter (Fig 6C). These data therefore demonstrate a fundamental difference in signalling by CLEC-2 and the ITAM receptor, GPVI-FcR γ-chain complex, in that activation by the lectin receptor is only partially dependent on the SLP-76/BLNK family of adapter proteins.

Figure 6
BLNK/SLP-76 plays a differential role in signalling by GPVI and CLEC-2. (A) Wild-type (WT) or BLNK-deficient (BLNK-/-) DT40 and (B) SLP-76 deficient Jurkat (J14) or J14 reconstituted with SLP-76 (J14-76) cells were transfected with either pcDNA3-GPVI ...

The lectin receptor Dectin-1 signals through a similar pathway to CLEC-2

Dectin-1 is also a member of the C-type lectin receptor family which has recently been shown to activate Syk via a single YXXL motif in its cytosolic tail in both dendritic cells and in macrophages (16, 48). However, the signalling events downstream of Syk activation in response to Dectin-1 stimulation have not been characterised. A series of studies were therefore undertaken in transfected DT40 and Jurkat cells to compare the signalling pathway used by Dectin-1 to that used by CLEC-2 using zymosan as the activating ligand.

Transfection of DT40 and Jurkat cells with Dectin-1 leads to expression of the lectin receptor on the cell surface as measured by flow cytometry using a goat α-Dectin-1 antibody (Fig 7A). In comparison, there was no specific binding of the antibody to mock-transfected cells suggesting that Dectin-1 is not endogenously expressed on either cell line (Fig 7A). Transfection of Dectin-1 conferred marked activation of luciferase in response to zymosan in DT40 cells (Fig 7B) and in Jurkat T-cells (data not shown), whereas there was no response in mock-transfected cells confirming the absence of expression of endogenous receptor (Fig 7B and data not shown). Stimulation with PMA and ionophore stimulated robust activation of luciferase in Dectin-1 and mock-transfected DT40 (Fig 7B) and Jurkat cells (data not shown), thereby confirming cell viability. These observations demonstrate that expression of Dectin-1 confers signalling responses to zymosan in both DT40 and Jurkat cell lines.

Figure 7
Role of Src, Syk and Tec kinases, PLCγ and SLP-76/BLNK in Dectin-1 signalling. Wild-type DT40 chicken B cells and Jurkat T cells were transiently transfected with NFAT-luciferase reporter construct, β-galactosidase construct and either ...

To investigate if the Dectin-1 signalling pathway shares the same characteristics as for CLEC-2, the lectin receptor was transfected into DT40 cells deficient in Syk, BLNK, Btk or PLCγ2, and in Jurkat cells deficient in the Src kinase Lck (JCaM1) or SLP-76 (J14). In all cases, Dectin-1 was cotransfected with the luciferase reporter construct and luciferase activity measured following stimulation with zymosan. Flow cytometry studies confirmed that similar levels of Dectin-1 were expressed in each cell line (data not shown).

As observed with CLEC-2, Dectin-1 signalling was completely inhibited in the absence of Syk, Btk and PLCγ2 in DT40 cells (Fig 7Ci) and in the absence of Lck in JCaM1 cells (Fig 7Cii). Further, Dectin-1 signalling was partially but not fully dependent on the adapters BLNK and SLP-76 in DT40 and Jurkat cells, respectively (Fig 7Ci+iii). In both cases, the response to zymosan was approximately 40% of that in the control (Fig 7Ci+iii). Dectin-1 signalling was also markedly inhibited in Syk-deficient DT40 cells transfected with either of the SH2 domain mutants of Syk described above relative to the response seen with transfection of wild type Syk (Fig 7Di). Mutation of the N-terminal (R37A) or C-terminal (R190A) SH2 domain of Syk reduced the response to Dectin-1 by approximately 80 % in both cases (Fig 7Di). Expression of similar amounts of Syk and the two Syk mutants in the DT40 cells was confirmed by western blotting (Fig 7Dii). The marked but partial inhibition of response to Dectin-1 observed with the Syk SH2 domain mutants contrasts with the complete abolition of response to CLEC-2 (Figure 5C).

These results demonstrate that CLEC-2 and Dectin-1 signal through a similar signalling cascade which can be distinguished from that used by ITAM receptors through the partial dependence on the SLP-76/BLNK family of adapter proteins. The CLEC-2 and Dectin-1 signalling pathways can be distinguished from each other by their complete or partial dependence on the phosphotyrosine-binding properties of the Syk SH2 domains.

