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J Biol Chem, Vol. 273, Issue 51, 34115-34119, December 18, 1998
From the Natural killer cell and T cell subsets express at
their cell surface a repertoire of receptors for MHC class I molecules, the natural killer cell receptors (NKRs). NKRs are characterized by the
existence of inhibitory and activating isoforms, which are encoded by
highly homologous but separate genes present in the same locus.
Inhibitory isoforms express an intracytoplasmic immunoreceptor
tyrosine-based inhibition motif, whereas activating isoforms lack any
immunoreceptor tyrosine-based inhibition motif but harbor a charged
amino acid residue in their transmembrane domain. We previously
characterized KARAP (killer cell activating receptor-associated
protein), a novel disulfide-linked tyrosine-phosphorylated dimer that
selectively associates with the activating NKR isoforms. We report here
the identification of the mouse KARAP gene, its localization on chromosome 7 and its genomic organization in five exons. Point mutation and transfection studies revealed that KARAP is a
novel signaling transmembrane subunit whose transduction function
depends on the integrity of an intracytoplasmic immunoreceptor tyrosine-based activation motif. In contrast to previous members of the
immunoreceptor tyrosine-based activation motif polypeptide family,
KARAP is ubiquitously expressed on hematopoietic and nonhematopoietic cells, suggesting its association with a broad range of activating receptors in a variety of tissues.
Although NK1 cells have
been initially defined as non-MHC restricted large granular lymphocytes
(1), they have now been revealed as controlled in their effector
function (i.e. cytotoxicity and cytokine production) by MHC
class I molecules expressed at the surface of target cells (2).
Multigenic and multiallelic families of NK cell surface receptors for
classical and nonclassical MHC class I molecules have been identified
in human and mouse. Human NK receptors belong to two structurally
distinct groups: the immunoglobulin superfamily for the killer cell
inhibitory receptors (KIRs), and the C-type lectins for the CD94/NKG2
heterodimers (3-5). NK receptors are members of the ITIM-bearing
receptor family and recruit upon engagement with their cognate ligands, the intracytoplasmic SH2 tandem protein-tyrosine phosphatases, SHP-1
and SHP-2, which terminate NK cell activation programs (6). Isoforms of
NK receptors have been described that do not express intracytoplasmic
ITIM and propagate activating rather than inhibitory signals (7-10).
In this report, we identified and analyzed the structure and the
function of a novel ITAM-bearing molecule, KARAP, which selectively
associates with activating isoforms of KIRs, referred as to killer cell
activating receptor (KARs). In contrast to KARs that are restricted to
NK and T cell subsets, the KARAP transcription pattern revealed its
expression on a wide variety of cell types including nonhematopoietic
cells, such as neurons. Originally identified in lymphocytes,
macrophages, and mast cells (11, 12), ITAM-bearing molecules are
therefore more broadly distributed, because, in addition to the wide
spectrum of KARAP expression, members of this family can also be
encoded by viral genes or involved in platelet activation (13, 14).
Bio-informatics--
The "select_hits" program has been
developed to extract all potential KARAP sequences in a fully automatic
manner, from sequence data bases. This program has been written in C
(ANSI) and FORTRAN 77 and processes a data base in four consecutive
steps: step 1, for DNA data base, translation of entries in all reading
frames and selection of only potential peptides 50-200 amino acids in length; step 2, selection of entries with a predicted ITAM site (Y-X-X-[IL]-X(6,8)-Y-X-X-[IL]);
step 3, on this subset, selection of entries with a predicted
transmembrane region, as described (15); these regions must contain
more than 12 amino acids; and step 4, selection of entries with charged
amino acids in the transmembrane region (Asp, Glu, Arg, or Lys), a
cysteine residue between the transmembrane region and the N terminus,
and the ITAM between the transmembrane region and the C terminus.
Cell Transfection--
RBL-2H3 cell transfectants expressing
p50.2 KAR, i.e. KIR2DS2 (RBL-KAR: RTIIB.p50 cells), have
been previously described (16). Wild type mouse KARAP and human DAP-12
cDNAs were cloned in pNT-neo using PCR and cDNAs prepared from
mouse spleen and human NK cell clone total RNA, respectively (17).
