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The Journal of Immunology, 2006, 177: 2819-2830.
Copyright © 2006 by The American Association of Immunologists, Inc.

Molecular and Functional Characterization of CD300b, a New Activating Immunoglobulin Receptor Able to Transduce Signals through Two Different Pathways1

Águeda Martínez-Barriocanal and Joan Sayós2

Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, we describe the characterization of human CD300b, a novel member of the CMRF-35/immune receptor expressed by myeloid cell (IREM) multigene family of immune receptors. Immune receptor expressed by myeloid cell-3 cDNA was cloned from a PHA-activated PBMC library and RT-PCR revealed the gene to be expressed preferentially in cells of myeloid origin. The CD300b cDNA open reading frame encodes a 201-aa type I protein composed of a single extracellular Ig V-type domain followed by a transmembrane region containing a positively charged residue (lysine) which is a common feature among receptors that associate with activating adaptor proteins. Indeed, CD300b was able to associate with DNAX-activating protein of 12 kDa (DAP-12) and deliver different activating signals through this ITAM-based adaptor. Unusually for an activating receptor, the 29-aa cytoplasmic tail of CD300b contains a tyrosine-based motif that, upon c-Fyn phosphorylation, became a docking site for the intracellular signaling mediator growth factor receptor-bound protein 2. Moreover, in the absence of DAP-12, CD300b was able to activate NFAT/AP-1-dependent transcriptional activity in RBL-2H3 cells. This activity could be abolished only by mutating both the cytoplasmic tyrosine and the transmembrane lysine. Our data suggest the existence of an unidentified molecule capable of interacting with CD300b through a charged residue of the transmembrane region and allowing receptor signaling independent of DAP-12. Therefore, CD300b defines a nonclassical Ig receptor able to trigger signals by coupling distinct mediators and thus initiating different signaling pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune function is regulated by a balance between positive and negative signals delivered by activating and inhibitory receptors on the surface of leukocytes. These receptors are responsible for the correct transduction of information from the exterior to the interior of the cell and elicit a response that can range from reactivity to quiescence depending on the stimulus (1). Inhibitory receptors display long cytoplasmic tails presenting a variable number of ITIMs. Upon ligand binding, ITIMs are tyrosine phosphorylated by Src kinases and become docking sites for Src homology 2 (SH2)3-containing phosphatases, such as SHIP and SH2 domain-containing phosphatase 1 (SHP-1). Dephosphorylation of key signaling molecules will lead to inhibition of cellular activation (2, 3). In contrast, classical activating receptors display both a short cytoplasmic tail having no known signaling motifs, and a positively charged residue in the transmembrane region that allows the receptor to associate with transmembrane adaptor proteins. To date, two types of transmembrane adaptor proteins have been described: the ITAM-bearing adaptors, including DNAX-activating protein of 12 kDa (DAP-12)/killer cell-activating receptor-associated protein (KARAP), Fc{epsilon}RI{gamma}, and CD3{zeta}; and the PI3K-associated adaptor DAP-10. Engagement of activating receptors promotes tyrosine phosphorylation of transmembrane adaptors and subsequent recruitment of either protein tyrosine kinases containing SH2 domains such as Syk or Zap70, or PI3K that would contribute to activation of AKT and phospholipase C-{gamma} pathways. Activating signaling events will lead to secretion of cell mediators, migration, proliferation, and/or differentiation, among other processes (4, 5, 6, 7, 8).

We recently identified immune receptor expressed by myeloid cell (IREM)-1 and IREM-2, members of the CMRF-35/IREM family (CD300) of Ig receptors (9, 10). The genes of this family are clustered in a region on chromosome 17 (17q25.1) spanning ~250 kb, and code for both activating and inhibitory receptors. IREM-1 and IRp60 have been shown to recruit SHP-1/2 phosphatases and deliver inhibitory signals (9, 11). Oppositely, IREM-2 has been found to associate with the DAP-12 adaptor and therefore deliver activating signals (10). The first identified member, CMRF-35, has a short cytoplasmic tail and a transmembrane region containing a negatively charged residue, structural features that make its classification uncertain (12).

In the present study, we report the identification and functional characterization of a novel Ig-like receptor belonging to the CMRF-35/IREM family. CD300b, expressed mainly on myeloid cells, is a nonclassical activating receptor able to deliver signals not only by associating with the transmembrane adaptor protein DAP-12, but also by recruiting growth factor receptor-bound protein 2 (Grb-2) through a tyrosine-based motif present in the receptor’s cytoplasmic tail.


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

Human T (Jurkat, Molt-4, T-All 103/102), B (Ramos, Daudi, RPMI 8866), NK (YT, NKL), and myelomonocytic cell lines (U937, THP-1, HL60, MonoMac6), and KU812, P815, and Raw264.7 cell lines were maintained in RPMI 1640/L-glutamine medium supplemented with 10% heat-inactivated FBS, 25 mM HEPES, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. T-All 103/102 and NKL culture medium were additionally supplemented with 100 U/ml human IL-2 (BD Pharmingen). COS-7 and RBL-2H3 cells were grown in DMEM containing 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Human PBMC were obtained from heparinized venous blood of healthy donors by Ficoll-Hypaque gradient centrifugation. Human NK cells were isolated and cultured as described (13).

Anti-HA.11 mAb was obtained from Covance. Mouse anti-phosphotyrosine mixture coupled to HRP was obtained from Zymed Laboratories. Anti-FLAG M2 mAb and anti-DNP SPE7 mAb were obtained from Sigma-Aldrich. Anti-Grb2 mAb was obtained from BD Transduction Laboratories, and anti-Grb2 (C-23) polyclonal Ab was from Santa Cruz Biotechnology. HRP-conjugated goat anti-mouse Ab was from Amersham Biosciences. Streptavidin-HRP was purchased from Roche. Anti-rat high-affinity IgE receptor mAb was obtained from BD Pharmingen. Polyclonal rabbit anti-mouse FITC was from DakoCytomation. Z199 mAb, used as an isotypic control, and anti-HA12CA5 mAb were described previously (9, 14).