DC-SIGN cross-linking results in PLCγactivation, independent of tyrosine phosphorylation of its YXXL motif

Activation of a third member of the C-type lectin family, DC-SIGN, with specific antibodies has been reported to lead to ERK1/2, Akt and PLCγ phosphorylation and increases in intracellular calcium in dendritic cells (17). DC-SIGN also contains a single YXXL motif in its cytoplasmic tail, although it is not known whether this undergoes tyrosine phosphorylation upon activation. Experiments were therefore designed to compare the signalling pathway used by DC-SIGN with that of Dectin-1 and CLEC-2 and to investigate whether this requires the YXXL motif of DC-SIGN.

Jurkat cells co-transfected with the luciferase reporter construct and DC-SIGN conferred a marked increase in luciferase activity over mock-transfected cells in response to cross-linking with an antibody to DC-SIGN, whereas there was no increase in response to an isotype matched negative control (Fig 8A). Strikingly, cells transfected with a mutant form of DC-SIGN in which the Y residue of the YXXL sequence has been replaced by phenylalanine (Y31F DC-SIGN), gave a slightly greater response upon crosslinking of DC-SIGN relative to cells transfected with wild type receptor (Fig 8A). Expression of similar levels of the YXXL mutant of DC-SIGN and wild type DC-SIGN was confirmed by flow cytometry (data not shown). All cells exhibited a similar increase in expression of luciferase in response to PMA and ionomycin (data not shown). These results demonstrate that DC-SIGN signals through a distinct pathway to that used by CLEC-2 and Dectin-1.

Figure 8
DC-SIGN signalling is not dependent on the YXXL motif. (A) Jurkat cells were transiently transfected with NFAT-luciferase reporter construct, β-galactosidase construct and either pcDNA3-DC-SIGN, pcDNA3-Y31FDC-SIGN or empty pcDNA3 vector (mock). ...

In order to investigate the role of Src kinases in signalling by DC-SIGN, we transfected the Lck deficient Jurkat cell line JCam1 with DC-SIGN and stimulated them with α-DC-SIGN antibody as above. JCam1/Lck(+) cells were used as a control. DC-SIGN antibody induced a robust signal in JCam1/Lck(+) cells whereas in JCam1 cells DC-SIGN was unable to signal (Fig 8B). Treatment of either cell line with a non-specific IgG did not induce signalling. These results demonstrate that although DC-SIGN signalling is independent of the YXXL motif of the receptor it does require Src family kinases. Together these data demonstrate a marked difference in the mechanism of signalling of the C-type lectin receptor DC-SIGN relative to that used by the C-type lectin receptors CLEC-2 and Dectin-1 in that signalling by the former is independent of its YXXL motif.

CLEC-2 signalling is dependent on the DEDG sequence preceding its YXXL motif

Since CLEC-2 and Dectin-1 are both able to signal via their YXXL motifs and DC-SIGN is not, we have compared the sequences flanking the YXXL in all three receptors (Table 1). CLEC-2 and Dectin-1 have an identical 4 amino acids preceding their YXXL motifs, DEDG, whereas DC-SIGN does not have this sequence. In order to investigate the role of these amino acids in signalling by CLEC-2, we have made a series of mutants, substituting each amino acid in turn for alanine. These mutants were co-transfected into DT40 cells with the NFAT-luciferase reporter construct. Cells were stained with α-CLEC-2 antibody and analysed for receptor expression by flow cytometry. They were then stimulated with rhodocytin and signalling measured by luciferase assay. D3A, E4A and G6A were expressed at the surface of the cells at similar levels to WT CLEC-2 (Fig 9A). However, D5A CLEC-2 was not detected at the cell surface (Fig 9A). In response to rhodocytin, D3A and E4A CLEC-2 responded to the same degree as WT CLEC-2 (Fig 9B). D5A CLEC-2 transfected cells did not respond to rhodocytin consistent with the lack of surface expression of this mutant. Strikingly, signalling by the G6A mutant of CLEC-2 is significantly reduced to approximately 25% of WT CLEC-2.

Figure 9
CLEC-2 signalling is dependent on the DEDG sequence preceding its YXXL motif. Wild-type DT40 cells were transfected with empty pEF6 vector (mock), pEF6-MYC-CLEC-2, pEF6-MYC-D3ACLEC-2, pEF6-MYC-E4ACLEC-2, pEF6-MYC-D5ACLEC-2 or pEF6-MYC-G6ACLEC-2 together ...
Table 1
C-type lectin receptors containing YXXL motifs

In order to investigate if D5A CLEC-2 is retained in an intracellular compartment, WT CLEC-2 and D5A CLEC-2 were transfected into 293T cells and whole cell lysates were western blotted for CLEC-2. 293T cells were used for these experiments since they tolerate high levels of exogenous protein expression therefore facilitating analysis by western blotting. Consistent with the results in DT40 cells, CLEC-2 was detectable on the surface of 293T cells transfected with WT CLEC-2 but not on the surface of mock transfected or D5A CLEC-2 transfected cells. CLEC-2 was detectable by western blot in 293T cells transfected with WT CLEC-2 but not in cells transfected with empty vector or D5A CLEC-2. The multiple immunoreactive bands in the cells transfected with WT CLEC-2 likely reflect a combination of different glycosylation states (4) and breakdown products of the receptor.