Stable mouse KARAP and human DAP-12 transfectants of RBL-KAR cells were
established by electroporation and culture in the presence of G418 (1 mg/ml); representative clones were selected for further investigation.
Point mutation constructs were generated by PCR-directed mutagenesis of
the mouse KARAP and human DAP-12 constructs. Each point mutation
involved a tyrosine replacement by phenylalanine. Fidelity of the
constructs was verified by sequencing.
Cell Activation and Immunoblotting--
Cells were resuspended
at 10 × 106 cells/ml in phosphate-buffered saline and
pre-incubated for 10 min at 37 °C. Cells were then incubated for 5 min in the presence or absence of pervanadate (500 µM)
prepared as described (18). Cells were immediately lysed in digitonin
lysis buffer for 30 min on ice. After removing insoluble material by
centrifugation at 12,000 rpm for 15 min, samples were subjected to
immunoprecipitation for 2 h using indicated mAbs coupled to
protein G-Sepharose beads (Amersham Pharmacia Biotech). Samples were
then combined with reducing sample buffer (New England Biolabs) and
boiled prior to fractionation on SDS-PAGE and immunoblotting (18). RBL
cell serotonin release assays were performed as described previously
(16).
Genomic DNA Cloning--
A 129 mouse genomic DNA library
cloned in EMBL3 phage was kindly provided by M. Malissen (Centre
d'Immunologie INSERM/CNRS de Marseille-Luminy (CIML)); this
genomic DNA was extracted from E14 clone of embryonic stem cells
derived from 129/Ola substrain. The screening of the library was
performed as described previously by Sambrook et al. (19).
Mouse KARAP cDNA was labeled with [ Reverse Transcription-PCR Analysis--
Mouse DC27.1 T cell
hybridoma and mouse BW Fluorescence in Situ Hybridization--
Metaphase spreads were
prepared from a WMP male mouse, in which all autosomes except
chromosome 19 were in the form of metacentric Robertsonian
translocations. Concanavalin A-stimulated lymphocytes were cultured at
37 °C for 72 h with 5-bromodeoxyuridine (60 µg/ml) added to
the final 6 h of culture. The KARAP Bio-informatic Identification of the Mouse KARAP Gene as EST
AA734769--
We previously identified KARAP as a 12-kDa polypeptide
that selectively associates with KARs (23). Several biochemical
observations revealed that KARAP shares striking similarities with
members of the ITAM-bearing polypeptide family. First, KARAP is
expressed as disulfide-linked dimer. Second, KARAP associates with
KARs, which contain a charged amino acid residue in their transmembrane portion, similarly to ITAM-bearing polypeptides and their associated receptors (e.g. TCR, BCR, Fc Structure Function Analysis of Mouse and Human KARAPs--
We
originally reported that the stable transfection of RBL cells with KAR
cDNAs (i.e. KIR2DS2) leads to cell surface expression of
a nonfunctional KAR molecule (16). RBL-KAR transfectant cell lines were
then transfected with a set of cDNAs corresponding to wild type or
mutant mouse KARAP (AA734769) and human DAP-12. RBL transfectants
coated with mouse anti-KIR2DS2 mAb or mouse IgE as a positive control
were stimulated by the addition of goat anti-mouse antisera in a
standard serotonin release assay. As shown in Fig.
2A, both wild type mouse KARAP
(mKARAP) and human DAP-12 (hDAP-12) reconstituted the activating
property of KAR in RBL transfectants. Consistent with these results,
anti-phosphotyrosine immunoblots performed on anti-KAR
immunoprecipitates prepared from RBL-KAR + mKARAP transfectants
revealed that mKARAP co-precipitates with KAR as a
tyrosine-phosphorylated protein upon pervanadate stimulation (Fig.
2B), as well as upon KAR engagement (data not shown). In
contrast, single Tyr Genomic Organization and Chromosomal Localization of Mouse KARAP
Gene--
Southern blot analysis revealed that mouse KARAP
is a single gene, as is its human DAP-12 ortholog (data not
shown). An 18-kilobase mouse KARAP genomic clone was isolated by
screening a 129 mouse Ubiquitous Expression of Mouse KARAP--
ITAM-bearing molecules
have been documented only within the hematopoietic compartment. In
contrast, reverse transcription-PCR analysis of KARAP transcription in
a variety of cell lines revealed that KARAP transcripts can be detected
not only in NK cells (not shown) or in T and B lymphocytes but also in
mast, endothelial, and epithelial cell lines as well as in neuronal
cells lines (Fig. 4).