DNA reagents

Mammalian and yeast expression vectors pMES/hFyn (15), pFLAG-CMV2/Fc{epsilon}RI{gamma} (10), pSR{alpha}/CD3{zeta} (10), and pACT2/Grb-2 (16) were described previously. Sequence encoding CD300b lacking the signal peptide was amplified by PCR using oligos 1 and 2 (Table I) and cloned into the BglII/SalI sites of the pDisplay vector. pDisplay/CD300b substitution mutants were generated with sets of mutagenic oligos according to the instructions of the QuikChange Site-Directed Mutagenesis kit (Stratagene). pDisplay/CD300b Y188F and pDisplay/CD300b K158L mutants were obtained by annealing oligos 3–4 and 5–6, respectively, with the pDisplay/CD300b construct. The pDisplay/CD300b K158L Y188F double mutant was created by annealing oligos 5–6 with the pDisplay/CD300b Y188F construct. Presence of the introduced mutations was confirmed by DNA sequencing with an ABI PRISM Big Dyes Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems). Two deletion mutants of CD300b affecting the cytoplasmic tail were obtained by PCR and cloned into the BglII/SalI sites of pDisplay. CD300b {Delta}1 (del 178–201 aa) was amplified using oligos 1–7, and CD300b {Delta}2 (del 188–201 aa) was amplified using oligos 1–8. pCDNA3/FLAG-DAP10 was obtained by subcloning FLAG-DAP-10 of pMxNeo into the BamHI/EcoRI sites of pCDNA3 (17). A pCDNA3-FLAG vector was generated by cloning CD8 signal peptide followed by a FLAG epitope into the HindIII site of pCDNA3. Sequences encoding DAP-12 and CD3{zeta} transmembrane adaptor proteins without their signal peptides were amplified by PCR and cloned into the BamHI/EcoRI and EcoRI/XhoI sites, respectively, of pCDNA3-FLAG. DAP-12 was amplified from pJFE14-SR{alpha}/DAP-12 using oligos 9–10 and CD3{zeta} was amplified from pSR{alpha}/CD3{zeta} using oligos 11–12 (10). A pBabePuro-hemagglutinin (HA) expression vector was generated by cloning the Ig{kappa} signal peptide followed by an HA epitope into the BamHI/EcoRI sites of pBabePuro. CD300b wild-type (WT), CD300b Y188F, CD300b K158L, and CD300b K158L Y188F were cloned into the EcoRI/SalI sites of pBabePuro-HA vector by PCR amplification from the pDisplay constructs using oligos 13-2. For the three-hybrid system assay, sequence encoding the CD300b cytoplasmic tail, obtained using oligos 14–2, was cloned into the EcoRI/SalI sites of pBridge/c-Fyn420Y-F, 531Y-F, 176R-Q MCSI cassette described previously (15). The pBridge/CD300b Cyto/c-Fyn vector was digested with BglII endonuclease and religated to obtain a pBridge/CD300bCyto/c-Fyn catalytic mutant construct.


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  		Table I. Oligos used in this studya

 
Cloning strategy of CD300b and murine IREM-3 (mIREM-3)

A PCR strategy was used to amplify the full-length CD300b sequence from a PHA-activated PBMC cDNA library. Oligos 15–16 in Table I, mapping to the 5' and 3' untranslated regions of CD300b, were used for this purpose. PCR conditions were as follows: 94°C 5 min; 25 cycles of 94°C 1 min, 55°C 30 s, 72°C 2 min; and 72°C 10 min. mIREM-3 full-length sequence was amplified from Raw264.7 cDNA using oligos 17–18, which also map to the 5' and 3' untranslated regions of mIREM-3. PCR conditions were as follows: 94°C 5 min; 35 cycles of 94°C 1 min, 55°C 30 s, 72°C 1 min; and 72°C 10 min. DNA products were resolved in a 1% agarose gel and visualized by ethidium bromide staining. Expected size fragments were cloned into the pCR2.1-TOPO cloning vector (Invitrogen Life Technologies) and sequenced for confirmation using the universal T7 primer as described above.

RT-PCR

RNA extracted with TRIzol reagent (Invitrogen Life Technologies) from human polyclonal NK cells and hemopoietic cell lines was retrotranscribed using a ProtoScript First Strand cDNA Synthesis kit (New England Biolabs) according to the manufacturer’s instructions. CD300b amplification from human cell lines with oligos 15–16 was performed using 2 µg of cDNA as template. The conditions used were: 94°C 10 min; 40 cycles of 94°C 30 s, 60°C 30 s, 72°C 1 min; and 72°C 10 min. CD300b amplification from human isolated blood populations was performed using 10 ng of commercial cDNA (poly(A)+ RNA origin) (BD Clontech) or 2.5 µg of cDNA from polyclonal NK cells (total RNA origin). The conditions used were: 94°C 10 min; 35 cycles of 94°C 30 s, 60°C 30 s, 72°C 1 min; and 72°C 10 min. PCR products were cloned into pCR2.1-TOPO vector and sequenced for confirmation. GADPH amplification was conducted in parallel with oligos 19–20. Detection of CD300b whole transcript in human and fetal tissues was performed using a Human Rapid-Scan Gene Expression Panel (Origene Technologies) following the manufacturer’s recommendations. beta-actin amplification was used as an internal control. FastStart TaqDNA Polymerase (Roche) was used for all PCRs.

Cell transfections

COS-7 cells (106) were transiently transfected using the DEAE-dextran method (18) and lysed for the immunoprecipitation assay after 48 h. For the generation of RBL-2H3 stable transfectants, 20 x 106 cells were electroporated in the presence of 20 µg of linearized construct at 280 V and 950 µF in a Gene Pulser Electroporator (Bio-Rad). Transfectants were selected and maintained in culture with 1 mg/ml G418 (Invitrogen Life Technologies). RBL-2H3 double transfectants were generated sequentially and maintained with 1 µg/ml Puromycin (Sigma-Aldrich) in addition to G418. COS-7 cells (105) for flow cytometry analysis were transfected with Fugene 6 reagent (Roche) according to the manufacturer’s instructions.

Flow cytometry

Cell surface expression of the desired molecules was checked by flow cytometry with the corresponding Abs following standard techniques (19). Analysis was performed using a FACScan instrument and CellQuest software (BD Pharmingen).

Immunoprecipitation and Western blotting

Cells were lysed at 4°C for 15 min using 1% Triton X-100 (Tx-100)-containing buffer described previously (15). Cell lysates were clarified by centrifugation at 14,000 x g for 15 min at 4°C and the crude lysates were precleared for 1 h at 4°C using 20 µl of protein G-Sepharose beads (Amersham Biosciences) and 5 µg of mouse or rabbit IgG (Sigma-Aldrich). Two additional preclearings were conducted for 30 min at 4°C with 20 µl of protein G-Sepharose beads. For immunoprecipitations, precleared lysates were incubated with 30 µl of protein G-Sepharose beads and 1 µg of Ab for 3 h at 4°C. Proteins in the crude lysates and immunoprecipitates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) filters (Millipore). Filters were blocked for 1 h with 5% skim-milk or 3% BSA (Sigma-Aldrich) and then probed with the indicated Abs. Bound Abs were detected using ECL reagent (Pierce).

Luciferase assays

RBL-2H3 transfectants were transiently electroporated with a luciferase reporter plasmid (pT81Luc) containing three tandem copies of the distal NFAT/AP-1 site of the murine IL-2 promoter (20) (0.5 µg/106cells) and a TK Renilla construct (Promega) (0.1 µg/106 cells). Twenty-four hours posttransfection, 1.5 x 106 cells were stimulated for 7 h with 5 µg/ml anti-HA12CA5 or a negative isotypic Ab using the murine mastocytoma P815 line as presenting cell (1 x 106). Plastic-coated anti-DNP IgE (5 µg/ml) was used as positive control for stimulation and the P815 cell line in supplemented RPMI 1640/L-glutamine was used as negative control. Postnuclear lysates were obtained as described (20) and luciferase activity was measured according to the Dual Luciferase Reporter kit manual (Promega).

Hexosaminidase release assays

A total of 5 x 105 RBL-2H3 cells resuspended in 50 µl of Tyrode’s buffer (21) was stimulated for 1 h at 37°C by plastic-coated Abs (5 µg/ml). Supernatants (20 µl) were collected and incubated for an additional hour at 37°C with 1 mM 4-nitrophenyl N-acetyl-beta-D-glucosaminide in 0.05 M citrate buffer (pH 4.5) (50 µl; Sigma-Aldrich). Reactions were stopped by adding 0.2 M glycine buffer (pH 10.7) (150 µl), and the OD was measured at 405 nm. For determining the maximum degree of hexosaminidase release, cells were lysed with Tx-100 (1%) before incubation with the substrate.

