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International Immunology Advance Access originally published online on August 28, 2006
International Immunology 2006 18(10):1499-1508; doi:10.1093/intimm/dxl083
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

CMRF-35-like molecule-5 constitutes novel paired receptors, with CMRF-35-like molecule-1, to transduce activation signal upon association with FcR{gamma}

Masanori Fujimoto1,2, Hiroyuki Takatsu1,2 and Hiroshi Ohno1,2

1 Laboratory for Epithelial Immunobiology, Research Center for Allergy and Immunology, RIKEN, Yokohama 230-0045, Japan
2 Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan

Correspondence to: H. Ohno; E-mail: ohno{at}rcai.riken.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The murine CMRF-35-like molecule (CLM) family belongs to the Ig superfamily, and consists of nine mapped genes. Here we report that CLM-5 and CLM-1 constitute novel paired Ig-like receptors (PIRs). CLM-1 and CLM-5 genes are encoded in close vicinity on chromosome 11. CLM-1 has been reported to possess immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic region and to inhibit the differentiation of osteoclast. CLM-5 has an Ig domain that is highly homologous with that of CLM-1 in its extracellular domain, and possesses a negatively charged residue in its transmembrane domain. CLM-5, like CLM-1, is expressed in myeloid cells, such as dendritic cells, macrophages and granulocytes. We also show that CLM-5 interacts with FcR{gamma} to be expressed on the plasma membrane and that the cross-linking of CLM-5 leads to tyrosine phosphorylation of proteins, including FcR{gamma}, in the monocyte/macrophage cell line. Taken together, these characteristics suggest that CLM-1 and CLM-5 constitute novel PIRs and that CLM-5 may transduce activation signals as the activating receptor in myeloid cells.

Keywords: immunoglobulin superfamily, myeloid cells, RAW264.7, tyrosine phosphorylation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Arrays of paired activating and inhibitory cell-surface receptors have been discovered in recent years (1–6), and the activating and inhibitory signals transduced by them play important roles in regulating immune responses (5). A pair of activating and inhibitory receptors shares homologous extracellular domains, and their genes reside adjacently on the genome (7). Two distinct groups of paired receptors have been reported on the basis of their structures: One belongs to the Ig superfamily and the other to the C-type lectin family (8).

In general, inhibitory receptors have long cytoplasmic tails containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (9). Upon binding of a ligand to an inhibitory receptor, ITIM phosphorylation is induced to recruit Src homology 2 (SH2)-containing tyrosine phosphatases, such as SHP-1, SHP-2 and SHIP, to terminate activation signals by dephosphorylating intracellular substrates involved in activation responses (10, 11). In contrast, activating receptors usually have short cytoplasmic tails without any motif sequences. Instead, they have a charged amino acid in their transmembrane domains that can interact with adaptor proteins possessing immunoreceptor tyrosine-based activation motifs (ITAMs), such as FcR{gamma} (12), CD3{zeta} (13) and DAP12 (14). Ligand-induced phosphorylation of ITAM recruits tandem SH2-containing tyrosine kinases, such as spleen tyrosine kinase and {zeta} chain-associated protein-70, to transduce activation signals (15). In some cases, the activating receptors interact with another adaptor protein, DAP10 (16), which has a phosphatidylinositol 3-kinase docking site in the cytoplasmic tail.

The murine CMRF-35-like molecule (CLM) family was first described by Chung et al. (17) as a cluster of nine genes on chromosome 11. Among them, CLM-8 and CLM-4 have been reported as paired receptors, myeloid-associated Ig-like receptor-I (MAIR-I) and MAIR-II, respectively (18). MAIR-I inhibits degranulation of mast cells, whereas MAIR-II is involved in cytokine production by peritoneal macrophages. Another CLM family member, CLM-1, has been shown to possess ITIMs and to inhibit osteoclast formation (17).

In this paper, we report that CLM-5 and CLM-1 constitute novel paired Ig-like receptors (PIRs). CLM-5, like CLM-1, is expressed on dendritic cells, macrophages and granulocytes. We also show that CLM-5 is associated with FcR{gamma}, and could transduce activation signals via the ITAM of FcR{gamma}, in myeloid cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6J (6–7 weeks old) mice were obtained from CLEA Japan and Charles River Laboratories Japan. They were maintained under specific pathogen-free conditions in RIKEN animal facilities until use in experiments for 1–8 days. All animal experiments were approved by the Animal Research Committee of RIKEN Yokohama Research.

