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INTRODUCTION |
It is well recognized that the first level regulation of
activation or inhibition of an immune response occurs at the cell surface receptor site (1). The signals sensed by the receptors are
relayed through their cytoplasmic signaling modules or adaptor molecules to regulate various cellular activities. The immunoreceptor tyrosine-based activation motif
(ITAM)1 is one of such signal
modules present in cytoplasmic tails of many antigen and Fc receptors
such as T cell receptor (TCR), B cell receptor (BCR), Fc
RI
, and
Fc
RI
. The consensus of ITAM (YXX(L/I)X6-8YXX(L/I))
has been deduced from sequence analysis of existing
ITAM-containing receptors (2). In T cells, following the antigen
binding to TCR
and
chain, the two tyrosine residues in the ITAM
are phosphorylated by Src-family protein tyrosine kinase, Lck or Fyn
(1). The phosphotyrosines, in turn, participate in and physically
interact with the signal-amplifying kinase, Syk/ZAP-70, as well as
other SH2 domain-containing proteins, which leads to the activation of
downstream signaling pathways (1). Analogous events occur in B cells,
basophils, mast cells, and other immune cells (3).
Nuclear factors of activated T cells (NFAT) are a family of
transcription factors expressed in a diverse cell types of immune systems (4-8). NFAT has been implicated in the activation of mast
cells (9, 10), B and T lymphocytes (11, 12), and NK cells (13, 14), and
plays a key role in the regulation of transcription of a wide variety
of cytokines and cell surface receptors that mediate important
immune functions, including interleukin (IL)-2 (15), IL-4
(16), IL-5 (17), IL-13 (18), interferon-
(19), tumor necrosis factor
(TNF)-
(20), and granulocyte-macrophage colony-stimulating factor
(20), as well as the CD40L (16), and cytotoxic T
lymphocyte-associated antigen 4 (21). NFAT is activated by stimulation
of receptors coupled to calcium/calcineurin signals, such as the
antigen receptors on T and B cells (5, 22), Fc
receptors on mast
cells and basophils (9), the Fc
receptors on macrophages and NK
cells (23), and receptors coupled to heterotrimeric G proteins
(24).
During a comprehensive search for novel activating receptors expressed
on cell surface using the bioinformatics approach, we identified a
human gene that encodes a novel ITAM-containing protein. Expression of
this protein in HMC-1 cells activated transcription of IL-13 and
TNF-
promoters, which was mediated through the
calcineurin/NFAT-signaling pathway. Therefore, the newly identified
protein was named calcineurin/NFAT activating and ITAM-containing
protein, or CNAIP. We report here its identification, gene
structure and stimulatory role for NFAT.
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EXPERIMENTAL PROCEDURES |
Identification and in Silico Characterization of
CNAIP--
The protein data base compiled by International Protein
Index (IPI) (www.ensembl.org/IPI/) containing ~65,000
protein sequences (as of March 2002) was used as data source in
this study. A hidden Markov model (HMM)-based method was employed for
Ig-domain search against IPI data base. The HMM, which was built from
an alignment of 113 confident Ig domains and calibrated using the
program HMMER, was obtained from the Pfam (version 6.6) data base (25).
To identify ITAM-containing proteins, a PROSITE-formatted motif profile was first constructed based on the common features of ITAM motif, and
the software "seedtop" (NCBI) was used to perform the search. Large-scale transmembrane region prediction for all of the IPI proteins
was carried out by using software TMHMM version 2.0 (26) (www.cbs.dtu.dk/services/TMHMM/). The three sets of genes generated above were then overlapped to identify molecules with all of the common
features by a relational data base system. In silico cloning was carried out as follows: the starting sequence was blasted against
the human database of expressed sequence tag (NBCI) data base,
the resulting hits were assembled as a contig, which was then reblasted
against database of expressed sequence tag. This process was proceeded
in a cyclic fashion until no more new expressed sequence tag
hits could be found. Protein signal peptide prediction was performed by
using both SignalP (27) and SOSUIsignal (28). Potential glycosylation
sites were identified by PROSITE (29). Protein fold recognition
was performed by using 3D-PSSM (30, 36).