Three-hybrid system assay

To characterize the interaction between the cytoplasmic tail of CD300b and Grb-2, a three-hybrid system assay was conducted by cotransforming CG1945 yeast with pGAD424, either alone or containing Grb-2, and pBridge/c-Fyn420, 531Y-F, 176R-Q or pBridge/c-Fyn catalytic mutant containing the cytoplasmic tail of CD300b. Transformants were plated on synthetic dropout medium lacking tryptophan, leucine, and methionine. Clones were tested by beta-galactosidase liquid culture assay using o-nitrophenyl-beta-D-galactopyranoside as a substrate as described (15).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cloning and sequence analysis of CD300b and its murine ortholog mIREM-3

Following a three-hybrid system strategy in yeast, we recently described a novel cell surface inhibitory receptor termed IREM-1/CD300f (AY303545) (9). This immune receptor is a member of a multigene family of activating/inhibitory receptors located on human chromosome region 17q25.1 which includes the previously identified molecules CMRF-35/CD300c and CMRF35-H/IRp60/CD300a (11, 12, 22, 23, 24). With the aim of identifying new members of the same family, we searched for sequences having homology to IREM-1 and CMRF-35 in the Ensembl genome database (Ensembl BLAST server at <www.ensembl.org>). This yielded a cDNA coding for the activating receptor IREM-2/CD300e (AF395839) (10). In the process, we identified a second cDNA sequence that shared homology not only with IREM-1 and CMRF-35 but also with IREM-2, that we termed IREM-3. The sequence was found in the database as triggering receptor expressed on myeloid cell (TREM)-5 (AF427618, AY359025, BC028091) and recently named CD300b (25). We designed primers for amplification of the predicted cDNA from a PHA-activated PBMC cDNA library. The obtained 732-bp fragment contains an open reading frame of 606 bp that encodes a protein of 201 aa with a predicted molecular mass of 22.7 kDa (AY646929) (Fig. 1A). Sequence analysis revealed CD300b to be a type I transmembrane protein driven by a signal peptide 17 aa in length (SignalP 3.0 server at <www.cbs.dtu.dk/services/SignalP>). The extracellular region of CD300b displays a single Ig V-type domain followed by a 29-aa membrane-proximal region containing a high proportion of proline, serine, and threonine residues that would confer on the molecule an extended open conformation (26). Additionally, the Ig domain stem presents many potential O-glycosylation sites. The transmembrane domain, which has a positively charged residue (lysine) in a central position, is followed by a short cytoplasmic tail of 29 residues in which a tyrosine-based motif can be distinguished (Fig. 1A).


Figure 1
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  		FIGURE 1. A, Predicted amino acid sequence of CD300b (AY646929). The putative signal peptide is underlined (dotted line). The Ig-like domain is marked in bold and the transmembrane domain is underlined (single line). Cysteine residues involved in the Ig-like domain fold are circled and the transmembrane-charged lysine residue is boxed. The tyrosine-based motif located in the cytoplasmic tail is in bold and underlined (double line). B, CD300b gene organization. Schematic organization of the CMRF-35/IREM locus at chromosomal region 17q25.1. CD300b genomic organization is shown below. Exons are represented by boxes (respective lengths in coding amino acids and domain architecture are shown); introns are represented by connecting lines.

 
The CD300b gene is located at chromosomal region 17q25.1. Through alignment of cDNA and genomic sequences, we determined the organization of the CD300b gene (Fig. 1B). The GT-AC rule was used to define intron-exon boundaries (27). The CD300b gene spans ~10 kb and contains four exons: exon 1 encodes the 5' untranslated region and the protein’s signal peptide; exon 2 encodes the Ig domain; exon 3 encodes the membrane-proximal and transmembrane regions; and exon 4 encodes the entire cytoplasmic tail.

Alignment of residues of the extracellular Ig domain of CD300b with sequences of the CMRF-35/IREM family and other closely related immune receptors showed high levels of homology among the V-type Ig folds: 77% with IREM-1, 57% with IREM-2, 58% with IRp60, 60% with CMRF-35 and 44% with NKp44 (Fig. 2A). A lower, although significant, degree of protein sequence homology was detected between the Ig domains of CD300b and TREM-1 and TREM-2 (41 and 42%, respectively, alignments not shown). The conservation of not only the pair of cysteine residues describing the Ig-V-type fold (Cys36 and Cys104) but also the second pair of cysteines also in the Ig domain (Cys50 and Cys58) is noteworthy. It has been proposed that the additional disulfide bond created by the second cysteine pair could define a discrete evolutionary group of receptors stemming from duplication of a common ancestral V-type Ig domain gene (28).


Figure 2
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  		FIGURE 2. CD300b protein homologies. A, Sequence alignment of the CD300b Ig-like domain with Ig domains from closely related immune receptors (IREM-1, IREM-2, CMRF35, CMRF35-H/IRp60, and NKp44). Identical residues are shown on a black background and similar residues are shown on a gray background. Conserved cysteine residues are marked with asterisks (*). B, Sequence alignment of CD300b with its murine ortholog, mIREM-3 (AY996128). The entire molecule is represented. Identical amino acids are shown on a black background and cons ervative changes are shown on a gray background.

 
To identify mouse orthologs of CD300b, we used the human CD300b cDNA sequence to search the Ensembl mouse genome database. We identified a cDNA sequence highly homologous but not identical with CML-7, a member of a recently described family of murine myeloid receptors (29). Amplification of the mIREM-3 full-length transcript was performed using cDNA from the murine myelomonocytic cell line Raw264.7 as template. From a complex pattern of bands we cloned a cDNA fragment 684-bp long containing an open reading frame of 630 bp. The cDNA codes for a 209-aa protein with a predicted molecular mass of 23.7 kDa that we termed mIREM-3/CD300b (AY996128). Alignment of the cDNA to the mouse genome sequence located the mIREM-3 gene to chromosome 11, which is considered syntenic to human chromosome 17. We cloned four transcript variants of the gene, but, with the exception of mIREM-3, they all specified incomplete mRNAs (data not shown). The human and murine forms of CD300b share 73% amino acid identity (Fig. 2B). It is striking that the tyrosine-based motif of the cytoplasmic tail is lost from the murine molecule while the charged amino acid of the transmembrane domain is retained.

CD300b is expressed mainly in myelomonocytic cells

To determine the distribution of CD300b transcript, we performed RT-PCR on human hemopoietic cell lines, PBMC, and purified blood populations. CD300b transcript was found abundantly in all human myelomonocytic cell lines tested (U937, THP-1, MM6, and HL60), and weakly in both the NK cell line NKL and the B cell line RPMI 8866 (Fig. 3A). However, when assessing the presence of the transcript in human hemopoietic isolated populations, CD300b was found exclusively in peripheral blood monocytes (Fig. 3B). No specific cDNA amplification was observed in activated NK and B cells. To ensure that the detected transcripts did indeed correspond to CD300b, the PCR products were cloned and sequenced. Based on these data, it seems that expression of CD300b is restricted to myeloid lineages, as we observed previously for IREM-1 and IREM-2 (9, 10).


Figure 3
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  		FIGURE 3. CD300b mRNA is expressed mainly in human myelomonocytic cells. RT-PCRs using RNA extracted from human hemopoietic cell lines (A), fresh PBMC and purified blood populations (B), and adult and fetal tissues (C) were performed to amplify full-length cDNAs from the CD300b transcript (upper panels). RT-PCRs of GADPH (A and B) or beta-actin (C) were performed as amplification controls (lower panels). DNA size markers are shown on the left (base pair).