Antibodies
PE-conjugated anti-CD3{varepsilon} mAb, FITC-conjugated anti-CD19 mAb, FITC-conjugated anti-CD11c mAb, PE-conjugated anti-NK1.1 mAb and PE-conjugated anti-Gr-1 mAb were purchased from BD PharMingen; FITC-conjugated anti-F4/80 mAb, purified anti-CD16/CD32 mAb and isotype-matched control mouse IgG1 mAb were purchased from eBioscience; anti-phosphotyrosine mAb 4G10 and anti-FcR{gamma} polyclonal antibody were purchased from Upstate Biotechnology and anti-FLAG mAb M2 was purchased from Sigma. PE-conjugated donkey anti-mouse IgG, PE-conjugated donkey anti-rabbit IgG and the F(ab')2 fragment of goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories. Phospho-mitogen-activated protein kinase (MAPK) Family Antibody Sampler Kit was purchased from Cell Signaling Technology.

Cells
Cell lines HEK293T (19) and RAW264.7 (20) were cultured in DMEM (Sigma) supplemented with 10% heat-inactivated FBS (Sigma), 100 U ml–1 penicillin and 100 µg ml–1 streptomycin (Sigma). WEHI-3 (21) was cultured in RPMI1640 (Sigma) supplemented with 10% heat-inactivated FBS, 100 U ml–1 penicillin and 100 µg ml–1 streptomycin and 1x MEM non-essential amino acids (Sigma). To obtain various hematopoietic cell lineages, splenocytes of C57BL/6J mice were stained with mAbs recognizing CD3{varepsilon} (for T cells), CD19 (for B cells), NK1.1 (for NK cells) or CD11c (for dendritic cells), and the positively stained cells were collected with FACSVantageTM (Becton Dickinson). Macrophages and granulocytes were isolated as Gr-1–F4/80+ and Gr-1+F4/80– cells, respectively (22). To obtain mast cells, bone marrow (BM) cells from C57BL/6J mice were cultured with the indicated 50% WEHI-3 culture medium and 50% WEHI-3 cell-conditioned medium. The BM-derived non-adherent cells were transferred weekly into new culture dishes for 5 weeks. The obtained cells were checked by Kimura staining (23).

Quantitative real-time PCR
Total RNA was isolated from cells with an RNeasy Mini Kit (Qiagen). Reverse transcription of RNA was performed with ReverTra Ace-{alpha}- (TOYOBO). PCR was performed with a SYBR® Premix Ex TaqTM (TaKaRa) and analyzed with an iCycler (Bio-Rad Laboratories). The cycling conditions were as follows: 95°C for 1 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. Melting curves of the PCR products were determined according to the manufacturer's instructions. Primers used for amplification are as follows—CLM-1 forward: 5'-GCCTCGCTCTTTGCTTGG-3', reverse: 5'-GTCAGAGCGGCATATGAAACC-3'; CLM-5 forward: 5'-ACTGGTGCCGAGGAGTTCC-3', reverse: 5'-GTGTTCTCAGCGCTTGGTTG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 5'-ATCAACGACCCCTTCATTGACC-3', reverse: 5'-CCAGTAGACTCCACGACATACTCAGC-3'; DAP12 forward: 5'-CAAGATGCGACTGTTCTTCCG-3', reverse: 5'-GGTCTCTGACCCTGAAGCTCC-3'; DAP10 forward: 5'-CCCCCCAGGCTACCTCC-3', reverse: 5'-TGACATGACCGCATCTGCA-3', and FcR{gamma} forward: 5'-GCCGTGATCTTGTTCTTGCTC-3', reverse: 5'-CTGCCTTTCGGACCTGGAT-3'. The transcription levels of CLM-1, CLM-5, DAP12, DAP10 and FcR{gamma} were normalized to that of GAPDH. The expression levels of transcripts in various organs and cells were compared, and the amount of expression was shown relative to the highest amount of expression observed in each panel. We confirmed that the melting curves of the PCR products showed single peaks.