Together, these results demonstrate that Aspartate 5 and Glycine 6 are important for CLEC-2 signalling. Whether the analagous residues of Dectin-1 are important for signalling and how these residues effect CLEC-2 and Dectin-1 signalling are worthy of further investigation.

DISCUSSION

In this study, we have used a specific antibody to CLEC-2 and the snake venom toxin rhodocytin to dissect the signalling pathway used by the lectin receptor in a physiologically relevant system, the platelet, and in two transfected cell lines. We have further compared the molecular basis of signalling by CLEC-2 with that of the two C-type lectin receptors Dectin-1 and DC-SIGN, both of which have a YXXL motif in their cytosolic tail. The results support a model in which CLEC-2 and Dectin-1 signal through Src, Syk and Tec tyrosine kinases leading to activation of PLCγ downstream of tyrosine phosphorylation of the YXXL motif. In striking contrast, signalling by DC-SIGN is independent of its YXXL motif.

The signalling pathway used by CLEC-2 and Dectin-1 resembles that regulated by ITAM receptors in haematopoietic cells. However, the signalling pathway used by the two lectin receptors can be distinguished from that used by ITAM receptors by its partial rather than absolute dependency on the SLP-76/BLNK family of adapter proteins and by the presence of a single rather than dual YXXL motif within their cytoplasmic tail. Many of the signalling proteins used by CLEC-2 and Dectin-1 also participate in the regulation of PLCγ2 by integrin receptors in haematopoietic cells, but the integrin pathway can be distinguished by the complete dependency on activation of the BLNK/SLP-76 family of adapters and by the fact that integrin receptors regulate Syk independent of a YXXL or an equivalent motif (49).

For ITAM receptors, phosphorylation of the two tyrosines in the dual YXXL motif is essential for activation of the Syk/ZAP-70 family of tyrosine kinases by virtue of binding to their tandem SH2 domains. Mutagenesis studies in T cells have shown that activation of the Syk-family kinase ZAP-70 by the T cell receptor requires a doubly phosphorylated ITAM to bind the tandem SH2 domains of ZAP-70 (7, 8, 50). The individual SH2 domains of ZAP-70 do not bind appreciably with phosphorylated ITAM peptides (51) and the tandem SH2 domains of ZAP-70 bind to monophosphorylated ITAM peptides with a 100-1000 fold lower affinity than to the corresponding doubly phosphorylated peptides (52). Similar studies in B cells have shown that a small degree of Syk activation can be seen following phosphorylation of the N-terminal ITAM tyrosine alone, but marked activation of Syk requires phosphorylation of both ITAM tyrosine residues and the integrity of both Syk SH2 domains (40, 41, 53). The coupling of CLEC-2 and Dectin-1 to Syk is also dependent on tyrosine phosphorylation of the YXXL motif within their cytosolic tail (present study and (16, 54)), but it is not known how a single YXXL motif is sufficient to cause robust activation of the Syk family of tyrosine kinases.

One of the striking features of the YXXL motifs in the cytosolic tails of CLEC-2 and Dectin-1 is conservation of the four upstream amino acids, namely DEDG. Moreover, although eight other C-type lectin receptors contain a cytosolic YXXL sequence, none of these contain the preceding sequence DEDG (Table 1). The sequence DEDGYXXL is also absent from ITAM receptors, although an acidic residue is often found three amino acids upstream of the first ITAM tyrosine (Table 2), and from ITIM receptors (not shown).

Table 2
ITAM receptor motifs

The alanine scan of the DEDG sequence in CLEC-2 in this study implicates the D5 and G6 residues as playing an important role in CLEC-2 stability and signalling. These studies suggest that the upstream amino acids are important in allowing the YXXL to confer signalling and that the YXXL motif alone is not sufficient to confer the ability to signal. Consistent with this is the observation in the present study that DC-SIGN does not signal via its YXXL motif. These observations raise the possibility that the sequence DEDGYXXL is sufficient to activate the Syk family of tyrosine kinases. Studies are currently underway to investigate this possibility.