Cell Culture--
Human HMC-1 and 293T cells were routinely
cultured in Iscove's modified Dulbecco's medium and Dulbecco's
modified Eagle's medium, respectively, supplemented with 10% fetal
calf serum (Invitrogen), 1 µg/ml penicillin and streptomycin.
Cyclosporin A (Calbiochem, San Diego, CA) was added to the culture
medium at the indicated concentration.
The cDNA Cloning and Expression--
The cDNA encompassing
CNAIP coding region was amplified by PCR and cloned into
pcDNA3.1D/V5-His-TOPO (Invitrogen) by using two oligonucleotide primers
derived from the predicted cDNA sequence. The cDNA sequence was
verified by sequencing the entire insert, and the construct was termed
CNAIP-V5C. An alternative CNAIP expression plasmid termed CNAIP-V5N was
constructed by subcloning a PCR fragment into pSecTag/FRT/V5-His-TOPO
vector (Invitrogen), which contains the truncated CNAIP coding region
from the 43rd amino acid to the stop codon and a V5 tag fused at the
N-terminal truncation site. Mutagenesis of tyrosine residues in the
ITAM was generated on CNAIP-V5N backbone by PCR SOEing as described
(31), and verified by sequencing. A full-length construct for
expression of wild type CNAIP was generated by cloning the entire
coding region sequence into pcDNA3.1. The cDNA sequence was
verified by sequencing the entire insert on both strands.
Transfection and Western Blotting--
For transient
transfection, six to eight µg of protein expression plasmids were
transfected into 293T or HMC-1 cells in a 50-mm dish with LipofectAMINE
2000 (Invitrogen) according to manufacturer's instructions. The
transfected cells were cultured for 40-48 h and lysed in either 1×
sample buffer directly or protein extraction buffer (20 mM
Tris-HCl, 1 mM EDTA, 70 mM KCl, pH 7.6) through freeze-thaw cycles. The membrane protein was separated from the soluble
proteins by high-speed centrifugation. The protein extracts produced by
cell lysis were separated by SDS-PAGE and transferred to polyvinylidene
difluoride membrane, which was blocked with 5% dry milk in TBST (10 mM Tris, pH 7.8, 150 mM NaCl, with 0.05% Tween), then incubated with horseradish peroxidase-labeled anti-V5 monoclonal antibody (Invitrogen), and the signal was developed by
enhanced chemiluminescence (ECL) assay kit (Amersham
Biosciences).
First-strand cDNA Synthesis--
Total RNA samples of
various human tissues were obtained from Clontech,
and total RNAs from cultured cells were purified by using the RNeasy
mini kit (Qiagen, Valencia, CA). First-strand cDNA was synthesized
using the total RNA and SuperScript II reagent (Invitrogen). Briefly,
the reaction was set in a volume of 19 µl with 1.5 µg of total RNA,
2 pmol of each primer, 4 µl of 5× first-strand buffer (10 mM dithiothreitol and 0.2 mM dNTP). The mixture
was heated to 70 °C for 10 min and then cooled to 42 °C when 1 µl (200 units) SuperScript II was added. The mixture was then
incubated for 1 h at 42 °C and then heated to 70 °C for 15 min. RT-PCR was performed as duplex RT-PCR using
-actin as a standard.
Real-time Quantitative PCR--
Oligonucleotide primers were
designed using Primer Express 2.0 (Applied Biosystems, Inc., Foster
City, CA) and were synthesized and used in RT-PCR reactions to monitor
the real-time expression of CNAIP. RNA samples were isolated from the
following tissues and cells: brain, heart, kidney, liver, lung, spleen,
monocytes, Daudi (a Burkitt's lymphoma cell line), HPB-ALL (a T cell
leukemia cell line), THP-1 (acute monocytic leukemia; lymphocytes),
Jurkat (a T cell leukemia cell line), HMC-1 (a mast cell line), human vascular endothelial cells (primary human vascular endothelial cells), neutrophils, peripheral blood mononuclear cells, and four different batches of in vitro cultured cord-blood-derived
mast cell samples. Real-time quantitative PCR was performed with the ABI Prism 7900 (Applied Biosystems, Inc.) sequence detection system, using Taqman reagents, according to the manufacture's instructions. Equal amounts of each of the RNAs from the tissues and cell lines indicated above were used as PCR templates in reactions to obtain the
threshold cycle (Ct), and the Ct was normalized
using the known Ct from 18 S RNAs to obtain
Ct. To compare relative levels of gene expression of
CNAIP in different tissues and cell lines, 
Ct values
were calculated by using the lowest expression level as the base, which
were then converted to real fold expression difference values.