Expression constructs and transfection
CLM-1 and CLM-5 cDNA were obtained from IMAGE Consortium (clone ID 1246922 and 1136486) and confirmed by sequencing. The cDNA fragment of wild-type or E185Q mutant (in which glutamic acid at position 185 was replaced with glutamine) CLM-5 lacking the signal sequence was created by PCR and sub-cloned into the pMx-IRES-GFP vector containing the mouse signaling lymphocyte activation molecule 1-signal sequence, followed by the FLAG epitope (a gift from Hiroshi Watarai, RIKEN) to generate FLAG-CLM-1/pMx-IRES-GFP, FLAG-CLM-5/pMx-IRES-GFP or FLAG-CLM-5-E185Q/pMx-IRES-GFP. DAP12, DAP10, FcR{gamma} and its mutant FcR{gamma}R45V (in which arginine at position 45 was substituted with valine) were created by PCR and sub-cloned into the pcDNA3 vector with hemagglutinin (HA) epitope (24). These expression vectors were transiently co-transfected into HEK293T using LipofectamineTM 2000 (Invitrogen). To obtain stable FLAG-CLM-5/RAW264.7, FLAG-CLM-5/pMx-IRES-GFP was transfected into RAW264.7 using LipofectamineTM 2000 and collected with FACSVantageTM, using green fluorescent protein (GFP) fluorescence as marker. After sorting, GFP-positive cells were cloned. Three clones were used in the experiments.

Flow cytometric analysis
To detect FLAG-CLM-5 on the cell surface, cells were pre-treated with anti-CD16/CD32 mAb to block Fc{gamma}Rs, stained with anti-FLAG mAb followed by PE-conjugated donkey anti-mouse IgG and analyzed with a FACSCaliburTM (Becton Dickinson). Live cell gating was performed with propidium iodide (Sigma). Data were analyzed with CellQuest software (BD Biosciences).

To examine the activation of MAPK, FLAG-CLM-5/RAW264.7 cells were stimulated with anti-FLAG mAb cross-linking as described in the next section. Cells were fixed and permeabilized with Cytofix/CytopermTM and Perm/WashTM (BD Biosciences) according to the manufacturer's instructions. Cells were then stained with anti-phospho-p38, anti-phospho-extracellular-related kinase (ERK)1/2 or anti-phospho-c-Jun N-terminal kinase (JNK) (Phospho-MAPK Family Antibody Sampler Kit, Cell Signaling Technology) in antibody diluent (Dako) followed by PE-conjugated donkey anti-rabbit IgG, and analyzed with a FACSCaliburTM (Becton Dickinson).

Cell stimulation, immunoprecipitation and immunoblotting
FLAG-CLM-5/RAW264.7 cells were stimulated by cross-linking with control IgG1 or anti-FLAG mAb, followed by F(ab')2 goat anti-mouse IgG. Cells with or without stimulation were solubilized in cold lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 15% glycerol, 20 mM NaF, 2 mM Na3VO4), containing CompleteTM protease inhibitor mixture (Roche Applied Science). After 30 min incubation on ice, the cell lysates were cleared by centrifugation at 14 000 r.p.m. for 20 min at 4°C. Then, the cell lysates were subjected to SDS-PAGE, and transferred to polyvinylidene fluoride membrane (Millipore) using a semidry blotting apparatus (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk (Yukijirushi)/PBS–Tween (0.1%) or Blocking One-P (Nacalai Tesque), probed with the indicated antibodies followed by HRP-conjugated secondary antibody and detected using the SuperSignal chemiluminescent substrate (Pierce). In some experiments, cell lysates were immunoprecipitated with anti-FcR{gamma} polyclonal antibody and nProtein A Sepharose 4 Fast Flow (Amersham Biosciences), and subjected to SDS-PAGE and immunoblotting as described above. In some experiments, cells stimulated with anti-FLAG mAb or control IgG1 immobilized on a plastic culture plate, or with 1 µg ml–1 of LPS (Escherichia coli, clone 055:B5, Sigma) for 24 h, were observed and photographed with an Olympus IX71 microscope and an Olympus DP70 digital camera.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
CLM-5 and CLM-1 constitute novel PIRs
We initially identified mouse CLM-5 as a candidate for the novel FcR for IgA, in the course of database search for genes showing sequence homology with the polymeric IgR and the FcR{alpha}/µ. However, we failed to detect CLM-5 binding to any class of Igs (data not shown). The homology search also revealed a gene named CLM-1 that is highly homologous to CLM-5. CLM-1 and CLM-5 are encoded in close proximity as members of the CLM family in the 11E2 region of mouse chromosome 11 (Fig. 1A). Comparison of the sequences of the Ig domains revealed that the homology of CLM-1 and CLM-5 is the highest among the CLM family, along with CLM-4 and CLM-8, also known as paired activating and inhibitory receptors, MAIR-II and MAIR-I, respectively (Fig. 1B and C) (18). The Ig domain of CLM-5 showed 91% identity and 97% similarity at the amino acid level with that of CLM-1.