A feature of the ITIM-bearing molecules such as KIRs is the
existence of activating counterparts devoid of intracytoplasmic ITIMs
and characterized by the presence of a charged amino acid residue in
their transmembrane domain (28). We report here that the activating
isoforms of KIRs associate with KARAPs, which function as transducing
polypeptides coupling the engagement of KAR to the signaling machinery
leading to RBL degranulation. The model of KAR reconstitution in RBL
cells is directly relevant to the activation of lymphocyte-mediated
cell cytotoxicity by KARs, because NK cells and cytotoxic T lymphocytes
express intracytoplasmic granules that undergo regulated exocytosis
upon interaction with target cells. Our molecular cloning strategy also
revealed that mouse KARAP is the ortholog of human DAP-12 and is highly
similar to the AA242315 mouse EST, which was identified during the course of our studies (26). We will thus refer to KARAP/DAP-12 for both
human and mouse polypeptides hereafter.
KARAP/DAP-12 is a canonical ITAM-bearing polypeptide closer to CD3 CD3 Similarly to other ITAM-bearing polypeptides, the integrity of
KARAP/DAP-12 ITAM is mandatory to its transducing properties (32), as
judged by receptor-induced RBL serotonin assays (Fig. 2A).
The SH2-containing protein-tyrosine kinases ZAP-70 and Syk are the only
effector molecules that associate in vivo with the phosphorylated form of ITAM-bearing molecules (33). The requirement of
both tyrosines residues for complete KARAP/DAP-12 transducing function
is consistent with the structure of ZAP-70 tandem SH2 domains, which
dictates the simultaneous recruitment of both ITAM phosphotyrosines to
a ZAP-70 SH2 tandem (34, 35). The recently reported in vitro
association between KARAP/DAP-12 phosphorylated peptides and ZAP-70/Syk
is consistent with our in vivo point mutation analysis (26).
More surprisingly, no in vivo phosphorylation of
KARAP/DAP-12 can be detected when a single KARAP/DAP-12 tyrosine is
mutated (Fig. 2B). Similarly, analysis of Y-F Ig A major contrast between KARAP/DAP-12 and all other ITAM-bearing
polypeptides is the almost ubiquitous expression of KARAP, as judged by
its transcription pattern (Fig. 4). KARAP/DAP-12 is the first
ITAM-bearing polypeptide to be expressed outside of the hematopoietic
compartment. The ubiquitous distribution of ITIM-bearing molecules and
their activating counterparts is therefore consistent with the
association of KARAP/DAP-12 with a variety of activating isoforms of
ITIM-bearing molecules. In particular, the co-expression of SIRP- We thank Bernard Malissen (CIML) for
continuous encouragement and insightful advice, as well as Corinne
Béziers La Fosse (CIML) for graphic expertise.
*
This work was supported by institutional grants from INSERM,
CNRS, and Ministère de l'Enseignement Supérieur et de la
Recherche and specific grants from Association pour la Recherche contre le Cancer (to E. V.), Ligue Nationale contre le Cancer (to E. T.), "Axe Immunologie des Tumeurs" de la Ligue Nationale contre le
Cancer (to E. V.), ZENECA (to F. V.), and from the Training and Mobility of Researcher Program (to L. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF077829.
The abbreviations used are:
NK, natural killer; KIR, killer cell inhibitory receptor; ITIM, immunoreceptor
tyrosine-based inhibition motif; ITAM, immunoreceptor tyrosine-based
activation motif; KAR, killer cell activating receptor; PCR, polymerase
chain reaction; mAb, monoclonal antibody; EST, expressed sequences tag; bp, base pair(s).
