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The Journal of Immunology, 2007, 178: 808-816.
Copyright © 2007 by The American Association of Immunologists, Inc.

The IREM-1 (CD300f) Inhibitory Receptor Associates with the p85{alpha} Subunit of Phosphoinositide 3-Kinase1

Damiana Álvarez-Errico, Joan Sayós2,3 and Miguel López-Botet2,3

Molecular Immunopathology Unit, Department de Ciéncies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune receptor expressed by myeloid cell 1 (IREM-1) (CD300f) inhibitory receptor displays five cytoplasmic tyrosine residues, two of them (Y205 and Y249) fit with ITIMs, whereas Y236 and Y263 constitute putative binding sites for PI3K. In the present study, immunoprecipitation analysis revealed that both the p85{alpha} subunit of PI3K and Src homology region 2 domain-containing phosphatase-1 could be recruited by IREM-1 in transfected cells as well as in the U937 monocytic leukemia cells, which constitutively express the receptor. By assaying the ability of different IREM-1 mutants to regulate the secretion of beta-hexosaminidase induced via FcR{epsilon}I in rat basophilic leukemia cells, both Y205 and Y249 appeared crucial for IREM-1-mediated inhibition. Remarkably, engagement of an IREM-1 mutant (Y205,249,284F), which did not recruit Src homology region 2 domain-containing phosphatase-1 and lost its inhibitory function, induced rat basophilic leukemia cell degranulation. This effect was dependent on the recruitment of PI3K, requiring the integrity of Y236 and Y263, and was blocked by PI3K inhibitors (i.e., wortmannin and LY-294002). Altogether, these data reveal a putative functional duality of the IREM-1 myeloid cell receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leukocyte function depends on the balance between opposite signals triggered by activating and inhibitory receptors. Leukocyte inhibitory receptors are characterized by cytoplasmic ITIMs. Upon receptor engagement, ITIMs become tyrosine phosphorylated recruiting and activating Src homology 2 (SH2)4 domain-containing phosphatases, which mediate the inhibitory signals. Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 dephosphorylate tyrosine residues in molecules that play a central role in the initiation and amplification of activating pathways (1). SHIP has been shown to regulate B cell activation by decreasing the concentration of phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5 P3) and inhibiting activation of Tec family kinases (2).

Immune receptor expressed by myeloid cell 1 (IREM-1) is an inhibitory receptor encoded by the CD300LF (CD300 Ag-like member F) gene located in chromosome 17q24.25 (3, 4). Thus far, four additional members of the CD300L gene family, termed CMRF-35 or CD300c (5), CMRF-35H or CD300a, also IRp60 (6, 7), IREM-2 or CD300e (8), and very recently IREM-3 or CD300b (9), have been characterized at the protein level. These molecules have a single extracellular Ig-like domain, display structural homology with TREM-2 and NKp44 receptors (10, 11), and their expression pattern appears restricted to different hemopoietic cell lineages (3, 5, 7, 8). IREM-1 is detected on different myeloid cells, and IREM-2 is specifically expressed by the monocytic lineage. IREM-2 is able to deliver activating signals, whereas IRp60 (7, 12, 13) and IREM-1 (3) act as inhibitory receptors; thus far, the function of CMRF-35 remains uncertain.

IREM-1 displays a long cytoplasmic tail with five tyrosine-based motifs; Y205 (LCYADL) and Y249 (ISYASL) fit with the ITIM consensus structure. Though tyrosine residue 284 (TEYSTI) does not integrate an ITIM (S/I/V/LxYxxI/V/L) (14) according to the residue present at position –2, a functional role has been proposed for such ITIM-like motifs in other inhibitory receptors (15). We previously described that Y205 constitutes the main docking site for SHP-1 (3). Interestingly, Y236 (YVTM) and Y263 (YCNM) conform putative binding motifs for the p85 regulatory subunit of PI3K (16). In the present work, we analyzed the molecular basis for signal transduction via IREM-1. Our results indicate that this receptor is able to interact with both SHP-1 and p85{alpha}, involving distinct tyrosine-based binding sites. Moreover, functional analysis of an IREM-1 mutant, which did not bind SHP-1 and lost its inhibitory activity, supports that PI3K may be also involved in signaling, thus revealing a remarkable putative functional duality of this leukocyte receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and Abs