Figure 1
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  		Fig. 1 Chromosomal location and cluster analysis of CLM family genes. (A) Physical map of the region constituting the CLM family gene cluster within mouse chromosome 11E2. The diagram was drawn based on data from the National Center for Biotechnology Information Map Viewer site as reference. (B) The amino acid sequences of all CLM family genes were aligned using GENETYX software. Identical amino acids among all CLM family genes are indicated by asterisks. (C) Phylogenetic tree generated based on the deduced amino acid sequences of the Ig domain of the murine CLM family genes using GENETYX software and Kimura's two-parameter method. The GenBank accession codes used are as follows: CLM-1, AY457047; CLM-2, AY457048; CLM-3, AY457049; CLM-4, AY457050; CLM-5, AY457051; CLM-6, AY457052; CLM-7, AY457053; CLM-8, AY457054, and CLM-9, AY457055.

 
Figure 2 depicts the deduced amino acid sequences of CLM-1 and CLM-5. Both are presumably type I integral membrane proteins having an N-terminal signal sequence, an extracellular domain with a single Ig domain, a transmembrane domain and a cytoplasmic tail. CLM-5 possesses a negatively charged glutamic acid in its transmembrane domain, and has a short cytoplasmic tail without any motif sequences. In contrast, CLM-1 possesses a relatively long cytoplasmic tail containing two consensus sequences for the ITIMs. Taken together, the above data suggest that CLM-1 and CLM-5 are paired inhibitory and activating receptors.


Figure 2
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  		Fig. 2 Sequence analysis of CLM-1 and CLM-5. Alignment of murine CLM-1 (AY457047) and CLM-5 (AY457051) deduced amino acid sequences. The putative signal sequence is shown in lowercase and the transmembrane domain is enclosed in a box, where the glutamic acid residue in CLM-5 is boldfaced and italicized. The Ig-like domain is in bold letters. The ITIM sequences in the cytoplasmic domain of CLM-1 are indicated by underlined bold letters. Identical amino acids between CLM-1 and CLM-5 are indicated by asterisks.

 
CLM-5, like CLM-1, is expressed in myeloid lineage cells
CLM-1 has been reported to be expressed mainly in myeloid lineage cells (17). To determine which cell types express CLM-5, we first examined the tissue distribution of the CLM-5 transcript by quantitative real-time PCR and compared it with that of CLM-1. As shown in Fig. 3(A), both CLM-5 and CLM-1 genes were predominantly expressed in the spleen and moderately expressed in the lung. To further analyze the expression of CLM-1 and CLM-5 in hematopoietic cells, quantitative real-time PCR was applied to RNA obtained from various lineage-committed cells purified by flow cytometry from the spleen, or mast cells derived from BM cell culture, of C57BL/6J mice. Both CLM-1 and CLM-5 transcripts were expressed in myeloid lineage cells, such as dendritic cells, macrophages and granulocytes, and were barely detectable, if any, in lymphoid lineage cells (Fig. 3B), consistent with the previous study of CLM-1 expression (17). Mast cells expressed only CLM-1.