Luciferase Reporter Assay--
Luciferase reporter plasmids,
NFAT-luc, NF-
B-luc, and AP-1-luc were purchased from
Clontech. The luciferase reporter plasmids, IL-13-luc, TNF-
-luc, and Fc
RI
-luc, were constructed by
inserting 1.7 kb, 2.4 kb and 1.5 kb promoter sequences derived from
IL-13, TNF-
, and Fc
RI
genes into pTA-Luc vector
(Clontech), respectively. All of the promoter
sequences used were starting from a putative transcription start site
extending to the 5' upstream region. HMC-1 cells were seeded onto a
24-well culture plate at the density of 0.2 million cells per
milliliter of medium. Three plasmids, a firefly luciferase reporter
with a defined promoter, CNAIP expression plasmid, and
Renilla luciferase control plasmid, pRL-SV40 (Promega, Inc.,
Madison, WI), were co-transfected into HMC-1 cells. Cells were
harvested 40-46 h after transfection and lysed in lysis buffer. Both
firefly and Renila luciferase activities were assayed with the dual luciferase assay kit (Promega). The fluorescent light emission
was determined by TD-20 luminometer (Turner Design, Sunnylvale, CA).
Immunostaining--
The transfected 293T cells were washed and
pre-incubated at 4 °C for 20 min in the enzyme-free cell
dissociation buffer containing 1% bovine serum albumin (Invitrogen).
Cells were then incubated with fluorescein isothiocyanate-conjugated
anti-V5 monoclonal antibody (10 µg/ml) (Invitrogen) in the same
buffer for 30 min. After three washes, cells were fixed in 1 × phosphate-buffered saline with 1% paraformaldehyde. Alternatively, the
cells were first fixed in methanol for 5 min at room temperature and
rinsed three times before the immunostaining. The samples were analyzed by FACScan (BD Biosciences) or fluorescence microscopy.
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RESULTS |
Identification and Molecular Cloning of CNAIP--
Non-redundant
human protein data base IPI was searched for novel molecules containing
1) Ig domain, 2) ITAM, and 3) transmembrane region. Those are common
features shared by many signal-activating receptors mediating immune
system functions, including components of TCR, BCR, Fc
RI, and
several other recently identified activating receptors (2, 3, 32-35).
A hypothetical protein sequence labeled as "IPI00086590" was
identified that satisfied all three criteria. In silico
cDNA-cloning procedure was used to derive its full-length cDNA sequence.
To verify this cDNA, we designed two oligo primers encompassing the
putative starting methionine codon and stop codon, and amplified the
cDNA from human monocytes by RT-PCR. Two cDNA clones were
isolated, sequenced, and found to be identical to the in silico cloning-derived coding region sequence (Fig.
1A). The analysis of immediate 5'
flanking sequence to the coding region revealed a perfect Kozak motif,
and several in-frame stop codons preceding the predicted initiation
methionine. Furthermore, this cDNA contains a putative signal
peptide starting from the initiation methionine with a predicted
cleavage site between amino acids 42 and 43. Based on the structural
features and functionality that we elucidated, we designated this
protein calcineurin/NFAT-activating
and ITAM-containing protein, or CNAIP.

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Fig. 1.
CNAIP amino acid sequence, ITAM alignment,
and gene structure. A, protein sequence of CNAIP. The
putative signal peptide and transmembrane region were indicated by
single and double underlines, respectively, and
the ITAM was shaded. B, alignment of ITAM
sequences of selected human proteins. C, schematic
representation of CNAIP gene structure.