The U937 myelomonocytic cell line was grown in RPMI 1640/Glutamax medium supplemented with 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (growing culture medium). COS-7 and the rat basophilic leukemia (RBL) RBL-2H3 cells were grown in DMEM with 10% heat-inactivated FCS, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured at 37°C with 5% CO2 atmosphere in growing medium. Biotinylated anti-hemagglutinin (HA) mAb 12CA5 was described before (17). Anti-SHP-1 rabbit serum was obtained from Santa Cruz Biotechnology. Anti-p85{alpha} Ab was purchased from Upstate Biotechnology. HRP-conjugated anti-phosphotyrosine mAb mixture (PY-7E1, PY-1B2, and PY20) was from Zymed Laboratories. Anti-IREM-1 mAbs (UP-D1 and UP-D2) have been described previously (3). HRP-conjugated secondary Abs were from Amersham Biosciences. HRP-conjugated streptavidin was from Roche. Purified anti-rat Fc{epsilon}RI was from BD Pharmingen. Anti-SHIP-1 rabbit polyclonal Abs and anti-SHP-2 mAb were from were from Upstate Biotechnology. Anti-Grb-2 rabbit polyclonal Abs were purchased from Santa Cruz Biotechnology and anti-Grb-2 mAb from BD Transduction Laboratories.

DNA reagents

Full-length wild-type IREM-1 was cloned in pDisplay, SHP1 in pCDNA3, c-Fyn in pMES, IREM-1 cytoplasmic tail in pBridge, and c-Fyn420, 531Y-F, 176R-Q/IREM-1 in pBridge were previously described (3); additionally, SAP cloned in pGAD424 (18) and 3BP2 in pACT2 (19) were used. PI3K regulatory subunit cloned in pSR{alpha} was provided by Dr. J. Sancho (Instituto de Parasitologia y Biomedicina Lopez-Nevra, Granada, Spain) (20). To analyze the contribution of IREM-1 cytoplasmic tail tyrosine residues, PCR site-directed mutagenesis was developed. Mutants Y205F, Y249F, and double Y205,249F were described previously (3). HA-tagged IREM-1 mutants were obtained by cloning IREM-1 in pDisplay vector (Invitrogen Life Technologies) between BglII and SalI restriction sites and primers used for mutagenesis are described in Table I. The integrity of all constructs was confirmed by sequencing with ABI PRISM Bigs Dyes Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) and universal T7 primer sites. p85{alpha} was cloned into pGAD424 for a three-hybrid system using the BamHI sites. p85{alpha} was amplified from the vector pSR{alpha} containing p85{alpha} described above, using the primers shown in Table I, and PCR conditions for amplification were 94°C for 5 min and 30 cycles (94°C for 1 min, 55°C for 30s, and 72°C for 2 min) and 72°C for 10 min.


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  		Table I. Primers used in this study

 
Cell transfection

COS-7 cells were transiently transfected with the DEAE-dextran method and then lysed for immunoprecipitation assays as described previously (21). RBL-2H3 transfectants expressing constructs encoding wild-type and mutated IREM-1 were developed as follows. A total of 107 cells was electroporated with 20 µg of DNA at 280 V and 950 µF in a Gene Pulser Electroporator (Bio-Rad), left overnight to recover without antibiotics and then selected with G418 (1 mg/ml). IREM-1+ cells were further selected with anti-HA mAb (12CA5) using sheep anti-mouse IgG magnetic beads (Dynabeads).

beta-Hexosaminidase release assay

IgE dependent degranulation assays with RBL-2H3 cells were a modification of a previously described protocol (22). A total of 5 x 104 cells (RBL-2H3/IREM-1 wild type and mutants) was incubated in 96-well plates, precoated with mouse IgE (Sigma-Aldrich), mouse IgE and anti-IREM-1 mAb UP-D2, or IgE and anti-HLA class I mAb HP-1F7 in triplicates. Cells diluted in 50 µl of Tyrode’s buffer were incubated with the immobilized Abs for 1 h at 37°C. In some experiments, cells were pretreated with the PI3K inhibitors wortmannin or LY-294002 before incubation with the immobilized Abs. Cell supernatants (20 µl) were harvested and incubated with 50 µl of 1 mM p-nitrophenyl-N-acetyl-beta-D-glucosamine (Sigma-Aldrich) in 0.05 M citrate (pH 4.5) for 1 h at 37°C. Color was developed by adding 100 µl of 0.2 M glycine (pH 10.7). Absorbance (abs) was measured at 405 nm, and the percentage of beta-hexosaminidase release was calculated as (abs sample – abs spontaneous release)/(abs maximum release – abs spontaneous release) x 100, maximum release was determined by lysing cells with 1% Triton X-100.

Flow cytometry analysis

IREM-1 surface expression was tested by immunofluorescence and flow cytometry analysis (FACScan; BD Biosciences) as described previously (15). Cells were stained by indirect immunofluorescence with anti-IREM-1 UP-D2 mAb or isotype-matched control Abs and a secondary FITC-conjugated goat anti-mouse (DakoCytomation).