 
Next, we determined the distribution of CD300b transcript in human tissues. A wide set of tissues was positive for the presence of CD300b (Fig. 3C). We were able to amplify CD300b from colon and lung, where mature differentiated myeloid cells can be found, and from placenta, bone marrow, and fetal liver that constitute the main reservoirs for myeloid precursors (30). A small amount of transcript was amplified from spleen. In the future, CD300b protein distribution will be assessed with mAbs to correlate transcript detection with cell surface expression of the molecule.

Biochemical characterization of CD300b

COS-7 cells were transiently transfected with CD300b tagged with an HA epitope. Forty-eight hours posttransfection, the cells were lysed and subjected to anti-HA immunoprecipitation and SDS-PAGE analysis under both reducing and nonreducing conditions. Despite CD300b having a predicted molecular mass of 22.7 kDa, the molecule appeared as three discrete bands ranging in size from 26 to 32 kDa (Fig. 4A), suggesting the occurrence of posttranslational modifications to the mature protein. The equivalent electrophoretic mobilities observed under reducing and nonreducing conditions indicates that the receptor was not forming dimers/multimers through disulfide bridges (Fig. 4A). The lack of asparagine residues in an N-glycosylation context suggests the possibility that the different bands may correspond to O-glycosylated and/or Ser/Thr-phosphorylated forms of the molecule. Treatment of the immunoprecipitates with a mixture containing the most common O-glycosidases or with alkaline phosphatase had no effect on the number and size of bands (data not shown). Expression of the molecule without its cytoplasmic tail led to a reduction in molecular mass of the bands but did not alter the triplet pattern (Fig. 4B), indicating that whatever the modification may be, it affects the extracellular portion of the molecule. Similar results were obtained when the molecule was expressed in RBL-2H3 cells (data not shown).


Figure 4
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  		FIGURE 4. Biochemical characterization of CD300b. A, COS-7 cells transfected with an empty vector or with a CD300b-HA construct were subjected to immunoprecipitation using anti-HA.11 mAb. Samples were analyzed by 11% SDS-PAGE under reducing and nonreducing conditions. Proteins were transferred to a PVDF membrane and probed with anti-HA12CA5 mAb coupled to biotin and developed with streptavidin-HRP. B, HA-tagged CD300b WT and {Delta}1 mutant were expressed in COS-7 cells and resolved under reducing conditions as described.

 
CD300b associates with DAP-12/KARAP and becomes tyrosine phosphorylated in transfected COS-7 cells

The presence of a positively charged amino acid in the transmembrane region of an immune receptor strongly suggests binding to a signaling transmembrane adaptor molecule (5, 6). Considering that CD300b has a lysine residue in its transmembrane portion, we wanted to test whether the molecule was able to associate with any ITAM-bearing adaptors such as DAP-12/KARAP, Fc{epsilon}RI{gamma}, and CD3{zeta}. For this purpose, COS-7 cells were transiently transfected with CD300b-HA and the different ITAM-bearing adaptor molecules in both the presence and absence of c-Fyn kinase. CD300b was able to interact with DAP-12 only (Fig. 5A). Furthermore, Western blot analysis with anti-phosphotyrosine mAb revealed phosphorylation of the CD300b receptor. A low level of phosphorylation of CD300b was observed in the presence of the c-Fyn kinase alone and none when the kinase was absent, but it increased dramatically when c-Fyn was coexpressed with DAP-12 (Fig. 5A). Some receptors such as signal regulatory protein-beta1 and murine NKG2D are able to couple not only DAP-12 but also the PI3K-associated adaptor DAP-10 (31, 32). We tested CD300b for its ability to recruit FLAG-tagged DAP-10. CD300b did not associate with DAP-10 nor did it become efficiently tyrosine phosphorylated (data not shown).


Figure 5
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  		FIGURE 5. CD300b phosphorylation and association with DAP-12. A and C, COS-7 cells were transiently transfected using the DEAE-dextran method with HA-tagged CD300b WT or mutant forms alone or in combination with c-Fyn, DAP-12 (or DAP12-Flag), Fc{epsilon}RI{gamma}, or CD3{zeta}. Cell lysates were immunoprecipitated with anti-HA.11 mAb and analyzed by 12% SDS-PAGE under reducing conditions. Proteins were transferred to a PVDF filter and probed with the indicated Abs. B, COS-7 cells were transiently transfected using Fugene 6 reagent with HA-tagged CD300b WT or K mutant in combination with FLAG-tagged adaptor molecules. Surface expression was monitored by flow cytometry using HA12CA5 mAb for the receptor (gray histogram), FLAG M2 mAb for the adaptors (data not shown), and an isotypic mAb as a negative control (white histogram). The results show a representative experiment of three performed.

 
It has been suggested that DAP-12 acts not only as a signaling molecule but also as a chaperone for certain immune receptors, preventing their retention in the endoplasmic reticulum and intracellular degradation by forming stable receptor-DAP-12 complexes (33, 34). To determine whether the presence of DAP-12 was necessary for CD300b surface expression, COS-7 cells were transfected with CD300b-HA in presence or absence of DAP12-FLAG. Surface expression of CD300b was evaluated after 48 h by flow cytometry with anti-HA mAb. CD300b was detected on the cell surface independently of DAP-12 expression, indicating that CD300b does not require its adaptor counterpart for membrane localization (Fig. 5B). Nevertheless, CD300b expression was enhanced in the presence of DAP-12 (25.87 ± 7.64% mean of expression = 39.22 ± 2.34 vs 41.08 ± 6.66% mean of expression = 76.60 ± 4.37) (Fig. 5B). Oppositely, DAP-12 expression was not modified by coexpression of the receptor (data not shown). Increased expression of CD300b was not detected when the receptor was cotransfected with any other transmembrane adaptor protein and it was abolished when a CD300b mutant affecting transmembrane lysine 158 (K158L) was used (Fig. 5B and data not shown).

Analysis of CD300b/DAP-12 complex formation and CD300b phosphorylation

The association between an activating immune receptor and its adaptor counterpart relies on the formation of a membrane-embedded salt bridge between a basic residue on the receptor (lysine or arginine) and an acidic residue on the adaptor protein (aspartate) (5, 6). As CD300b phosphorylation was occurring only in the presence of both c-Fyn and DAP-12, we addressed whether tyrosine phosphorylation was required for association of CD300b and DAP-12. For this purpose, we generated a set of HA-tagged CD300b mutants affecting the transmembrane lysine (K158L), the cytoplasmic tyrosine (Y188F) or both residues. The CD300b Lys mutant cotransfected into COS-7 cells with c-Fyn kinase and DAP-12 was unable to recruit the adaptor molecule but, unexpectedly, it retained the ability to become tyrosine phosphorylated (Fig. 5C). Therefore, in this system, tyrosine phosphorylation of CD300b depends on the expression of DAP-12 but does not require its binding. The CD300b Tyr mutant recruited DAP-12 as efficiently as the wild-type form (WT), indicating that CD300b tyrosine phosphorylation is not needed for recruitment of the adaptor (Fig. 5C). As expected, the CD300b double mutant could neither recruit DAP-12 nor become phosphorylated in the presence of c-Fyn kinase (Fig. 5C). Association between CD300b and DAP-12 was maintained in absence of the c-Fyn kinase, indicating that phosphorylation of the adaptor was not essential for the formation of the complex (data not shown). Immunoprecipitation of DAP-12 resulted in coimmunoprecipitation of the three bands corresponding to CD300b, indicating that modification to the extracellular portion of the immune receptor does not affect its ability to recruit adaptor (data not shown).