Figure 3
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  		Fig. 3 Expression of the transcripts of CLM-1, CLM-5 and adaptor proteins in various tissues and hematopoietic cells. (A) Expression levels of CLM-1 and CLM-5 mRNAs in various tissues were analyzed by real-time PCR, as described in Methods. (B) Expression levels of CLM-1, CLM-5 and adaptor protein mRNAs in the indicated cell populations were analyzed by real-time PCR, as described in Methods. The amount of expression was shown relative to the highest amount of expression observed in each panel. The data are the results of one experiment.

 
CLM-5 associates specifically with FcR{gamma} among adaptor proteins
Paired activating receptors usually cannot express efficiently on the cell surface by themselves in the absence of the signaling motif-bearing adaptor proteins (3, 6, 25, 26). We therefore examined the cell-surface expression of CLM-5 by transiently expressing N-terminal FLAG epitope-tagged CLM-1 or CLM-5 (FLAG-CLM-1 or FLAG-CLM-5) in HEK293T cells. As control, FLAG-CLM-1 was readily detectable on the cell surface (Fig. 4), consistent with previous observation (17). On the other hand, the cell-surface expression of FLAG-CLM-5 was marginal (Fig. 4). Immunofluorescence staining indicated that a substantial amount of FLAG-CLM-5 was retained intracellularly (data not shown). Taken together with the structural characteristics of CLM-5 mentioned above as a putative activating receptor, the data suggest that CLM-5 likely needs to associate with a signaling motif-bearing adaptor protein for it to be expressed efficiently on the cell surface. To identify the adaptor protein that can associate with CLM-5 for its cell-surface expression, we first examined the expression of DAP12, DAP10 and FcR{gamma} transcripts in various hematopoietic cell lineages. We neglected CD3{zeta} from the analysis because its expression is known to be restricted to T cells and NK cells that do not virtually express CLM-5 (27). Quantitative real-time PCR analysis indicated that DAP12 and FcR{gamma}, but not DAP10, showed a similar expression profile to CLM-5 (Fig. 3B). We next tested whether these adaptor proteins could enhance the cell-surface expression of FLAG-CLM-5. To this end, FLAG-CLM-5 was expressed together with DAP12, DAP10 or FcR{gamma} in HEK293T cells, and the expression level of CLM-5 on the cell surface was analyzed by flow cytometry. Respective adaptors, sub-cloned into the pcDNA3 vector with HA epitopes, were checked for their expression ability by immunoblotting (data not shown). Among the three adaptors, only FcR{gamma} was able to induce the efficient surface expression of CLM-5 when co-expressed (Fig. 4).


Figure 4
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  		Fig. 4 Cell-surface expression of CLM-5 in association with FcR{gamma} in HEK293T cells. HEK293T cells were transiently transfected with the indicated expression construct, and the cell-surface expression of FLAG epitope-tagged CLM-1 and CLM-5 was analyzed by flow cytometry using control IgG1 and anti-FLAG mAb followed by PE-conjugated donkey anti-mouse IgG, as described in Methods. Histograms of the cells stained with anti-FLAG mAb were represented as filled, whereas those of control IgG1 were as blank. The data are representative of more than three independent experiments.

 
The interaction between charged amino acid residues in the transmembrane domains is thought to be important for the association of activating receptors with adaptor proteins (27–29). There are two charged amino acid residues, a negatively charged aspartic acid and a positively charged arginine, in the transmembrane domain of FcR{gamma} (30). Therefore, we hypothesized that the arginine residue of FcR{gamma} might play a role, by interacting with glutamic acid in the transmembrane domain of CLM-5, in the association of the two molecules. To test the hypothesis, we generated mutant constructs for these charged residues, in which arginine was substituted with valine in FcR{gamma} and glutamic acid with glutamine in CLM-5. As shown in Fig. 4, both mutations could still support the surface expression of CLM-5, similar to the wild-type molecules, suggesting that neither of the charged residues is involved in the interaction between CLM-5 and FcR{gamma}.

These results indicate that CLM-5 specifically associates with FcR{gamma} among the adaptor proteins tested, for it to be efficiently expressed on the cell surface, and the interaction seems to occur independent of the charged residues in the transmembrane domains of the two molecules.