Three-hybrid system assay

To analyze IREM-1 the interaction with p85{alpha}, a three-hybrid assay was conducted by cotransforming CG1945 yeast strain with the constructs pGAD10/p85{alpha} or PACT2/CSK and pBridge/IREM-1 or pBridge/c-Fyn420, 531Y-F, 176R-Q/IREM-1. Transformants were plated in SD medium supplemented with a -Trp, -Leu, and -Met dropout. Clones were tested by beta-galactosidase liquid culture assay using o-nitrophenyl-beta-D-galactopyranoside as a substrate as described before (23).

Pervanadate treatment, immunoprecipitation, and Western blot analysis

As previously described (3), cells were incubated with 1 mM sodium pervanadate for 15 min at 37°C and then lysed with lysis buffer with Triton X-100 1%, 1 mM PMSF (Sigma-Aldrich), and 1% of a mixture of protease inhibitors consisting of 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin (Sigma-Aldrich). Cell lysates were centrifuged at 22,000 x g for 15 min at 4°C, and supernatants were used for precipitation. Preclearings were done with 30 µl of protein G-Sepharose beads and 5 µl of normal rabbit or mouse serum for 1 h at 4°C. Immunoprecipitation with anti-IREM-1 mAb was performed for 3 h at 4°C. Beads were washed three times with lysis buffer, sample buffer was added, and samples were denatured under reducing and non reducing conditions by heating 6 min at 100°C. Samples were resolved by SDS-PAGE in a 12% acrylamide gel and transferred onto polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% skim milk or 3% BSA in TTBS (20 mM Tris (pH 7.6) and 150 mM NaCl with 0.2% Tween 20) for 1 h and then probed with the indicated Abs. HRP-conjugated secondary Abs at appropriate dilutions were used to detect bound Abs. Blots were developed with West Pico SuperSignal kit (Pierce) and visualized on Hyperfilm (Amersham Biosciences).

Cross-linking of IREM-1 in U937 and RBL cells

U937 cells (200 x 106 cells) or monocytes (20 x 106 cells) were resuspended in incomplete RPMI 1640/Glutamax at 4°C and split in five aliquots of 100 µl. Cells were stimulated with 10 µg/ml UP-D2 mAb followed by 5 µg/ml sheep anti-mouse (Sigma-Aldrich). RBL transfectants (20 x 106 cells) were detached from culture vessels with PBS 5 mM EDTA, washed, and split in five aliquots in incomplete DMEM. Subsequently, 5 µg/ml rat IgE (Sigma-Aldrich) and either UP-D2 (10 µg/ml) or a control mAb of the same isotype were added, followed by 5 µg/ml F(ab')2 sheep anti-mouse IgG. In every case, cells were incubated at 37°C, and the reaction was stopped at different time points (0, 5, 10, 20, and 30 min) by centrifugation after addition of 1 ml of ice-cold PBS; pellets were lysed with 1 ml of 1% Triton lysis buffer (described above). After preclearing with IgG bound to Sepharose (Amersham Biosciences), immunoprecipitation with UP-D2 mAb and Western blots were performed as described above. Whole cell lysates were analyzed by Western blot with anti Akt and anti-phospho-Akt Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IREM-1 inhibits FcR{epsilon}I-mediated degranulation

We previously described that engagement of IREM-1 transfected in the RBL cell line inhibited Fc{epsilon}RI-induced activation of a luciferase reporter under the control of a NFAT-dependent promoter, which is consistent with the ability of the receptor to recruit SHP-1 (3). To further characterize IREM-1 function, different mutants bearing an HA epitope were generated and transfected in RBL cells (Fig. 1); their ability to inhibit Fc{epsilon}RI-dependent degranulation was evaluated measuring beta-hexosaminidase release as a readout. In this assay, engagement of Fc{epsilon}RI with IgE either alone or in the presence of a control anti-HLA class I mAb (HP-1F7) comparably induced RBL cell degranulation. By contrast, coengagement of IREM-1 with the UP-D2 anti-IREM-1 mAb impaired the secretion of beta-hexosaminidase triggered via Fc{epsilon}RI (Fig. 2). When RBL cells that expressed an IREM-1 deletion mutant missing the cytoplasmic tail (IREM-1 {Delta}cyt) were activated via Fc{epsilon}RI, no differences in degranulation were detected in the presence of either UP-D2 or HP-1F7 Abs. The inhibitory function of IREM-1 was also abrogated in RBL cells expressing a mutant (IREM-1 5YF) in which all tyrosines were changed to phenylalanine (Fig. 2).


Figure 1
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  		FIGURE 1. Structure of IREM-1 mutants. IREM-1 mutants were generated either by sequence deletion or site-directed mutagenesis of tyrosine residues (Y205, 236, 249, 263, and 284) to phenylalanine (F). All sequences were tagged with an N-terminal HA epitope.