Gene Structure, Expression Pattern, and Biological Activity of
Mouse Killer Cell Activating Receptor-associated Protein
(KARAP)/DAP-12*
,,,,,,
, and**
Centre d'Immunologie INSERM/CNRS de
Marseille-Luminy, Case 906, 13288 Marseille cedex 09, France, the
§ IBCP, CNRS UPR 412, 69 367 Lyon cedex 07, France, the
¶ CNRS, E.P.91, 13402 Marseille cedex 20, France, the
INSERM U491, Faculté de Médecine de la Timone,
Marseille, France, and the ** Institut Universitaire de France,
Paris, 75005 France
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-32P]dCTP using
Klenow enzyme and then utilized as radiolabeled probe.
thymoma cell lines were gifts of B. Malissen
(CIML); the mouse T cell line 2M2 (gift of J.-P. Kinet, Harvard,
Boston, MA), IIA1.6 B cell line, and P815 mastocytoma have been
previously described (20, 21). Mouse 3T3 fibroblasts, T end
(endothelial cell line), 3.19 (perivasal cell line), and 1D (thymic
epithelial cell line) were provided by P. Naquet (CIML). The mouse
fibroblast cell line LTK was a gift of M. D. Cooper and H. Kubagawa (Birmingham, AL). Mouse cell lines N2A (neuroblastoma) and
AZT20 (derived from a pituitary axis tumor) were provided by G. Rougon
(IBDM, Marseille, France). Total RNA was prepared using TRIzol (Life
Technologies, Inc.) according to the manufacturer's protocol.
Oligo(dT) primers and Moloney murine leukemia virus reverse
transcriptase were used for cDNA conversion in a total volume of 20 µl. PCR were performed in a 50-µl total volume with 5 µl of
cDNA template. Primer used were as follows: KARAP forward
(5'-GGCTCTGGAGCCCTCCTGGTGC-3') and KARAP reverse
(5'-CTGTGTGTTGAGGTCACTGTA-3'). PCR was performed as follows: 94 °C
for 1 min, 55 °C for 1 min, and 72 °C for 1 min for a total of 26 cycles. 
-Actin was used as a template control with the following
primers: -actin forward (5'-TACCACTGGCATCGTGATGGACT-3') and
-actin reverse (5'-TCCTTCTGCATCCTGCGGCAAT-3'). DNA was subsequently denatured in a NaOH 0.4% solution and transferred under alkaline conditions onto a Hybond N+ membrane. This membrane was hybridized with
mouse KARAP cDNA, previously labeled with
[-32P]dCTP and then revealed by autoradiography. For
-actin, a mouse -actin PCR product was labeled with
[-32P]dCTP and used as probe.
phage was biotinylated by
nick translation with biotin-16-dUTP according to manufacturer's protocol (Boehringer Mannheim). Hybridization to chromosome spreads was
performed with standard protocols (22). The biotin-labeled DNA was
mixed with hybridization solution at a final concentration of 10 µg/ml and used at 150 ng per slide. Before hybridization, the labeled
probe was annealed for 45 min at 37 °C with a 150-fold excess amount
of murine Cot-1 DNA (Life Technologies, Inc.) to compete with
nonspecific repetitive sequences. The hybridized probe was detected by
means of fluorescein isothiocyanate-conjugated avidin (Vector
Laboratories). Chromosomes were counterstained and R-banded with
propidium iodide diluted in anti-fade solution at pH 11.0.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
RI, and Fc
Rs) (24). Third,
KARAP co-precipitates with a protein-tyrosine kinase activity and is tyrosine-phosphorylated, a critical signaling feature shared with ITAM-bearing polypeptides. We reasoned that these biochemical characteristics would enable us to elaborate a bio-informatic strategy
in an attempt to identify the KARAP cDNA. The "select_hits" program was then generated as indicated under "Materials and
Methods" and applied on ESTs. As reported in Table
I, 467 potential candidates were
detected. Among these hits, 131 were discarded because they present
significant similarity with protein sequences of known function. On the
subset of 336 hits with unknown function, we subsequently searched for
the most similar entries with the CD3
and FcRI polypeptides by
using the FASTA program set on default parameters (25). The most
similar entry to CD3 and FcRI was the mouse EST AA734769.