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CNAIP encodes a polypeptide with 270 amino acid residues and a
calculated molecular mass of ~30 kDa. It is predicted to be a type I
transmembrane protein, which contains a putative signal peptide at the
N-terminal (amino acids 1-42), an Ig-domain (amino acids
50-150) in the extracellular region, a transmembrane domain (amino
acids 164-186), and an ITAM (amino acids 220-235) in the cytoplasmic
region (Fig. 1B). One potential N-glycosylation
site was found in the extracellular region (amino acids 107-110). The Ig-domain most likely adopts a V-type fold based on 3D-PSSM fold recognition algorithm (30, 36). CNAIP has been mapped to chromosome 22q13.2 by sequence similarity search. Alignment of cDNA with genomic sequence showed that the coding region of CNAIP comprises six
exons (Fig. 1C). Sequence similarity search against various public data bases showed that CNAIP does not share statistically significant similarity with any known proteins.
Tissue Distribution of CNAIP--
The CNAIP mRNA expression
levels in a number of human cells and tissues were assessed using real
time quantitative RT-PCR. The results showed that CNAIP was highly
expressed in neutrophils, primary monocytes and monocytic cell lines
(THP-1), lymphocytes, and in vitro cultured mast cells
derived from cord blood (Fig. 2). The
expression in spleen and lung was also evident. In contrast, the CNAIP
expression in all other tissues and cells (brain, heart, kidney, liver,
Daudi, HPB-ALL, Jurkat, HMC-1, and HUVAC) was low, suggesting that the
primary role of CNAIP may be restricted to the immune system.
Interestingly, the CNAIP expression level in more mature mast cells
(cultured for 7 weeks and 86% tryptase-stained positive) is about
3-fold higher than that in the earlier stage of mast cells (cultured
for 4 weeks and 17% tryptase-stained positive).

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Fig. 2.
Expression profile of CNAIP transcript in
human tissues and immune related cells. The gene expression was
measured using real-time quantitative RT-PCR as described under
"Experimental Procedures" and normalized with 18 S RNAs. The
relative expression level of CNAIP is represented as real fold
difference with the lowest set to 1.
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Expression and Subcellular Localization of CNAIP Protein--
To
characterize the CNAIP gene product, we made two expression constructs,
one with a V5 tag fused in frame to the C terminus of CNAIP coding
region (CNAIP-V5C) and the other with the native signal peptide
replaced by a heterologous signal peptide in the vector, which is
immediately followed by a V5 tag fused to the N-terminal region of the
truncated CNAIP (CNAIP-V5N). Transient transfection of CNAIP-V5C into
293T cells showed two protein bands of ~33 and 36 kDa in Western
blot, which are absent in cells transfected with the empty vector (data
not shown). In protein fractions prepared by freeze-thaw cycle and
high-speed centrifugation, the 33 and 36 kDa proteins were only
detected in the insoluble membrane fraction, not in the soluble cytosol
fraction (Fig. 3), indicating that CNAIP is a
membrane-associated protein.

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Fig. 3.
Western blot analysis of recombinant
CNAIP. 293T cell were transfected with CNAIP-V5C. The cells were
lysed and separated into soluble and insoluble fractions and subjected
to Western blotting with anti-V5 monoclonal antibody as described under
"Experimental Procedures."
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Immunofluorescence staining was then performed to further determine the
subcellular localization and the orientation of CNAIP in the membrane.
FACS analysis of 293T cells transfected with CNAIP-V5N or CNAIP-V5C
showed that N terminus-tagged CNAIP was detected only in the living
cells transfected with CNAIP-V5N (Fig. 4) but
not in the living cells transfected with CNAIP-V5C (data not shown).
However, fixation of the cells transfected with the two expression
constructs resulted in unambiguous detection of both the N terminus-
and C terminus-tagged protein by FACS and fluorescent microscopy (data
not shown). These results indicated that CNAIP is a transmembrane
protein with the N terminus exposed to the outside of the cellular
membrane.

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Fig. 4.
FACS analysis of 293T cells expressing
CNAIP. 293T cells were transfected with either vector control or
CNAIP expression plasmid. Cells were stained with fluorescein
isothiocyanate-conjugated anti-V5 monoclonal antibody.
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CNAIP Activates the Calcineurin/NFAT-signaling
Pathway--
The co-existence of Ig domain and ITAM motif in CNAIP,
along with the preferential expression in immune cells, strongly
suggest that CNAIP may function as an activating receptor in immune
system. To test if CNAIP can activate the transcription of cytokine
genes, we co-transfected CNAIP-V5N with luciferase reporter constructs that were linked to IL-13, TNF-
, or Fc
RI
promoter in HMC-1 cells. The luciferase reporter assays showed that CNAIP increased IL-13
and TNF-
promoter activities by ~14- and ~5-fold, respectively, as compared with the expression vector control. In contrast, the Fc
RI
promoter-linked luciferase activity was not changed
(Fig. 5A).