 

Figure 2
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  		FIGURE 2. Engagement of IREM-1 inhibits RBL cell degranulation. A, RBL stable transfectants expressing IREM-1 wild-type, {Delta} cyt, and 5YF IREM-1 (see Fig. 1) were stained by indirect immunofluorescence with the anti-IREM-1 UP-D2 mAb (open histogram) or an isotype-matched control Ig (gray-filled histogram) and analyzed by flow cytometry. B, Degranulation in RBL cells expressing wild-type, {Delta} cyt, or 5YF IREM-1 after coengagement of Fc{epsilon}RI alone, represented in {blacksquare}, or together with IREM-1, represented in {square}, or in the presence of an isotype-matched control Ig (Figure 2), was assessed measuring beta-hexosaminidase release. Data (mean ± SD) are representative of three independent experiments.

 
To more precisely analyze the contribution of each tyrosine residue to the IREM-1 inhibitory function, we analyzed the different mutants transfected in RBL cells. First, we focused on residues Y205 and Y249, which are in an ITIM context, as well as on Y284 that fits with an ITIM-like motif. As compared with the inhibition mediated by wild-type IREM-1, single mutants (Y205F and Y249F) only partially reduced Fc{epsilon}RI-induced beta-hexosaminidase release (Fig. 3). By contrast, the inhibitory function of IREM-1 was markedly impaired in RBL transfectants expressing the Y205,249F double mutant and was abrogated when Y205, Y249, and Y284 were substituted by phenylalanine (Fig. 3).


Figure 3
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  		FIGURE 3. Involvement of IREM-1 ITIMs in the inhibition of IgE-mediated degranulation. A, RBL stable transfectants expressing wild-type IREM-1 or different mutants (Fig. 1) were analyzed for IREM-1 expression. Cells were stained by indirect immunofluorescence with the UP-D2 anti-IREM-1 mAb or an isotype-matched control Ig and analyzed by flow cytometry. B, Hexosaminidase secretion by RBL cells was measured after coengagement of Fc{epsilon}RI and IREM-1 ({square}) or in the presence of an isotype-matched control Ig ({blacksquare}); inhibition of secretion was calculated as described in Materials and Methods. Data (mean ± SD) are representative of three independent experiments.

 
IREM-1 binds the p85{alpha} regulatory subunit of PI3K

Y236 (YVTM) and Y263 (YCNM) fit with the consensus motif reported to bind the p85{alpha} regulatory subunit of PI3K (16). To study whether IREM-1 is able to recruit p85{alpha}, we cotransfected the CG1945 yeast strain with IREM-1 and p85{alpha} in the presence or absence of the c-Fyn kinase. In this assay, binding of the IREM-1 cytoplasmic tail to p85{alpha} was detected by the increase of beta-galactosidase activity (Fig. 4A). The association between IREM-1 and p85{alpha} was only perceived in the presence of c-Fyn, suggesting that tyrosine phosphorylation of IREM-1 cytoplasmic tail of IREM-1 was required for the interaction. The specificity was confirmed by the lack of association between IREM-1 and the SH2 containing adaptors 3BP2 and SAP (Fig. 4A).


Figure 4
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  		FIGURE 4. IREM-1 binds the p85{alpha} subunit of PI3K. A, The putative association between IREM-1 (cytoplasmic tail) and the indicated molecules was assessed in a three-hybrid system assay in yeast, in the presence or absence of c-Fyn; beta-galactosidase activity was measured with o-nitrophenyl-beta-D-galactopyranoside as a substrate and expressed as described in Materials and Methods. Data are expressed in arbitrary units and correspond to the mean SD of three independent clones; results are representative of three different experiments. B, COS-7 cells were cotransfected with wild-type IREM-1 and p85{alpha}, both HA tagged, in the presence or absence of c-Fyn. Cell lysates were immunoprecipitated with anti-IREM-1 mAb (UP-D2). Western blots were sequentially performed with anti-PTyr, anti-HA, and anti-p85 Abs; whole cell lysates were included as controls.

 
To confirm this observation in mammalian cells, COS-7 cells were cotransfected with IREM-1 and p85{alpha}, both tagged with an HA epitope, in the presence or absence of c-Fyn. Immunoprecipitation was performed with the UP-D2 anti-IREM-1 mAb, and Western blots were sequentially conducted with different Abs; p85{alpha} was detected with both anti-HA and an anti-p85{alpha} mAb, whereas precipitated IREM-1 was assessed by anti-HA. UP-D2 mAb coprecipitated both IREM-1 and p85{alpha} when IREM-1 was phosphorylated in the presence of c-Fyn; under these conditions, p85{alpha} was also phosphorylated (Fig. 4B). It is of note that some IREM-1 phosphorylation and p85{alpha} coprecipitation were also detectable when IREM-1 and p85{alpha} were cotransfected in the absence of c-Fyn.