An unexpected observation in the present study was that only the Syk tandem SH2 domains and not the single SH2 domains were able to precipitate CLEC-2 from stimulated platelets and that site directed mutagenesis of the single SH2 domains in Syk abrogated signalling by CLEC-2 and inhibited that by Dectin-1 by approximately 80%. The crystal structure of ZAP-70 interacting with an ITAM peptide maps the binding sites for the ITAM tyrosine residues to the ZAP-70 SH2 domains (55). The binding site for one of the tyrosines of the ITAM lies within the C-terminal SH2 domain of the kinase, whereas the binding site for the other ITAM-tyrosine lies in the interface between the two SH2 domains. The observation that association of the CLEC-2 YXXL motif with Syk requires both SH2 domains could be explained by the binding taking place in the interface between the two SH2 domains of Syk, as is the case for ZAP-70. However, this does not explain the abolition or marked reduction in signalling observed following the introduction of point mutations into the N-terminal and C-terminal SH2 domains of Syk. It is possible that the two SH2 domains of Syk bind to two CLEC-2 receptors and that this is necessary for its activation. However, if this were the case it would be expected that either SH2 domain of Syk alone would bind CLEC-2 and this does not seem to be the case. Crystallisation and structural studies of the signalling complex between Syk and tyrosine phosphorylated CLEC-2 or Dectin-1 would provide valuable information on this.

A further surprising difference between the downstream signalling pathway of the lectin receptors and that used by ITAM receptors is their dependency on the SLP-76/BLNK adapter family. Signalling through the GPVI/FcR γ-chain complex is completely abrogated in SLP-76- and BLNK-deficient Jurkat and DT40 cells, respectively, whereas the response to CLEC-2 or Dectin-1 is only reduced by 60-70% in both cases. Further examples of the absolute dependency of ITAM signalling on SLP-76/BLNK, include the complete block in T-cell development in SLP-76-deficient mice as a result of a failure of signal transduction through the pre-TCR (44) (45) and abrogation of B-cell signalling in the absence of the SLP-76 homologue BLNK (46). It is therefore of interest to establish the way in which the signalling pathway used by CLEC-2 and Dectin-1 differs from that used by ITAM receptors. It is possible that spatial separation of signalling pathways in membrane microdomains such as membrane rafts or tetraspanin webs (56) may be responsible for this difference. Alternatively, CLEC-2 and Dectin-1 may be capable of using one or more adapter proteins that function as alternatives to SLP-76 or BLNK, such as Clnk or Slnk (57). Interestingly, a very recent study has also highlighted a difference in the proteins used by ITAM receptors and Dectin-1 in regulating NF-κB in immune cells (58). The caspase recruitment domain family adaptor protein, Card9, plays a critical role in the activation of NF-κB by Dectin-1 but is dispensable for NF-κB regulation by the T cell and B cell antigen receptors (58). Further analysis of the signalling pathways activated downstream of these receptors may lead to identification of other differences in signalling by this class of receptor and novel signalling proteins.

The ability of CLEC-2 to activate platelets represents a novel role for this family of proteins in platelet function. To date C-type lectin receptors are known to play important roles in pathogen recognition within the immune system. For example, Dectin-1 is known to recognise β-glucan bearing pathogens and is responsible for phagocytosis of various fungi into macrophages (59-61). As well as its role in pathogen internalisation, Dectin-1 has been shown to initiate signalling responses leading to production of cytokines IL-2 and IL-10 in dendritic cells and reactive oxygen species in macrophages (16, 48). The possibility therefore that CLEC-2 may also bind one or more exogenous ligands such as bacterial or yeast envelope proteins or viral coat proteins, as well as rhodocytin, is worthy of consideration. Indeed we have recently reported a role for CLEC-2 in platelet binding to HIV and transmission of the virus to permissive cells in culture (62). It is noteworthy that many systemic infections such as sepsis or HIV result in platelet-based complications such as thrombocytopenia or thrombosis (63, 64). The role of CLEC-2 in mediating responses of platelets and other blood cells to pathogens and its role in infection induced haemostatic complications is therefore worthy of investigation.

ACKNOWLEDGEMENTS

This work was funded by the British Heart Foundation and the Wellcome Trust. GLJF is in receipt of a British Heart Foundation Studentship. SPW is a British Heart Foundation Research Chair. JAE is financially supported by the German Research Council (grant EB177/3-3 of SPP 1086). MGT is funded by a MRC New Investigator Award. This study is supported in part by the grants from Ministry of Education, Culture, Sports, Science and Technology of Japan (grant No.16790533).

The authors would like to thank Dr Tomohiro Kurosaki for providing the DT40 cell lines, Prof Arthur Weiss for the luciferase expression vector and the Jurkat cell lines and derivatives and Dr Daniel Billadeau for the α–Vav3 antibodies. Thanks also to Greg Parsonage, Chris Buckley and Yotis Senis for their support in this work.

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