Table I indicates that four other ESTs were found to share high
pairwise similarities (at least 96% identity) with AA734769. PCR
primers were generated to obtain the full-length coding sequence
corresponding to AA734769 from mouse spleen RNA. The nucleotide
sequence corresponding to this PCR product revealed a 342-bp open
reading frame (Fig. 1). The predicted
amino acid analysis indicated the features of a type I transmembrane
protein, including a 27-amino acid leader peptide (Met
27
to Ala1), a 16-amino acid extracytoplasmic domain
(Gln1 to Gly16), a 24-amino acid transmembrane
domain (Val17 to Gly40), and a 47-amino acid
long intracytoplasmic domain (Arg41 to Arg87).
Consistent with our bio-informatic search strategy, the predicted mouse
KARAP is a 9.6-kDa molecular mass mature polypeptide that contains
N-terminal cysteine residues, a charged transmembrane amino acid
(Asp25), and a typical ITAM based on Tyr65 and
Tyr76: Y65QELQGQRPEVY76SDLN.
Computer-based sequence alignment revealed a 73% amino acid identity
of the AA734769 predicted protein with the recently described human
DAP-12 ITAM polypeptide, which was found to associate with KARs
(KIR2DS2) in transfected cells (26). A rabbit antiserum (KP1) was
raised against an amino acid stretch present in human DAP-12
intracytoplasmic domain
(I57TETESPYQELQGQRSDVYSDLNTQR81), which is
highly homologous to that present in AA734769
(I57AETESPYQELQGQRPEVYSDLNTQR81).
Immunoblotting of anti-KAR immunoprecipitates prepared from human
interleukin-2 activated NK cells revealed a 12-kDa band reactive with
KP1 antibodies (data not shown). Taken together, these results show
that DAP-12 corresponds to human KARAP and that AA734769 corresponds to
mouse KARAP.
Bio-informatics search results
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Fig. 1.
Nucleotide sequence and amino acid sequence
corresponding to mouse KARAP cDNA. One-letter codes are used
for the amino acid translation of EST AA734769. Lengths of various
KARAP domains are: leader peptide (dash), 27 amino acids;
extracellular region (double line), 16 amino acids;
transmembrane region (single line), 24 amino acids;
intracytoplasmic region, 47 amino acids. The dot indicates a
stop codon.
Phe point mutants of mouse KARAP, mKARAP Y65F
and mKARAP Y76F, as well as of human DAP-12, hDAP-12 Y75F, are unable
to couple KAR engagement to the signaling machinery that drives RBL
serotonin release (Fig. 2A). In parallel, no phosphorylation of mKARAP Y65F (Fig. 2B), mKARAP Y76F, or hDAP-12 Y75F (data
not shown) could be detected in anti-KAR immunoprecipitates prepared from RBL transfectants upon pervanadate stimulation. Of interest, a
50-60-kDa tyrosine-phosphorylated protein was repetitively observed to
selectively co-precipitate with mKARAP Y65F, mKARAP Y76F, or hDAP-12
Y75F but not with wild type mKARAP or hDAP-12 (data not shown).
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Fig. 2.
Reconstitution of KAR activating function in
RBL transfectants in the presence of mouse KARAP or human DAP-12.
A, indicated stable transfectants were incubated 1 h at
37 °C either with mouse IgE (2682-I, 1:100 dilution of straight
hybridoma supernatant, stippled bars), anti-KAR mAb (GL183,
10 ng/ml, filled bars), or medium alone (open
bars). After being washed, cells were challenged for 30 min at
37 °C with 50 µg/ml goat anti-mouse antisera F(ab')2,
and serotonin released in supernatants was measured. Cytometric
analysis of all RBL transfectants revealed comparable cell surface
expression of KAR and Fc RI using anti-KAR (GL183) and
anti-FcRI (BC4) mAb, respectively. B, indicated stable
transfectants were stimulated (+) or not (
) using pervanadate. Cell
lysates (15 × 106 cells/sample) were separated on a
13% SDS-PAGE under reducing conditions after immunoprecipitation using
indicated mAb, transferred to Immobilon P (Millipore), and
immunoblotted using anti-phosphotyrosine mAb (4G10). GL183 mAb was used
for anti-KAR immunoprecipitations and a mouse anti-V8.2 mAb (F23.2,
IgG1) was used as a negative control mAb (c).
phage library with mouse KARAP cDNA. This
KARAP phage served as a probe for determining the chromosomal
localization of the mouse KARAP gene using fluorescence
in situ hybridization. A total of 50 metaphase cells were
analyzed, and 90% of them showed specific fluorescent signal in the B
band of murine chromosome 7. These results are consistent with the
localization of human DAP-12 to chromosome 19q13, a region
syntenic to mouse chromosome 7. The structure of the mouse
KARAP gene was then determined by generating a set of
primers spanning the corresponding cDNA sequence (Fig. 3A). As shown in Fig.