Tyrosine residues Y236 and Y263 mediate p85{alpha} binding

To verify whether Y236 and Y263 were involved in the interaction with p85{alpha}, we tested the corresponding tyrosine to phenylalanine mutants. Single Y236F and Y263F, as well as double Y236,263F IREM-1, mutants were cotransfected in COS-7 cells together with c-Fyn and p85{alpha}; immunoprecipitation was conducted with UP-D2 anti-IREM-1 mAb. Western blots revealed that both single mutants were still able to recruit p85{alpha}, whereas binding of PI3K to the IREM-1 Y236,263F mutant was abolished (Fig. 5). It is noteworthy that even though both residues bound p85{alpha}, its interaction with tyrosine 236 appeared consistently stronger, according to the relative intensity of the bands (Fig. 5). When the mutant lacking all tyrosine residues (IREM-1 5YF) was coexpressed with c-Fyn and p85{alpha} neither IREM-1 phosphorylation nor coprecipitated p85{alpha} were detectable, supporting that their association required tyrosine phosphorylation.


Figure 5
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  		FIGURE 5. Both IREM-1 YxxM motifs recruit p85{alpha}. COS-7 cells were transiently cotransfected either with HA-tagged wild-type IREM-1 or its mutants (Y236F, Y263F, Y236,263F, 5YF) together with p85{alpha}, in the presence or absence of c-Fyn. Cell lysates were immunoprecipitated with anti IREM-1 mAb, and Western blots were conducted with anti-PTyr, anti-HA, and anti-p85 Abs. Whole cell lysates were included as controls.

 
SHP-1 does not block p85{alpha} recruitment by IREM-1

To analyze whether IREM-1 was able to bind p85{alpha} in the presence of SHP-1, COS-7 cells were cotransfected with IREM-1, p85{alpha} and SHP-1 in different combinations, together with c-Fyn. In the absence of SHP-1, both IREM-1 and coprecipitated p85{alpha} were tyrosine phosphorylated (Fig. 6A). SHP-1 and p85{alpha} transfected together with IREM-1 coprecipitated with the receptor; yet, under these conditions, phosphorylation of p85{alpha} was markedly inhibited (Fig. 6A). It is of note that the amount of SHP-1 associated to IREM-1 appeared reproducibly reduced as compared with that bound to the receptor in the absence of p85{alpha} (Fig. 6A), thus suggesting that PI3K may partially interfere with SHP-1 recruitment.


Figure 6
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  		FIGURE 6. IREM-1 coprecipitates SHP-1 and p85{alpha}. A, COS-7 cells were transiently transfected with IREM-1, either alone or with p85 and/or SHP-1, in the presence of c-Fyn. Cells were lysed, immunoprecipitated with anti-IREM-1 mAb, and analyzed by Western blot with anti-phosphotyrosine, anti-HA, anti-p85, and anti-SHP-1 Abs. B, U937 monocytic cells were surface labeled with biotin, treated in the presence or absence of sodium pervanadate, lysed, and immunoprecipitated with anti-IREM-1 mAb or an isotypic negative control. Western blots were conducted with the indicated Abs. Molecular mass markers are displayed. C, IREM-1 in U937 cells was cross-linked with a specific mAb, and at the indicated times, cells were lysed and IREM-1 was immunoprecipitated with the UP-D2 mAb; Western blots were performed with the indicated Abs. Molecular mass markers are displayed.

 
To verify whether the interaction with p85 takes place in cells constitutively expressing IREM-1, experiments were conducted with the U937 monocytic cell line. Cells were surface labeled with biotin and subsequently treated with sodium pervanadate to induce tyrosine phosphorylation (Fig. 6B); IREM-1 was detected with HRP-labeled streptavidin. Anti-SHP-1 and anti-p85 Western blot analysis revealed that both endogenous molecules associated with phosphorylated IREM-1 in pervanadate-treated cells; it is noteworthy that no tyrosine phosphorylation was detected in coprecipitated p85{alpha} (Fig. 6B). These results indicated that IREM-1 and p85{alpha} may interact in human monocytic cells, and further confirmed that SHP-1 did not block p85{alpha} recruitment by IREM-1.

To further assess the recruitment of intracellular signaling mediators upon ligand engagement of IREM-1, the surface receptor was cross-linked with a specific Ab evaluating at different time points (0, 2, 5, 10, and 20 min) its phosphorylation status as well as the presence of coprecipitated molecules. As shown in Fig. 6C, IREM-1 phosphorylation reached maximum levels after 5 min and gradually decreased, persisting at 20 min after stimulation. Consistent with IREM-1 phosphorylation, Western blots revealed that the receptor was coprecipitated with both p85 and SHP-1. By contrast, neither SHP-2 nor SHIP-1 were detected in immunoprecipitates under these conditions (Fig. 6C).