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Fig. 5.
Luciferase reporter assay of CNAIP.
A, luciferase reporter assay of CNAIP-V5N and its mutants in
HMC-1. The assays were carried out as described under "Experimental
Procedures." The luciferase reporter constructs are indicated at
bottom of the graph, and the five protein
expression constructs (including vector) are indicated in the bar
graph. The firefly luciferase reporter activity was normalized by
Renilla luciferase activity and expressed as arbitrary unit
at y-axis. B, Western blot of the wild type and
mutant CNAIPs expressed in HMC-1 cells. About 1.5 million cells
transfected with each of the wild type and mutant CNAIP constructs were
lysed by freeze-thaw cycles. The membrane fractions were separated by
centrifugation and equal amount of protein was loaded in each lane.
C, luciferase reporter assay of full-length CNAIP,
CNAIP-V5N, and Y220A/Y231A mutant in HMC-1. The assays were carried out
as described under "Experimental Procedures" and
A.
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It was well documented that NFAT is involved in transcriptional
activation of cytokine genes, such as IL-13 (18) and TNF-
(20), and
that ITAM-containing receptors, upon ligand binding, can lead to the
activation of several transcription factors including NFAT (37),
NF-
B (38), and AP-1 (39). To test whether NFAT, NF-
B, and/or AP-1
are potential downstream targets of the CNAIP-signaling pathway, we
assessed the NFAT, NF-
B, or AP-1 luciferase reporter activity by
transient transfection with CNAIP-V5N in HMC-1 cells. Expression of
CNAIP elevated NFAT luciferase reporter activity by ~19-fold as
compared with the transfection with the vector only, whereas the
NF-
B or AP-1 luciferase reporter activities showed no significant
changes (Fig. 5A). To confirm that full-length CNAIP with
its native signal peptide can also activate NFAT, we co-expressed a
full-length construct of CNAIP with an NFAT luciferase reporter.
Expression of full-length CNAIP also elevated NFAT luciferase reporter
by ~9-fold as compared with the transfection with a control vector
(Fig. 5C). These data were reproducible in three separate experiments.
Because calcium flux and calcineurin activation are signaling
events upstream of NFAT activation (12), we tested whether the
CNAIP/calcineurin/NFAT-signaling pathway could be blocked by
cyclosporin A, a immune-suppressive agent which specifically inhibits
calcineurin activation. Addition of 1 µM of cyclosporin A
to the culture inhibited CNAIP-mediated NFAT activation by ~90% (Fig. 6), indicating that CNAIP can activate
calcineurin/NFAT-mediated signaling cascade and therefore activate
transcription of NFAT-regulated cytokine genes.

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Fig. 6.
Inhibition of the CNAIP-mediated luciferase
activity by cyclosporin A. HMC-1 cells were co-transfected with
NFAT-luc, CNAIP-V5N and pRL-SV40 as described under "Experimental
Procedures." Vehicle or 1 µM of cyclosporin was added
at 18 h after transfection and luciferase assays were performed as
described under "Experimental Procedures." The average inhibition
(%) was calculated from four independent experiments. The firefly
luciferase activity was first normalized by Renilla
luciferase activity.
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Calcineurin/NFAT Activation by CNAIP Is Mediated by ITAM
Motif--
Sequence analysis of CNAIP revealed a good match of ITAM in
the cytoplasmic tail (Fig. 1B). To determine whether the
putative ITAM motif mediates the signal transduction, we generated
three CNAIP mutants (Y220A, Y231A, and Y220A/Y231A) by replacing the two tyrosines (Tyr-220 and Tyr-231) in the ITAM to alanine
individually or in combination. Transfection of either of these mutants
into HMC-1 cells failed to activate IL-13, TNF-
, or NFAT luciferase reporter activity (Fig. 5). The relative luciferase reporter activity in these cells was comparable with that of cells transfected with vector control (Fig. 5A). Western blot analysis showed that
mutant proteins were expressed at similar level as that of wild type CNAIP (Fig. 5B), indicating that mutations do not affect the
expression and stability of CNAIP in these cells. These data indicated
that activation of the calcineurin/NFAT-signaling pathway by CNAIP is
mediated by ITAM, and both tyrosine residues are required for the
ITAM-mediated function in CNAIP.