CLM-5 induces tyrosine phosphorylation in monocyte/macrophage cell line
To assess the function of CLM-5 in myeloid cells, we stably transfected the RAW264.7 monocyte/macrophage cell line with FLAG-CLM-5 to create CLM-5/RAW264.7. We chose RAW264.7 cells because they express the CLM-5 transcript endogenously (Fig. 3B) and have been used to study the function of CLM-1 (17). Immunoblot analysis of CLM-5/RAW264.7 lysate under reducing and non-reducing conditions demonstrated that CLM-5 has two different molecular masses, ~35 and ~25 kDa, under both conditions (Fig. 5A). The ~35-kDa band likely represents the mature and glycosylated form of CLM-5, whereas the ~25-kDa band is the immature unglycosylated form, as discussed later.


Figure 5
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  		Fig. 5 Association of CLM-5 with endogenous FcR{gamma} in RAW264.7 cells. (A) RAW264.7 cells stably expressing the FLAG epitope-tagged CLM-5 (CLM-5/RAW264.7) or parental RAW264.7 cells were lysed and proteins were analyzed by immunoblotting with anti-FLAG mAb under reducing and non-reducing conditions, as described in Methods. Arrowheads indicate CLM-5. (B) CLM-5/RAW264.7 cells were analyzed by flow cytometry using anti-FLAG mAb (filled) and control IgG1 (blank) followed by PE-conjugated donkey anti-mouse IgG. The data were obtained using clone #22. The data are representative of three independent experiments. Two other clones, #3 and #9, gave similar results.

 
We next examined if CLM-5 could mediate signal transduction in RAW264.7 cells. When FLAG-CLM-5 on CLM-5/RAW264.7 cells was cross-linked with anti-FLAG mAb followed by the F(ab')2 fragment of goat anti-mouse IgG, tyrosine phosphorylation of some proteins was significantly enhanced (Fig. 6A). Importantly, tyrosine phosphorylation of FcR{gamma} was also induced upon cross-linking (Fig. 6B). In addition, two tyrosine-phosphorylated molecules of ~55 and 23 kDa were co-immunoprecipitated with FcR{gamma} only upon cross-linking with anti-FLAG mAb (Fig. 6C).


Figure 6
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  		Fig. 6 Tyrosine phosphorylation induced by CLM-5 cross-linking in RAW264.7 cells. (A) CLM-5/RAW264.7 cells were cross-linked and lysed, and proteins were analyzed by immunoblotting with anti-phosphotyrosine mAb, as described in Methods. (B) FcR{gamma} proteins were immunoprecipitated and analyzed by immunoblotting using anti-phosphotyrosine mAb, as described in Methods (upper panel). A duplicated membrane was probed with anti-FcR{gamma} (lower panel). (C) FcR{gamma} proteins were immunoprecipitated and analyzed by immunoblotting using anti-phosphotyrosine mAb, as described in the Methods. Arrowheads indicate the two tyrosine-phosphorylated molecules co-immunoprecipitated with FcR{gamma}. The data were obtained using clone #22. The data are representative of three independent experiments. Two other clones, #3 and #9, gave similar results.

 
Stimulation of anti-FLAG mAb cross-linking also induced a fibroblastic morphological change in CLM-5/RAW264.7 cells similar to that observed when RAW264.7 cells were stimulated with LPS (Fig. 7A). The fibroblastic morphology was not observed in parental RAW264.7 upon anti-FLAG mAb cross-linking but only upon LPS stimulation (Fig. 7A), suggesting that the change resulted from FLAG-CLM-5 cross-linking.


Figure 7
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  		Fig. 7 Cross-linking of FLAG-CLM-5 on CLM-5/RAW264.7 cells induces a morphological change and phosphorylation of MAPK. (A) CLM-5/RAW264.7 cells and RAW264.7 cells were stimulated with anti-FLAG mAb, control IgG1 or LPS (1 µg ml–1 for 24 h). Scale bars = 10 µm. (B) CLM-5/RAW264.7 cells were cross-linked for 30 min, as described in the Methods. The cells were then analyzed for phospho-p38, -ERK1/2 and -JNK (p-p38, p-ERK1/2 and p-JNK, respectively) after fixation and permeabilization using flow cytometry, as described in the Methods. Specific antibody staining of the cells stimulated with anti-FLAG mAb, thick line, and control IgG1, thin line. Control staining of the cells stimulated with anti-FLAG mAb, broken line, and control IgG1, fine-dotted line. The data were obtained using clone #22. The data are representative of three independent experiments. Two other clones, #3 and #9, gave similar results.