Given the presence of a putative cytoplasmic Grb-2 binding motif (YxN), we tested the ability of IREM-1 to bind this adaptor. To evaluate this possibility, COS cells were transfected with wild-type IREM-1 carrying an HA tag, in the presence of c-Fyn. Endogenous Grb-2 was immunoprecipitated with a specific rabbit polyclonal Ab, and coprecipitation of IREM-1 was checked by Western blots with anti-phosphotyrosine and anti-HA Abs. A small amount of phosphorylated IREM-1 coprecipitated with Grb-2 only in the presence of c-Fyn. (Fig. 7). However, we were unable to detect Grb-2 when IREM-1 was immunoprecipitated with UP-D2 Ab (data not shown). This constitutes first evidence that IREM-1 is able to associate with Grb-2, but further studies are required to confirm it in IREM-1 expressing primary cells and eventually to elucidate the physiological relevance of this interaction.


Figure 7
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  		FIGURE 7. IREM-1 binds Grb-2 in transfected COS cells. COS cells were transfected with HA-tagged IREM-1 and c-Fyn, c-Fyn alone, or the empty vector. Endogenous Grb-2 was immunoprecipitated from cell lysates, and coprecipitation of IREM-1 was checked by Western blots with biotinylated anti-HA mAb and an anti-phosphotyrosine mAb mixture. Grb-2 immunoprecipitation was confirmed with a specific mAb.

 
Engagement of the Y205,249,284F IREM-1 mutant activates RBL cell degranulation: evidence for PI3K involvement

Among the set of controls used in the functional assays with transfected RBL cells, IREM-1 was engaged with UP-D2 in the absence of IgE. No response was detected in cells expressing either IREM-1 or the Y205F, Y249F, Y205,249F mutants (Fig. 7A); by contrast, degranulation was consistently observed when the IREM-1 Y205,249,284F triple mutant was engaged with UP-D2 (Fig. 8A). Ligation of IREM-1 {Delta}cyt deletion mutant did not trigger beta-hexosaminidase release (Fig. 8B); furthermore, no degranulation was either observed upon engagement with UP-D2 mAb of the IREM-1 Y5F mutant that did not bind PI3K (Fig. 8B), thus indirectly supporting an involvement in the response of Y236 and Y263 that recruit p85{alpha}.


Figure 8
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  		FIGURE 8. IREM-1 Y205,249,284F triggers RBL degranulation. A, Hexosaminidase release was measured as described in Materials and Methods after engagement of wild-type and mutated IREM-1 expressed in RBL cells. {blacksquare} represents the secretion in the presence of a control Ig, and {square} corresponds to hexosaminidase release after engagement of IREM-1 with UP-D2 mAb. Data correspond to the mean ± SD of three independent experiments. B, RBL cells expressing IREM-1 wild-type, 5YF, {Delta} cyt, and Y205,249,284F were engaged with UP-D2 mAb or with a control Ig. C, RBL cells expressing IREM-1 wild-type or Y205,249,284F mutant were treated with wortmannin or LY-294002 at the indicated concentrations and incubated with UP-D2 or a control isotype-matched Ig. Hexosaminidase release was calculated as described in Materials and Methods. {blacksquare} represents IgE-triggered secretion and {square} the response upon IREM-1 coengagement. Data (mean ± SD) are representative of three independent experiments.

 
Next, we analyzed the effect of PI3K inhibitors in the degranulation induced by UP-D2 mAb in RBL cells expressing IREM-1 Y205–249-284F. Stimulation in the presence of either wortmannin or LY-294002 resulted in the inhibition of beta-hexosaminidase release (Fig. 8C). Taken together, these data further supported that PI3K is involved in the activation induced by the mutated IREM-1 Y205,249,284F receptor and indirectly suggested that the PI3K pathway may participate in physiological signaling via this leukocyte receptor.

Coengagement of IREM-1 and Fc{epsilon}RI leads to a partial inhibition of Akt phosphorylation in RBL cells

To further study the influence of IREM-1 signaling on PI3K activity, we evaluated the effect of IREM-1 engagement on the phosphorylation of Akt, a well-characterized PI3K substrate. Cross-linking of IREM-1 did not induce any detectable change in the phosphorylation of Akt in RBL/IREM-1 transfectants nor U937 cells, as compared with the effect of an isotypic control mAb (data not shown). By contrast, cross-linking of IREM-1-inhibited Akt phosphorylation induced upon coengagement of Fc{epsilon}RI in the RBL/IREM-1 transfectant (Fig. 9). These results are consistent with a dominant inhibitory function of IREM-1 as supported by the inhibition of Fc{epsilon}RI-mediated degranulation, described above.