CD300b engagement is able to activate transcriptional activity in the absence of DAP-12

Signal propagation to the intracellular compartment by activating receptors requires their association with transmembrane adaptor proteins (5, 6, 7). CD300b, unlike other activating immune receptors described to date, has a tyrosine-based motif in its cytoplasmic tail that could potentially deliver positive signals independently of DAP-12. To evaluate the activating capacity of the receptor, we stably expressed CD300b-HA on the surface of the RBL-2H3 cell line, in which DAP-12 is absent (31) (Fig. 6A). The cell line was transiently transfected with a vector containing three copies of the NFAT/AP-1 response element fused to the firefly luciferase gene. Transfected cells were stimulated with anti-HA mAb that would mimic CD300b natural ligand, as shown previously for IREM-2 (10). An increase in promoter activity, comparable to the one delivered by Fc{epsilon}RI stimulation, was achieved when the cells were stimulated with anti-HA mAb (Fig. 6B). To determine the importance of the tyrosine-based motif in activating the 3xNFAT/AP-1 reporter gene, we generated an RBL-2H3 stable transfectant expressing the CD300b Tyr mutant form of the molecule. Substitution of the tyrosine residue caused a marked decrease in the activating capacity of the receptor but did not abolish it (Fig. 6C). To address the possibility that other residues of CD300b were important for recruitment of signaling molecules, we generated a wider set of RBL-2H3 CD300b-HA stably transfected mutants (Fig. 6A). The set included the previously described substitution mutants CD300b K158L and CD300b K158L+Y188F, and two deletion forms affecting the cytoplasmic tail: CD300b {Delta}1 (del 178–201) in which the whole cytoplasmic tail was removed, and CD300b {Delta}2 (del 188–201) in which the amino acids preceding the tyrosine-based motif were maintained. The polyproline motif in the truncated cytoplasmic tail of CD300b {Delta}2 resembled a potential docking site for Src homology 3 (SH3) domain-containing molecules. However, both deletion mutants showed an activating capacity similar to the CD300b Y188F mutant, suggesting there are no other motifs in the cytoplasmic region able to contribute to reporter activation. Surprisingly, the CD300b K158L mutant caused a dramatic reduction in reporter activity, thus also establishing a role for the receptor’s transmembrane region in signaling in the absence of DAP-12 (Fig. 6C). Complete abrogation of reporter activity was achieved only when both residues, K158 and Y188, were mutated simultaneously (Fig. 6C). No differences were observed among transfectants in the activating capacity and cell surface levels of Fc{epsilon}RI (≥99% expression, data not shown).


Figure 6
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  		FIGURE 6. CD300b delivers an activating signal in the absence of DAP-12. A, RBL cells were stably transfected with HA-tagged forms of CD300b. Cell surface expression of CD300b molecules was checked by flow cytometry using anti-HA12CA5 mAb (gray histogram) or an isotypic mAb as a negative control (black histogram). B and C, RBL transfectants were transiently transfected with 3xNFAT/AP1-luciferase and TK-Renilla plasmids. Luciferase activity was measured after stimulation for 7 h with the indicated Abs. Absolute luciferase units are shown in B. Data in C are normalized and expressed as a percentage of luciferase activity considering IgE stimulation as the top threshold of activation. Duplicates were performed for all the stimulations. The results are representative of three and two independent experiments, respectively.

 
CD300b tyrosine does not mediate transcriptional activity in the presence of DAP-12

To explore the stimulatory function of CD300b in the presence of DAP-12, we stably expressed both molecules at the cell surface of the RBL-2H3 cells, which do not express either protein endogenously (Fig. 7A). NFAT/AP-1-dependent transcriptional activity was enhanced more efficiently in the presence of DAP-12 (Fig. 7B). To test whether the observed increase could be due to the convergence of two pathways initiated by CD300b, we performed a comparative analysis among RBL-2H3 cells expressing on the cell surface DAP-12 and mutants of CD300b altered at K158, Y188, or at both residues (Fig. 7A). Mutation of the cytoplasmic tyrosine did not reduce transcriptional activity, indicating that the signal observed was generated solely from the association of CD300b with DAP-12 (Fig. 7C). Additionally, we can conclude that the signal induced by the receptor-adaptor complex was stronger than that produced by the receptor alone (Fig. 7B). Consistent with these interpretations, mutation of the transmembrane lysine of CD300b, alone or in combination with mutation of the tyrosine residue, completely eliminated NFAT/AP-1-mediated transcriptional activity (Fig. 7C).


Figure 7
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  		FIGURE 7. DAP-12 disrupts the transcriptional activation generated by the CD300b cytoplasmic tail. A, RBL cells were first stably transfected with DAP12-FLAG and then with HA-tagged CD300b WT or mutants. Cell surface expression was checked by flow cytometry using anti-FLAG M2 mAb for DAP-12 (light gray histogram) and anti-HA12CA5 mAb for CD300b molecules (dark gray histogram). Isotypic mAb was used as a negative control (black histogram). B and C, RBL double transfectants were transiently transfected with 3xNFAT-AP1-luciferase and TK-Renilla plasmids. Luciferase activity was measured after stimulation for 7 h with the indicated Abs. Results are normalized and expressed as a percentage of luciferase activity considering IgE stimulation as the top threshold of activation. Duplicates were performed for all stimulations. The results are representative of three independent experiments.

 
CD300b induces hexosaminidase granule release only in the presence of DAP-12

NFAT/AP-1-dependent transcription was activated in RBL-2H3 cells by engagement of CD300b in the presence and absence of DAP-12 adaptor protein. We decided to look for additional pathways activated by CD300b either on its own or in association with DAP-12. Mast cells, when encountering a proper stimulus, are able to secrete proteases stored in cytoplasmic secretory granules (35). For instance, RBL-2H3 cells accumulate beta-hexosaminidase protease whose release can be induced upon Fc{epsilon}RI cross-linking (36). Stimulation of RBL-2H3 transfectants expressing CD300b alone failed to elicit a hexosaminidase exocytotic response, whereas coexpression of CD300b and DAP-12 resulted in a level of hexosaminidase release comparable to that induced by IgE cross-linking (Fig. 8A). Expression of a mutant form of CD300b in which the transmembrane lysine was mutated to a noncharged amino acid resulted in a reduction in protease release (Fig. 8B), confirming the dependence of this process on DAP-12. In contrast, CD300b carrying a substitution of the key cytoplasmic tyrosine elicited levels of hexosaminidase release comparable to WT CD300b (Fig. 8B), indicating that signaling through CD300b’s tyrosine-based motif is not involved in the exocytotic pathway.


Figure 8
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  		FIGURE 8. Hexosaminidase release is mediated by CD300b through its association with DAP-12. A and B, RBL cells expressing CD300b-HA alone or in combination with DAP-12 were stimulated with the indicated Abs. Percentage of hexosaminidase release was assessed by incubating the supernatant with 4-nitrophenyl N-acetyl-beta-D-glucosaminide substrate. Results are expressed as a percentage of specific hexosaminidase release: (Abs test – Abs spont)/(Abs max – Abs spont) x 100, where Abs spont is the spontaneous release obtained with cells incubated in absence of Abs and Abs max is the maximum release obtained with cells lysed using Tx-100. Spontaneous and maximum release did not differ markedly between transfectants. Each assay was set up in triplicate. The results are representative of three independent experiments.