 
We next examined whether FLAG-CLM-5 cross-linking leads to MAPK activation, since MAPKs are reported to be activated downstream of FcR{gamma} (31). We employed flow cytometric analysis for detection of MAPK phosphorylation as described by Perez and Nolan (32). As shown in Fig. 7(B), phosphorylation of p38, ERK1/2 and JNK was significantly augmented upon FLAG-CLM-5 cross-linking compared with the background level of staining seen after control IgG1 cross-linking, suggesting MAPK is activated downstream of CLM-5. Non-stimulated cells showed an equivalent staining as control IgG1 cross-linking (data not shown).

Taken altogether, these results suggest that CLM-5 is expressed on the cell surface and transduces activation signals upon cross-linking through association with FcR{gamma} in the monocyte/macrophage cell line.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The CLM family consists of nine members and their genes are encoded on mouse chromosome 11. CLM-1 to CLM-8 reside in close proximity in the ~250-kb region of 11E2, whereas CLM-9 is separated from the other family members to be located in 11D, near the boundary with 11E1. We have identified that two members of the CLM family, CLM-1 and CLM-5, constitute a novel inhibitory and activating receptor pair of the Ig superfamily. Comparison of the Ig domains of the CLM family revealed two pairs of genes with high degrees of homology. One of them is CLM-4 and CLM-8, also known as MAIR-II and MAIR-I, respectively, whose Ig domains show 91.6% identity at the amino acid level (18). MAIR-I inhibits degranulation of mast cells, whereas MAIR-II is involved in cytokine production by peritoneal macrophages. The other pair is CLM-1 and CLM-5. The inhibitory receptor, CLM-1, has been reported to be expressed on myeloid cells as a monomeric ~60-kDa protein and to inhibit osteoclast differentiation (17). In the present study, we characterized the putative activating receptor, CLM-5. The Ig domain of CLM-5 showed 91% identity and 97% similarity at the amino acid level with that of CLM-1. Like CLM-1, CLM-5 is predominantly expressed in myeloid cells, such as dendritic cells, macrophages and granulocytes, but not in lymphoid cells. Both CLM-1 and CLM-5, together with FcR{gamma}, are highly expressed in granulocytes; it is intriguing to hypothesize, therefore, that CLM-1 and CLM-5 may play an important role in granulocyte differentiation and/or function, although we have no proof to support this claim at present. Although our effort to raise Abs recognizing CLM-5 has so far been unsuccessful, we have recently been able establish anti-CLM-1 mAbs, which could facilitate our understanding of the function of these CLM proteins.

Whereas CLM-1 has a relatively long cytoplasmic tail with two putative ITIMs, CLM-5 has a short cytoplasmic tail without any known motif sequences. In addition, the transmembrane domain of CLM-5 possesses a negatively charged glutamic acid. These characteristics suggest that CLM-5 is an activating receptor interacting with a signaling motif-bearing adaptor molecule.

We identified FcR{gamma} as the CLM-5-binding adaptor molecule from among DAP12, DAP10 and FcR{gamma}: CLM-5 was efficiently expressed on the cell surface only in the presence of FcR{gamma} and not the other two adaptor proteins. In many instances, the interaction between activating receptors and adaptor proteins is thought to be mediated by the interaction of a positively charged amino acid in receptors and a negatively charged amino acid in adaptors of their transmembrane domains (27–29, 33). However, CLM-5 uniquely possesses a negatively charged glutamic acid in its transmembrane domain. As there are two charged amino acid residues, the negatively charged aspartic acid and the positively charged arginine, in the transmembrane domain of FcR{gamma} (30), it is possible that the glutamic acid in the transmembrane domain of CLM-5 plays a role in the association of the two molecules by interacting with the arginine residue of FcR{gamma}. Indeed, asparagine of FcR{gamma}I and aspartic acid of FcR{gamma}III have been shown to be involved in their interaction with FcR{gamma}, where aspartic acid mediates a much stronger interaction than asparagine (33). However, neither the replacement of glutamic acid with glutamine in CLM-5 nor the replacement of arginine with valine in FcR{gamma} perturbed the efficient transport of CLM-5 to the cell surface, suggesting that the interaction of CLM-5 with FcR{gamma} is independent of the charged amino acids in their transmembrane domains and may be mediated by domains other than transmembrane domain, although it is possible that the non-charged but polar amino acid glutamine still plays some role in the place of glutamic acid in this interaction. It is also possible that CLM-5 and FcR{gamma} may associate indirectly via interaction with a third membrane protein.