Figure 9
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  		FIGURE 9. Coengagement of Fc{epsilon}RI and IREM-1 partially inhibits Akt phosphorylation. Fc{epsilon}RI was engaged in IREM-1-transfected RBL cells with rat IgE in the presence of an anti-IREM-1 mAb or an isotype control Ig, and an F(ab')2 anti-mouse IgG antiserum. Akt phosphorylation in cell lysates was analyzed at the indicated time points by Western blot. Levels of total Akt are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we report that IREM-1 (CD300f), a myeloid cell inhibitory receptor that interacts with the SHP-1 tyrosine phosphatase, also recruits the p85{alpha} regulatory subunit of PI3K. Evidence is provided supporting that both enzymes may be involved in IREM-1 signaling, thus revealing a remarkable functional duality of this receptor molecule.

IREM-1 contains five cytoplasmic tyrosine residues, two in the context of ITIMs (Y205 and Y249) and two constituting putative PI3K binding motifs (Y236 and Y263); a systematic study of their role in IREM-1 signaling was conducted. Biochemical and functional analysis of different IREM-1 mutants transfected in RBL cells indicated that the inhibitory function of the receptor, assessed by its ability to regulate Fc{epsilon}R-induced secretion upon engagement with a specific mAb, required the integrity of both ITIMs. As previously reported, Y205 constitutes the main docking site for SHP-1; yet, the Y205F IREM-1 mutant was still able to partially inhibit Fc{epsilon}R-induced degranulation. Functional data supported the participation of Y249 and, marginally, of Y284 integrated in an ITIM-like motif. The possibility that other mediators might participate in inhibitory signaling has been considered. Thus far, there is no evidence for SHP-2 binding to IREM-1; by contrast, coprecipitation of SHIP-1 with IREM-1 was reported previously (24).

The prediction that IREM-1 might interact with PI3K, based on the existence of two cytoplasmic YxxM motifs, was confirmed in transfected RBL cells and, more importantly, in the U937 monocytic cell line that does constitutively express the receptor. Class I PI3K are heterodimers consisting of a catalytic (p110) and a regulatory (p85) subunit (25). Binding of p85 through its SH2 domains to phosphorylated receptors or adaptors promotes the recruitment of the enzyme, followed by the dissociation of the p85-p110 complex, the activation of p110 and its translocation to the plasma membrane. p110 phosphorylates PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3, which interacts with proteins containing pleckstrin-homology (PH) domains (26). The PI3K pathway is known to be involved in signaling via different leukocyte receptors and to play a complex role regulating cell activation, proliferation and/or survival (27).

Coprecipitation of p85{alpha} with IREM-1 was detected upon induction of tyrosine phosphorylation, either by cotransfecting c-Fyn in COS-7 cells or, more importantly, upon pervanadate treatment of the U937 cell line. Under these conditions, SHP-1 was also coprecipitated with IREM-1 and tyrosine phosphorylation of p85{alpha} was undetectable. Consistent with previous studies showing that p85{alpha} constitutes a substrate for SHP-1, the data suggest that the phosphatase may regulate the function of PI3K bound to IREM-1. Yet, the stoichiometry of the interactions established between both enzymes with individual receptor molecules is uncertain. In this regard, the amount of SHP-1 that coprecipitated with IREM-1 was reduced upon cotransfection of p85, suggesting that recruitment of PI3K might interfere with docking of the phosphatase.

IREM-1 inhibited beta-hexosaminidase secretion triggered via the Fc{epsilon}R in transfected RBL cells; the repressor effect was markedly reduced only in IREM-1 mutants missing both ITIM motifs. These observations were consistent with a dominant inhibitory role of IREM-1 involving the tyrosine phosphatase, as shown for other ITIM-bearing receptors. It is of note that p85 associated to IREM-1 together with SHP-1 was not phosphorylated. On that basis, it can be hypothesized that recruitment of p85 to IREM-1 might locally decrease its availability for other activating receptors, thus potentially contributing to the inhibition. Yet, engagement of the Y205,249,284F mutant receptor triggered degranulation in RBL cells; moreover, the effect was blocked by PI3K inhibitors, thus supporting an involvement of the enzyme in signal transduction. A further potential complexity of IREM-1 signaling is conceivable considering that one of the YxxM motifs (Y263) corresponds to the consensus sequence (YxN) known to recruit the Grb2 adaptor (28). Our results in transfected COS cells shown in Fig. 7 provide a first evidence supporting the interaction between IREM-1 and Grb-2, yet further studies are required to verify it in myeloid cells.