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DISCUSSION |
In the present report, we describe the identification of a novel
ITAM-containing protein, CNAIP, that activates NFAT, IL-13, and TNF-
promoter activity. Our data suggest the CNAIP may function as an
activating receptor. ITAM-containing receptors are a divergent group of
cell surface membrane proteins that are expressed in immune-related
cells and regulate cell growth, maturation, apoptosis, and cell
activation. Members of this family include Ig
, Ig
, TCR-
,
TCR-
, CD3
, CD3
, CD3
, Fc
RI
, and Fc
RI
. Some of
them contain Ig or Ig-like domains in the extracellular region, which are involved in protein-protein interaction. Similar to those receptors, CNAIP contains an Ig domain in the extracellular region. Some ITAM-containing receptors transduce signal within a multi-subunit of immune receptor complexes such as TCR and BCR. It is not clear at
present whether CNAIP is a component of an immune receptor complex or
functions as a single unit. Interestingly, overexpression of CNAIP can
activate downstream effectors without ligand binding or antibody
cross-linking. The mechanisms by which CNAIP activates the signaling
pathway remain to be determined. However, it is possible that
overexpression of CNAIP proteins in the cells results in the
aggregation or clustering of the receptors on cell surface, which leads
to the recruitment of downstream effector to the receptor molecules
that subsequently lead to its activation.
The ITAM consensus sequence (YxxL/Ix6-8YxxL/I) contains
two appropriately spaced tyrosine residues (33). Following receptor
engagement, phosphorylation of the two tyrosines by Src family kinases
creates temporary anchors for SH2-containing signaling proteins that
recruit to the receptor site (3). In our studies, mutation of either of
the two tyrosine residues in the ITAM of CNAIP abolished the activating
activity mediated by CNAIP. Thus, like other ITAM-containing activating
receptors (40), the phosphorylation of the two tyrosine residues is
critical for its activity. It was well documented that in mast cells
the tyrosines in the ITAM of Fc
RI
and
subunits are rapidly
phosphorylated upon IgE cross-linking. The phosphotyrosines are then
engaged in recruiting and activating Src-related protein receptor
kinases, Lyn and Fyn, and signal amplifying kinase, Syk (33). The
reminiscent functionality of ITAMs found in other immunoreceptor
complexes was established through extensive experimental research, such
as those in TCR and BCR (1). It is conceivable that CNAIP may transduce
its activating signal through the ITAM in the manner similar to that of
the other ITAM-containing proteins. However, the divergence and
multiplicity of ITAM domains might determine which Src-related receptor
kinase(s) and SH2-containing protein would be recruited (3). Because
the ITAM sequence of CNAIP displays no close similarity to any existing
ITAMs except for the four conserved positions (Fig. 1B), it
is not known yet what kinase(s) or SH2-containing protein(s) are
recruited to the ITAM of CNAIP.
The gene structure and topology of CNAIP resemble other activating
molecules such as NKp44 and TREMs (triggering receptor expressed on
myeloid cells molecules). All those molecules are type I transmembrane
protein with a single Ig domain in the extracellular region, and
transduce signals via ITAM motif, which is contained in either the
cytoplasmic region of the molecule or its associated adaptor molecule
DAP12. Moreover, both TREMs and CNAIP are abundantly expressed in
neutrophils and monocytes. However, sequence similarity between CNAIP
and TREM and NKp44 is very low (<10%) and statistically insignificant. TREM-1 was recently characterized and found to function
as an activating receptor (41). It is expressed at high levels on
neutrophils and monocytes that infiltrate human tissues infected with
bacteria and plays a critical role in acute inflammatory responses to
bacteria (41). It is tempting to speculate that CNAIP may play similar
roles in acute inflammation, which are characterized by an
exudate of neutrophils and monocytes (42). Further studies are
aimed to identify the functional role of CNAIP in the immune responses.