 
CD300b cytoplasmic tail recruits the Grb-2 adaptor molecule in the presence of the Src-kinase c-Fyn

The cytoplasmic CD300b tyrosine-based motif matches the binding consensus sequence for the Grb-2 adaptor molecule (YxN) (37). To test for a phosphotyrosine-dependent interaction between CD300b and Grb-2, we performed a Gal4 three-hybrid system assay in the presence of either c-Fyn kinase or a c-Fyn kinase mutant lacking part of the catalytic domain. Interaction of CD300b and Grb-2 was detected only when c-Fyn kinase was fully active (Fig. 9A). No interaction was observed when CG1945 yeast was cotransfected with CD300b and other molecules containing SH2/SH3 domains, such as Csk or PI3K (p85{alpha} subunit), in the presence of c-Fyn (data not shown). The interaction between both molecules was also tested in a mammalian system by transfecting COS-7 cells with the receptor in the presence and absence of the c-Fyn kinase. Endogenous Grb-2 was found to be bound to CD300b only when the kinase was coexpressed in the cells (Fig. 9B).


Figure 9
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  		FIGURE 9. CD300b cytoplasmic tail recruits Grb-2 in a phosphotyrosine-dependent way. A, Three-hybrid system in yeast. Liquid culture assay with o-nitrophenyl-beta-D-galactopyranoside as substrate to measure beta-galactosidase activity was used to score the interaction of Grb-2 with the CD300b cytoplasmic tail in the presence of c-Fyn WT or a c-Fyn catalytic mutant. For each construct, three independent colonies were tested in the galactosidase assay. The results are representative of three independent experiments. B, COS-7 cells were transiently transfected with CD300b-HA alone or with c-Fyn kinase. Endogenous Grb-2 was immunoprecipitated. Western blots were conducted with the indicated Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
With the identification and cloning of immune receptors, new forms of activating and inhibitory receptors that defy traditional classification are being recognized. In this study, we describe the molecular cloning and characterization of a novel-activating immune receptor belonging to the CMRF-35/IREM family. CD300b has a basic residue in its transmembrane domain and a functional tyrosine-based motif in its short cytoplasmic tail (Fig. 1A). This structural arrangement confers on the molecule the ability to signal through two independent pathways: one through formation of a complex with an ITAM-bearing adaptor, and the other through the tyrosine residue in its cytoplasmic tail. No precedents are found in which an activating Ig receptor displays motifs involved in delivering activating signals. The closest similarity can be established with NKp44, an activating receptor expressed exclusively on activated NK cells. One of the three known NKp44 isoforms produced by alternative splicing contains both a lysine residue in its transmembrane region and a tyrosine-based sequence in its short cytoplasmic tail (AJ225109) that matches the ITIM consensus sequence (38, 39). Although able to be tyrosine phosphorylated, the ITIM on NKp44 does not recruit SHP-1/2 or SHIP phosphatases and, as a consequence, it has no inhibitory function (40).

CD300b by itself acts as a functional receptor in RBL-2H3 cells which endogenously lack DAP-12 (31). Stimulation of CD300b through the HA epitope promoted NFAT/AP-1-dependent transcriptional activity (Fig. 6B). The signal generated was transduced by the tyrosine-based motif on the receptor’s cytoplasmic tail but a cooperative effect involving the molecule’s transmembrane domain was needed (Fig. 7C). We hypothesize that in RBL-2H3 and in the absence of DAP-12, CD300b cytoplasmic phosphorylation requires the formation of a complex with an unknown molecule. This molecule would interact with CD300b through the positively charged residue in its transmembrane domain, similar to how ITAM-based adaptors or DAP-10 bind cell surface receptors. This could explain why substitution of the lysine residue of CD300b eliminated the transcriptional activity (Fig. 7C). The function of this unknown molecule, in the context of CD300b signaling, would be to recruit the kinase(s) responsible for the receptor’s tyrosine phosphorylation. It is noteworthy that the RBL-2H3 cell line expresses Fc{epsilon}RI{gamma} and DAP-10 adaptors, but neither was found to be associated with CD300b when assessed in COS-7 cells (31, 41). The proposed complex, when coupled to the receptor, seems able to indirectly activate NFAT/AP-1-dependent transcription. This may explain why CD300b tyrosine or deletion mutants still exhibit a low level activation of the reporter gene (Fig. 7C). Future studies on this system will focus on phosphorylation status of the receptor under basal conditions and upon stimulation, and how the process is influenced by the integrity of its transmembrane region. In addition, efforts will move toward identifying the postulated coupling molecule.

The tyrosine-based motif of CD300b matches the consensus sequence for the Grb-2 docking site (YxN) (37). As predicted, Grb-2 was able to interact in yeast and mammalian cells with the cytoplasmic tail of CD300b in a phosphotyrosine-dependent manner (Fig. 9). Grb-2 is a ubiquitous adaptor protein consisting of an SH3 and two SH2 domains lacking catalytic activity of its own. However, Grb-2 is able to link cell surface receptors to downstream signaling molecules (42). Upon receptor cross-linking, Grb-2 translocates from the cytosol to the cell surface where it binds phosphorylated tyrosines on receptors via its SH2 domains. Simultaneously, Grb-2 is thought to use its SH3 domain to recruit other signaling molecules that would mediate a change in the activation status of the cell (43). For instance, Grb-2 contributes to the activation of the Ras/MAPK signal transduction pathway in response to extracellular signals (16). This could explain the CD300b-mediated transcriptional activation observed in our system of study. It will be necessary to map the signaling events conduced by Grb-2 upon CD300b cross-linking and determine their relevance to the function of myeloid cells.

Despite the presence of the tyrosine residue in the cytoplasmic tail, CD300b is able to act as a classical immune receptor and associate with an ITAM-bearing adaptor molecule (Fig. 5A). Interaction with DAP-12 occurred independently of the tyrosine phosphorylation status of both the receptor and the adaptor (Fig. 5C and data not shown). The association was confirmed to be functional in the RBL-2H3 cell expression system. Release of preformed intracellular mediators was promoted upon receptor stimulation through its association with DAP-12 adaptor. No hexosaminidase release was observed in the absence of the adaptor molecule or when its recruitment was disrupted (Fig. 8). CD300b/DAP-12 complex was also able to activate NFAT/AP-1-dependent transcriptional activity (Fig. 7B). The observed signal was generated exclusively through DAP-12, and not as a combined effect of signals initiated by the ITAM-bearing adaptor and CD300b cytoplasmic tail (Fig. 7C). These data support the hypothesis of a complex recruited by the CD300b transmembrane region that is necessary for signaling through the receptor’s cytoplasmic tail. Even so, we cannot discount the possibility that both pathways could act simultaneously and in a complementary manner in other processes such as cytokine release, migration, cell proliferation, and/or differentiation (44). For example, our biochemical experiments in COS-7 cells show how CD300b presents a curious dependence on DAP-12 in terms of cell surface expression and tyrosine phosphorylation (Fig. 5, B and C). In fact, the presence of DAP-12 enhanced more efficiently the phosphorylation of CD300b, even when no interaction with the adaptor through the transmembrane lysine took place (Fig. 5C). These data suggest that in COS-7 cells, DAP-12 is able to modulate the level of tyrosine phosphorylation of CD300b indirectly through its interaction with other molecules, such as a second set of kinases. Another possibility is that the interaction between CD300b and DAP-12 does not rely exclusively on the transmembrane lysine. For instance, it has been demonstrated that the SH2 domain of the SLAM-associated protein adaptor molecule binds the c-Fyn SH3 domain and directly couples this kinase to the cytoplasmic tail of the immune receptor CD150, promoting CD150 tyrosine phosphorylation (45). New experiments are needed to fully understand the regulation of CD300b phosphorylation.