3B, the mouse KARAP gene spans 3.56 kilobases (from the start methionine residue to the stop codon) and is divided into 5 exons of varying length. The leader sequence is encoded by exon
1 and exon 2, the latter being a mini-exon of only 28 bp. Similar
features have been observed in KIR genes, as well as in
FcR genes, irrelevant of their presence on chromosome 19, such as FcR, or on chromosome 1, such as
FcRIa and FcRs (27). The first two amino
acids of the extracytoplasmic domain are also encoded by exon 2. The
rest of the extracytoplasmic domain, the transmembrane domain as well
as the first eleven amino acids of the intracytoplasmic domain are
encoded by exon 3. As a typical ITAM-bearing molecule, KARAP
gene harbors two exons, exon 4 and 5, which encode the ITAM and are
separated by a type 0 intron (11). All intron/exon boundaries for the
KARAP gene have classical donor/acceptor motifs GT-AG except
for intron 1, which appears to harbor a TA donor site (Fig.
3A).
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Fig. 3.
Genomic organization of mouse KARAP
gene. A, summary of exon/intron boundaries
showing flanking nucleotide sequences. Numbers above three-letter code
amino acids indicate the position of the residues flanking exon/intron
boundaries. B, schematic organization of mouse
KARAP gene. Untranslated exon regions are indicated in
hatched boxes, translated exons are indicated in
filled boxes, and introns are indicated in open
boxes. Lengths of KARAP exons (E) and introns
(I) are: E1 > 60 bp, I1 = 440 bp, E2 = 28 bp, I2 = 135 bp, E3 = 147 bp, I3 = 981 bp, E4 = 44 bp, I4 = 1666 bp, and E5 > 63 bp. Intron types are indicated
between exons. Correspondence with the schematic organization of the
mouse KARAP protein is also depicted. Amino acid lengths of each KARAP
protein domain are indicated in parentheses. LP, leader
peptide; EC, extracellular domain; TM,
transmembrane domain; IC, intracytoplasmic domain.
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Fig. 4.
Transcription pattern of mouse KARAP
gene. Reverse transcription-PCR analysis was performed on total
RNA extracted from indicated mouse cell lines using specific mouse
KARAP primers (upper panel) or -actin primers
(lower panel) as a positive control of reverse
transcription. After transfer, blots were revealed using the mouse
KARAP full-length cDNA (upper panel) or a -actin
probe (lower panel).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and FcRI than to any other ITAM-bearing molecule. CD3, CD3,
CD3, Ig, and Ig polypeptides harbor an Ig-like
extracytoplasmic domain, whereas FcRI spans four times the
membrane. In contrast KARAP/DAP-12 as well as CD3 and FcRI are
single transmembrane pass polypeptides that express a very short
extracytoplasmic domain (5 amino acids for FcRI, 9 amino acids
for CD3, and 16 amino acids for mouse KARAP/DAP-12), suggesting the
absence of a specific extracytoplasmic ligand. CD3 and
FcRI are both present on mouse and human chromosome 1, whereas KARAP/DAP-12 are present on mouse chromosome 7 and
human chromosome 19. It is thus tempting to speculate that genes
encoding for ITAM-bearing molecules and their associated receptors
might have originally evolved from the same locus, as FcRs, FcRI, and FcRI as
well as KARAP/DAP-12 and KARs are respectively
present in a close chromosomal vicinity.