CLM-5 was detected as ~35- and ~25-kDa proteins in RAW264.7 cells. The ~35-kDa band likely represents the mature and glycosylated form of CLM-5, whereas the ~25-kDa band represents the immature unglycosylated form as the molecular weight of CLM-5 calculated from the deduced primary structure devoid of the signal sequence is 22 923 Da. The glycosylation of CLM-5 is likely O-linked, considering no possible N-linked glycosylation sequence in the extracellular domain of CLM-5. That we observed a larger amount of immature CLM-5 than the mature form in the FLAG-CLM-5-transfected RAW264.7 cells seems reasonable, given that CLM-5 could be efficiently transported out of the endoplasmic reticulum (ER) for expression on the cell surface only upon association with FcR{gamma}, since the exogenously introduced FLAG-CLM-5 transgene could produce far excess amount of CLM-5 proteins of which a majority could not likely associate with the limited amount of endogenous FcR{gamma} and is consequently retained in the ER.

We have shown that the cross-linking of CLM-5 induces phosphorylation of intracellular substrates including FcR{gamma} and MAPK, as well as a morphological change in CLM-5/RAW264.7 cells, indicating that CLM-5 could mediate signal transduction as an activating receptor. Interestingly, two tyrosine-phosphorylated molecules were co-immunoprecipitated with FcR{gamma} upon FLAG-CLM-5 cross-linking. We hypothesize that these molecules may play important roles in signal transduction downstream of CLM-5, and the mass spectrometric analysis is underway to identify these molecules. Although the precise signaling cascade and the resulting function mediated by CLM-5 have yet to be elucidated, CLM-5 may be involved in the differentiation of osteoclasts, since CLM-1 has been reported to inhibit osteoclast formation (17). In this context, it is intriguing that other FcR{gamma}-associating molecules, PIR-A (26) and osteoclast-associated receptor (27, 34), also enhance osteoclast formation upon cross-linking with antibodies (35).

Human chromosome 17q25 harbors a susceptibility locus for psoriasis. The locus has been reported to overlap with loci for atopic dermatitis and rheumatoid arthritis. Of note is that the CMRF-35 family genes (36–39), the human counterpart of the mouse CLM family genes, have been mapped in this region, suggesting the correlation of CMRF-35/CLM with susceptibility to these diseases (40). Further analysis of the CLM family may provide new insights into the molecular mechanisms of these diseases.


   Acknowledgements
 
We would like to thank H. Watarai for generously providing the reagent, T. Ohno and S. Okumura for technical advice on mast cell preparation and T. Murakami, K. Kawano and A. Yoshino for critical reading of the manuscript. We also thank H. Fujimoto and Y. Hachiman for technical assistance in flow cytometry and K. Hashimoto and M. Ohmae for general technical assistance. This study was supported in part by Grants-in-Aid for Young Scientists (B) (H.T.), Scientific Research (H.O.) and Scientific Research in Priority Areas (H.O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Abbreviations
 
BM, bone marrow
CLM, CMRF-35-like molecule
ER, endoplasmic reticulum
ERK, extracellular-related kinase
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
GFP, green fluorescent protein
HA, hemagglutinin
ITAM, immunoreceptor tyrosine-based activation motif
ITIM, immunoreceptor tyrosine-based inhibitory motif
JNK, c-Jun N-terminal kinase
MAIR, myeloid-associated Ig-like receptor
MAPK, mitogen-activated protein kinase
PIR, paired Ig-like receptor
SH2, Src homology 2

   Notes
 
Transmitting editor: K. Okumura

Received 22 April 2006, accepted 26 July 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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