Some leukocyte receptors have been shown to alternatively function as inhibitory or activating molecules. For instance, KIR2DL4 displays a single cytoplasmic ITIM together with a transmembrane Arg, which allows its interaction with an ITAM-bearing adaptor (29); there is evidence that this receptor may either trigger or inhibit NK cell functions (30, 31). 2B4 is also able to recruit different signaling mediators and to deliver either activating or inhibitory signals in NK cells (32). Moreover, 2B4 binding to SAP depends on p85 recruitment and results in positive signaling (33), whereas inhibition is related to its association to phosphatases such as SHP-1 and SHP-2 or to Csk (32). The ability of IREM-1 to interact with PI3K appears exceptional among ITIM-bearing inhibitory receptors. CD300a (IRp60 and CMRF-35H9), a member of the same family that displays a high degree of homology with IREM-1, does conserve the ITIMs and inhibits the activation of NK cells, eosinophils, and mast cells (7, 12, 13, 34) but lacks YxxM motifs. Interestingly, the membrane proximal YxxM (Y236) of IREM-1 is conserved in its murine ortholog CLM-1, whereas methionine from the distal YxxM motif is absent in the murine molecule (35). This change disrupts the structure required for p85 binding, but the motif for putative association to Grb-2 is conserved. Thus far, no association of CLM-1 with p85{alpha} has been substantiated (35). To our knowledge, CTLA-4 constitutes the best example of a receptor involved in negative regulation of T cell functions capable of recruiting class 1A PI3K (36, 37), a property shared with its homologous CD28 and ICOS costimulatory molecules. Despite the fact that CTLA-4 does not display any cytoplasmic ITIM, it recruits SHP-2 and the PP2A serine phosphatase, which are believed to participate in inhibitory signaling (37). As emphasized by Rudd and Schneider (37), a key open issue is to what extent and how signaling via PI3K may be preserved from the inhibitory function of CTLA-4. By analogy, the dominant inhibitory function of IREM-1 in Fc{epsilon}R-I-mediated activation does not necessarily rule out a contribution of PI3K-dependent pathways in signaling. Different compartmentalization of SHP-1 and PI3K might determine that a fraction of IREM-1 molecules could become preferentially coupled to PI3K or SHP-1. Moreover, phosphorylation of ITIM and YxxM motifs, required respectively for SHP-1 and PI3K recruitment, may not be necessarily coordinated nor take place with the same kinetics. On a theoretical ground, variables such as the involvement of distinct tyrosine kinases and qualitative/quantitative differences in receptor-ligand interaction might be relevant. In this regard, all known members of the CD300 receptor family, both in human and mice, remain "orphan" because their ligands have not been thus far identified. It is conceivable that CD300f may interact with one or more self cell surface molecule(s), as shown for other well-characterized inhibitory receptors (i.e., killer Ig-related receptor, Ig-like transcript (ILT) 2, and ILT4). In this line, evidence has been obtained supporting the expression of ligand(s) for the IREM-1 murine homolog in a myeloid cell line and T cells (35, 38). In contrast, the existence of pathogen-associated ligands cannot be ruled out, as shown for the interaction of the LIR-1 (ILT2 and CD85j) inhibitory receptor with both HLA class I molecules and the UL18 CMV glycoprotein (39). Furthermore, the possibility that some IREM-1 isoforms might preferentially bind to PI3K should be also considered. In this regard, the first ITIM (Y205) and YxxM motif (Y236) are encoded by exon 8, whereas the second pair of ITIM (Y249) and YxxM (Y263) corresponds to exon 9. According to our results Y205 has a dominant role in engaging SHP-1, and thus, skipping exon 8 might generate a sequence preferentially binding to PI3K, yet such transcripts have not been reported thus far. According to recent observations, IREM-1 can be detected at distinct maturation stages of the myeloid cell lineage, from early CD34+ progenitors to mature macrophages. The possibility that the relative participation of PI3K and SHP-1 in signaling might depend on the differentiation stage of the cell needs to be addressed. Further studies are required to assess whether PI3K-dependent activation events can be detected upon engagement of the receptor in different cell types that constitutively express IREM-1.


   Acknowledgments
 
We thank Dr. Oscar Fornas for advice in flow cytometry analysis and Águeda Martínez-Barriocanal for helpful discussion and critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from Plan Nacional de I+D (SAF2001-0696). D.A.-E. is supported by a fellowship from the Ministerio de Ciencia y Tecnología, and J.S. is supported by a contract Ramon y Cajal from Ministerio de Ciencia y Tecnología. Back

2 J.S. and M.L.-B. share equal credit for senior authorship. Back

3 Address correspondence and reprint requests to Dr. Joan Sayós, Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain; E-mail address: joan.sayos{at}upf.edu or Dr. Miguel López-Botet, Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain; E-mail address: miguel.lopez-botet{at}upf.edu Back

4 Abbreviations used in this paper: SH, Src homology; SHP, Src homology region 2 domain-containing phosphatase; IREM, immune receptor expressed by myeloid cells; abs, absorbance; PtdIns-3,4,5 P3, phosphatidylinositol-3,4,5-trisphosphate; RBL, rat basophilic leukemia; HA, hemagglutinin; ILT, Ig-like transcript. Back

Received for publication March 27, 2006. Accepted for publication October 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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