To understand the function of CD300b it will be necessary to focus our studies on those cells in which both the receptor and DAP-12 are naturally expressed. DAP-12 has been shown to be expressed and functional in NK and myeloid cells (46, 47). CD300b expression at the mRNA level is restricted to monocytes and myelomonocytic cell lines, similar to what has been described for IREM-1 and IREM-2 (Fig. 3, A and B). In addition, data on the Gene Expression server (at <http://symatlas.gnf.org/SymAtlas>) for TREM-5/CD300b revealed the presence of the transcript in dendritic cells, which could help to explain some of the transcripts detected in the tissue scan panel (Fig. 3C). Nevertheless, these data will need to be confirmed using specific mAbs. Several DAP12-associated activating myeloid Ig-like receptors, such as paired Ig-like type 2 receptor-beta (48), signal regulatory protein-beta (49), IREM-2 (10), and TREMs (50) have recently been described. Therefore, the existence of a DAP-12-free cell membrane in which CD300b would signal exclusively through its cytoplasmic tail seems unlikely. We think that CD300b signaling pathways are more likely to be controlled by engagement of the immune receptor with different ligands, compartmentalization of the molecule at the cell surface, and/or changes in CD300b affinity for extracellular, intracellular, and transmembrane coupling molecules.

Based on the results obtained, comparative functional analyses between human and murine CD300b molecules would be very interesting. Despite the high degree of homology between the two receptors, murine CD300b lacks the tyrosine-based motif of the cytoplasmic tail (Fig. 2B). The murine form will be essential for addressing the relevance of transmembrane associations in activating immune receptors by mechanisms distinct from the recruitment of classical adaptor proteins.


   Acknowledgments
 
We thank Dr. Oscar Fornas for advice in flow cytometry analysis and Dr. Margarita Martin, Damiana Alvarez-Errico, and Jordi Minguillon for helpful discussion. We are grateful to Daryl Henderson for his comments on 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 grants from Plan Nacional de I+D (SAF2004-00972). A.M.-B. was supported by a fellowship from the Ministerio de Ciencia y Tecnología, and J.S. was supported by a contract Ramon y Cajal from Ministerio de Ciencia y Tecnología. Back

2 Address correspondence and reprint requests to Dr. Joan Sayós, Molecular Immunopathology Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain. E-mail address: joan.sayos{at}upf.edu Back

3 Abbreviations used in this paper: SH2, Src homology 2; Grb2, growth factor receptor-bound protein 2; HA, hemagglutinin; WT, wild type; mIREM, murine IREM; Tx-100, Triton X-100; TREM, triggering receptor expressed on myeloid cell; SH3, Src homology 3; SHP-1, SH2 domain-containing phosphatase 1; PVDF, polyvinylidene difluoride; DAP-12, DNAX-activating protein of 12 kDa; KARAP, killer cell-activating receptor-associated protein; IREM, immune receptor expressed by myeloid cell. Back