, FcRI, and other ITAM-bearing polypeptides have been shown
to be involved both in the assembly and in the transducing properties
of oligomeric complexes (11, 12). In contrast, the cell surface
expression of KARs appears independent of KARAP/DAP-12 association in
stable transfectants of RBL and Ba/F3 cell lines (16, 26). However,
KARAP/DAP-12 also form noncovalent complexes with various activating
counterparts of ITIM-bearing molecules, such as the lectin-like MHC
class I receptors, NKG2C/CD94 heterodimers in human as well as Ly-49H
and Ly-49D homodimers in the mouse (29-31). The cell surface
expression of these lectin-like receptors requires association with
KARAP/DAP-12 for efficient cell surface expression (30, 31). It thus
appears that KARAP/DAP-12 is required for the stable cell surface
expression of lectin-like dimers, in contrast to Ig-like molecules. The
expression of KARAP/DAP-12 in CD3-deficient T cell lines, BW and
2M2, reveals that KARAP/DAP-12 cannot substitute for CD3 or
FcRI for the assembly of the TCR complex, because KARAP/DAP-12
association with the TCR components would have restored its cell
surface expression (21). Reciprocally, the absence of KAR function in
RBL-KAR transfectants as well as analysis of anti-KAR
immunoprecipitates have shown that despite its transmembrane charged
amino acid residue, KAR cannot associate with FcRI in RBL cells
(16). Therefore, the selective interaction between KARAP/DAP-12 and its
associated receptors occurs through the interaction between charged
transmembrane amino acid residues (26, 29-31) but is ensured by
specific amino acid interaction motifs yet to be determined.
single
point mutants has revealed that Ig tyrosine phosphorylation is
dependent on the presence of both Ig ITAM tyrosines (36). Therefore, it is possible that for some ITAM-bearing molecules, such as
KARAP/DAP-12 and Ig, the phosphorylation of ITAM tyrosine residues
is a sequential process. In this model, upon engagement of the
receptor, a SH2-bearing protein-tyrosine kinase (e.g. Lck,
Fyn, and Lyn) phosphorylates a first ITAM tyrosine, which in turn
interacts with the protein-tyrosine kinase through its SH2 domains,
allowing the efficient phosphorylation of the second ITAM tyrosine
residue. Such a mechanism is consistent with the analysis of the
interaction between Lck and CD3 (37, 38), as well as between the
SH2-bearing protein-tyrosine kinase Hck and phosphorylated peptides
(39).
molecules and KARAP/DAP-12 in the brain is suggestive of their
association (40). However, the distribution of ZAP-70 and Syk has been
reported to be restricted to the hematopoietic compartment (33).
Therefore effector/adaptor molecules distinct from ZAP-70 and Syk might
associate with phosphorylated KARAP/DAP-12 ITAMs. Along this line, the
in vivo association of mouse and human KARAP/DAP-12 Y-F
point mutants with a 50-60-kDa tyrosine-phosphorylated protein (data
not shown) suggests that mono-phosphorylated KARAP/DAP-12 ITAM might be
involved in the transduction pathways engaged by
KARAP/DAP-12-associated receptors. In this regard, a single Tyr Phe
point mutation of Ig ITAM C-terminal tyrosine can support the
propagation of partial signals including p62 tyrosine phosphorylation
and reduced calcium mobilization (36). Therefore, it is tempting to
speculate that upon engagement of receptors associated with
ITAM-bearing polypeptides (i.e. not only activating isoforms
of ITIM-bearing polypeptides such as KARs, SIRP-, PIR-A, ILT-1,
NKG2C/CD94, Ly-49D, and Ly-49H molecules but also TCR, BCR, FcRI,
and FcRIII complexes), mono-phosphorylated form of ITAM-bearing
molecules can support the propagation of a partial activation sequence
(37). The extent of ITAM phosphorylation might integrate the
"strength" of receptor engagement, which is a reflection of the
receptor-ligand affinity (36). Because physiological ligands of
KARAP/DAP-12-associated receptors are still not fully characterized,
the implications of KARAP/DAP-12 phosphorylation patterns remain to be unveiled.
ACKNOWLEDGEMENTS
FOOTNOTES
Member of the Institut Universitaire de France. To whom
correspondence should be addressed: Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288 Marseille cedex 09, France. Tel.:
33-4-91269444; Fax: 33-4-91269430; E-mail:
vivier{at}ciml.univ-mrs.fr.
REFERENCES
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Abstract
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References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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