Received for publication February 21, 2006. Accepted for publication June 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Medzhitov, R., C. Janeway. 2000. Innate immune recognition: mechanisms and pathways. Immunol. Rev. 173: 89-97. [Medline]
  2. Ravetch, J. V., L. L. Lanier. 2000. Immune inhibitory receptors. Science 290: 84-89. [Abstract/Free Full Text]
  3. Veillette, A., S. Latour, D. Davidson. 2002. Negative regulation of immunoreceptor signaling. Annu. Rev. Immunol. 20: 669-707. [Medline]
  4. Diefenbach, A., D. H. Raulet. 2003. Innate immune recognition by stimulatory immunoreceptors. Curr. Opin. Immunol. 15: 37-44. [Medline]
  5. Vivier, E., J. A. Nunes, F. Vely. 2004. Natural killer cell signaling pathways. Science 306: 1517-1519. [Abstract/Free Full Text]
  6. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19: 197-223. [Medline]
  7. McVicar, D. W., L. S. Taylor, P. Gosselin, J. Willette-Brown, A. I. Mikhael, R. L. Geahlen, M. C. Nakamura, P. Linnemeyer, W. E. Seaman, S. K. Anderson, et al 1998. DAP12-mediated signal transduction in natural killer cells: a dominant role for the Syk protein-tyrosine kinase. J. Biol. Chem. 273: 32934-32942. [Abstract/Free Full Text]
  8. Chang, C., J. Dietrich, A. G. Harpur, J. A. Lindquist, A. Haude, Y. W. Loke, A. King, M. Colonna, J. Trowsdale, M. J. Wilson. 1999. Cutting edge: KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties. J. Immunol. 163: 4651-4654. [Abstract/Free Full Text]
  9. Alvarez-Errico, D., H. Aguilar, F. Kitzig, T. Brckalo, J. Sayos, M. Lopez-Botet. 2004. IREM-1 is a novel inhibitory receptor expressed by myeloid cells. Eur. J. Immunol. 34: 3690-3701. [Medline]
  10. Aguilar, H., D. Alvarez-Errico, A. C. Garcia-Montero, A. Orfao, J. Sayos, M. Lopez-Botet. 2004. Molecular characterization of a novel immune receptor restricted to the monocytic lineage. J. Immunol. 173: 6703-6711. [Abstract/Free Full Text]
  11. Cantoni, C., C. Bottino, R. Augugliaro, L. Morelli, E. Marcenaro, R. Castriconi, M. Vitale, D. Pende, S. Sivori, R. Millo, et al 1999. Molecular and functional characterization of IRp60, a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK cells. Eur. J. Immunol. 29: 3148-3159. [Medline]
  12. Daish, A., G. C. Starling, J. L. McKenzie, J. C. Nimmo, D. G. Jackson, D. N. Hart. 1993. Expression of the CMRF-35 antigen, a new member of the immunoglobulin gene superfamily, is differentially regulated on leucocytes. Immunology 79: 55-63. [Medline]
  13. Saborit-Villarroya, I., J. M. Del Valle, X. Romero, E. Esplugues, P. Lauzurica, P. Engel, M. Martin. 2005. The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing. J. Immunol. 175: 4226-4235. [Abstract/Free Full Text]
  14. Sivori, S., M. Vitale, L. Sanseverino, S. Parolini, M. Barbaresi, C. Bottino, A. Moretta. 1996. Inhibitory CD94 molecules identified by the Z199 monoclonal antibody recognize different HLA-class I molecules. Transplant. Proc. 28: 3199-3203. [Medline]
  15. Sayos, J., M. Martin, A. Chen, M. Simarro, D. Howie, M. Morra, P. Engel, C. Terhorst. 2001. Cell surface receptors Ly-9 and CD84 recruit the X-linked lymphoproliferative disease gene product SAP. Blood 97: 3867-3874. [Abstract/Free Full Text]
  16. Martin, M., J. M. Del Valle, I. Saborit, P. Engel. 2005. Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229. J. Immunol. 174: 5977-5986. [Abstract/Free Full Text]
  17. Wu, J., H. Cherwinski, T. Spies, J. H. Phillips, L. L. Lanier. 2000. DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J. Exp. Med. 192: 1059-1068. [Abstract/Free Full Text]
  18. Sambrook, J., P. MacCallum, D. Russel. 2002. Molecular Cloning Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  19. Harlow, E., D. Lane. 1988. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  20. Hedin, K. E., M. P. Bell, K. R. Kalli, C. J. Huntoon, B. M. Sharp, D. J. McKean. 1997. {delta}-Opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element. J. Immunol. 159: 5431-5440. [Abstract]
  21. Geng, L., S. Pfister, S. K. Kraeft, C. E. Rudd. 2001. Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain. Proc. Natl. Acad. Sci. USA 98: 11527-11532. [Abstract/Free Full Text]
  22. Green, B. J., G. J. Clark, D. N. Hart. 1998. The CMRF-35 mAb recognizes a second leukocyte membrane molecule with a domain similar to the poly Ig receptor. Int. Immunol. 10: 891-899. [Abstract/Free Full Text]
  23. Clark, G. J., B. Cooper, S. Fitzpatrick, B. J. Green, D. N. Hart. 2001. The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family. Tissue Antigens 57: 415-423. [Medline]
  24. Speckman, R. A., J. A. Wright Daw, C. Helms, S. Duan, L. Cao, P. Taillon-Miller, P. Y. Kwok, A. Menter, A. M. Bowcock. 2003. Novel immunoglobulin superfamily gene cluster, mapping to a region of human chromosome 17q25, linked to psoriasis susceptibility. Hum. Genet. 112: 34-41. [Medline]
  25. Clark, G., D. Munster, S. Yusuf, D. N. Hart. 2005. Eighth Leucocyte Differentiation Antigen Workshop DC section summary. Cell. Immunol. 236: 21-28. [Medline]
  26. Classon, B. J., M. H. Brown, D. Garnett, C. Somoza, A. N. Barclay, A. C. Willis, A. F. Williams. 1992. The hinge region of the CD8 {alpha} chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains. Int. Immunol. 4: 215-225. [Abstract/Free Full Text]
  27. Breathnach, R., P. Chambon. 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50: 349-383. [Medline]
  28. Cantoni, C., M. Ponassi, R. Biassoni, R. Conte, A. Spallarossa, A. Moretta, L. Moretta, M. Bolognesi, D. Bordo. 2003. The three-dimensional structure of the human NK cell receptor NKp44, a triggering partner in natural cytotoxicity. Structure 11: 725-734. [Medline]
  29. Chung, D. H., M. B. Humphrey, M. C. Nakamura, D. G. Ginzinger, W. E. Seaman, M. R. Daws. 2003. CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation. J. Immunol. 171: 6541-6548. [Abstract/Free Full Text]
  30. Lotze, M., A. Thomson. 2001. Dendritic Cells: Biology and Clinical Applications Academic Press, Cold Spring Harbor.
  31. Anfossi, N., M. Lucas, A. Diefenbach, H. J. Buhring, D. Raulet, E. Tomasello, E. Vivier. 2003. Contrasting roles of DAP10 and KARAP/DAP12 signaling adaptors in activation of the RBL-2H3 leukemic mast cell line. Eur. J. Immunol. 33: 3514-3522. [Medline]
  32. Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J. K. Hsia, E. Vivier, D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat. Immunol. 3: 1142-1149. [Medline]
  33. Lanier, L. L., B. Corliss, J. Wu, J. H. Phillips. 1998. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8: 693-701. [Medline]
  34. Smith, K. M., J. Wu, A. B. Bakker, J. H. Phillips, L. L. Lanier. 1998. Ly-49D and Ly-49H associate with mouse DAP12 and form activating receptors. J. Immunol. 161: 7-10. [Abstract/Free Full Text]
  35. Blank, U., J. Rivera. 2004. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol. 25: 266-273. [Medline]
  36. Aketani, S., R. Teshima, Y. Umezawa, J. Sawada. 2001. Correlation between cytosolic calcium concentration and degranulation in RBL-2H3 cells in the presence of various concentrations of antigen-specific IgEs. Immunol. Lett. 75: 185-189. [Medline]
  37. Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, T. Yi, et al 1994. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol. Cell. Biol. 14: 2777-2785. [Abstract/Free Full Text]
  38. Vitale, M., C. Bottino, S. Sivori, L. Sanseverino, R. Castriconi, E. Marcenaro, R. Augugliaro, L. Moretta, A. Moretta. 1998. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J. Exp. Med. 187: 2065-2072. [Abstract/Free Full Text]
  39. Cantoni, C., C. Bottino, M. Vitale, A. Pessino, R. Augugliaro, A. Malaspina, S. Parolini, L. Moretta, A. Moretta, R. Biassoni. 1999. NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J. Exp. Med. 189: 787-796. [Abstract/Free Full Text]
  40. Campbell, K. S., S. Yusa, A. Kikuchi-Maki, T. L. Catina. 2004. NKp44 triggers NK cell activation through DAP12 association that is not influenced by a putative cytoplasmic inhibitory sequence. J. Immunol. 172: 899-906. [Abstract/Free Full Text]
  41. Ono, M., T. Yuasa, C. Ra, T. Takai. 1999. Stimulatory function of paired immunoglobulin-like receptor-A in mast cell line by associating with subunits common to Fc receptors. J. Biol. Chem. 274: 30288-30296. [Abstract/Free Full Text]
  42. Lowenstein, E. J., R. J. Daly, A. G. Batzer, W. Li, B. Margolis, R. Lammers, A. Ullrich, E. Y. Skolnik, D. Bar-Sagi, J. Schlessinger. 1992. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70: 431-442. [Medline]
  43. Egan, S. E., B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, R. A. Weinberg. 1993. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363: 45-51. [Medline]
  44. Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez-Espinosa, Y. Furumoto, S. Saitoh, L. E. Samelson, J. J. O’Shea, J. Rivera. 2002. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat. Immunol. 3: 741-748. [Medline]
  45. Latour, S., R. Roncagalli, R. Chen, M. Bakinowski, X. Shi, P. L. Schwartzberg, D. Davidson, A. Veillette. 2003. Binding of SAP SH2 domain to FynT SH3 domain reveals a novel mechanism of receptor signalling in immune regulation. Nat. Cell Biol. 5: 149-154. [Medline]
  46. Lanier, L. L., B. C. Corliss, J. Wu, C. Leong, J. H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391: 703-707. [Medline]
  47. Lanier, L. L., A. B. Bakker. 2000. The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol. Today 21: 611-614. [Medline]
  48. Shiratori, I., K. Ogasawara, T. Saito, L. L. Lanier, H. Arase. 2004. Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor. J. Exp. Med. 199: 525-533. [Abstract/Free Full Text]
  49. Dietrich, J., M. Cella, M. Seiffert, H. J. Buhring, M. Colonna. 2000. Cutting edge: signal-regulatory protein beta1 is a DAP12-associated activating receptor expressed in myeloid cells. J. Immunol. 164: 9-12. [Abstract/Free Full Text]
  50. Colonna, M.. 2003. TREMs in the immune system and beyond. Nat. Rev. Immunol. 3: 445-453. [Medline]



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