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*
Centre for Molecular Medicine and Therapeutics, British Columbia Research Institute for Children’s and Women’s Health, and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada; and
Department of Infectious Diseases, Washington University School of Medicine, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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CT SHPS-1), resulted in the rapid formation of
macroscopic Ba/F3 cell aggregates. As the integrin-associated
protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we
investigated whether CD47 played a role in the aggregation of
SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate
formation was inhibited by an anti-CD47 Ab. Furthermore,
erythrocytes from control, but not from CD47-deficient mice, were able
to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes
do not express integrins, this result suggested that SHPS-1-CD47
interactions can take place in the absence of a CD47-integrin
association. We also present evidence that the amino-terminal Ig domain
of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47
binding likely triggers bidirectional intracellular signaling
processes, these results demonstrate that this interaction can also
mediate cell-cell adhesion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1/SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP)
substrate-1), were initially identified based on their ability to
become tyrosine phosphorylated and to interact with the
SH2-domain-containing protein-tyrosine phosphatase SHP-2 following
activation of growth factor-receptor protein-tyrosine kinases (1, 3). SHPS-1, for example, was cloned from insulin-stimulated rat
fibroblasts (1, 3). Other groups have also identified
SIRPs based on the ability of these molecules to function in neural
cell adhesion (P84, BIT), as macrophage fusion receptors, and as Ag
presentation-stimulating molecules (4, 5, 6, 7).
SHPS-1 encodes a 65-kDa protein that is heavily glycosylated, giving
rise to a variety of isoforms that differ in m.w. according to the cell
line or tissue type examined (1, 3). The extracellular
region of SHPS-1 consists of an amino-terminal Ig variable (V) type
domain and two Ig constant (C) type domains (3, 6, 8). The
112-aa intracellular domain of SHPS-1 contains four tyrosine residues
that include phosphorylation sites that are evident following
stimulation of cells with growth factors and other stimuli, including
platelet-derived growth factor, epidermal growth factor, insulin,
CSF-1, or lysophosphatidic acid (1, 3, 9, 10, 11, 12, 13, 14). Two of the
potential phosphorylation sites lie within ITIMs, the latter being
characterized by the consensus sequence I/LxYxxL/V. ITIMs, an essential
component of the inhibitory-receptor superfamily, are found within the
cytosolic domains of a variety of molecules, including killer cell
Ig-like receptors, C-type lectin inhibitory receptors, paired Ig-like
receptors, Fc
RIIB, and CD22 (15). Although SHPS-1 may
be directly phosphorylated by activated receptor protein-tyrosine
kinases, the presence of Src family kinases has been shown to be
necessary for SHPS-1 phosphorylation following integrin clustering, and
Src kinases were able to phosphorylate SHPS-1 in vitro (13, 16). Phosphorylation of ITIM-associated tyrosine residues
enables the recruitment of phosphatases, such as SHP-1, SHP-2, or SHIP
(SH2 domain-containing inositol phosphatase), which in turn
dephosphorylate specific protein substrates involved in mediating a
variety of physiological effects.
An important step in understanding SHPS-1 function was the identification of a ligand for SHPS-1 (P84) in neural cells. Using a mouse brain cDNA library for expression cloning, CD47 (also known as the integrin-associated protein, IAP) was found to interact with the extracellular region of P84 (17). P84 is localized to sites of synaptogenesis, suggesting it might be involved in regulation of synapse formation or maintenance (6, 18). CD47 is a 50-kDa protein expressed on most mammalian cells (19, 20, 21), featuring one Ig-like domain in the extracellular region and five transmembrane domains with a short cytoplasmic tail.
Because SHPS-1 is highly expressed in cells of the macrophage lineage and at lower levels in other hematopoietic cells (5, 8, 22), we investigated the consequences of SHPS-1 expression in a pro-B-cell line, Ba/F3, that does not express detectable levels of endogenous SHPS-1 protein. Expression of SHPS-1 in Ba/F3 cells resulted in the rapid formation of large cell aggregates. Using several different experimental approaches, we present evidence that CD47 is a ligand for SHPS-1 on Ba/F3 cells, and that the amino-terminal Ig domain of SHPS-1 mediates this interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine pro-B cell line Ba/F3 was kindly provided by Dr.
J. W. Schrader (Biomedical Research Centre, University of British
Columbia) and cultured in Ba/F3 medium (RPMI 1640 (Life Technologies,
Gaithersburg, MD) supplemented with 10% FBS (Intergen, Purchase, NY)
and 20% WEHI-3 conditioned medium containing IL-3, 50 µM 2-ME, 50
U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies)) at
37°C in 5% CO2. The rat IgG2a anti-mouse
CD47 Ab (mIAP301) has been described (23, 24). The rat
IgG2a anti-mouse CD8 Ab (Ly-2, clone 53-6.7; PharMingen, San
Diego, CA) served as the isotype control. The mouse monoclonal
anti-Flag (M2) Ab was obtained from Sigma (St. Louis, MO).
Plasmids and plasmid constructions
The rat full-length SHPS-1 cDNA, having an introduced
carboxyl-ter-minal Myc epitope tag, was generously provided by
Takashi Matozaki (Kobe University School of Medicine, Kobe, Japan)
(3). To generate an extracellular epitope tag that would
enable FACS analysis of transfected populations, sequences encoding the
Flag tag were introduced into the SHPS-1 cDNA 3' of the
signal peptide. A sense primer "Bam" spanning a region 5' of the
ATG (5'-TCTCTCTGGATCCGCGCGCTCACCGCCGATC-3') and the
antisense primer "FL2" at the end of the signal peptide
(5'-AGAGAAGCTTGTCATCGTCGTCCTTGTAGTCCTTCAGTTCTTTCC
C-3') were used in the PCR (italics in the sequences denote the
restriction enzyme sites). The PCR product was digested with
BamHI/HindIII. The remaining sequence of SHPS-1
including the Myc tag was generated using the sense primer "H2"
(5'-CTCTAAGCTTGTGACTCAGGCTGACAAATCAGTGTC-3') and the
antisense primer "FFS"
(5'-GTGTGAATTCTCAGAGGTCTTC-3'). This PCR product was
then digested with HindIII/EcoRI and ligated with
the BamHI/HindIII fragment into
BamHI/EcoRI-digested pBluescript (Stratagene, La
Jolla, CA). The BamHI/EcoRI SHPS-1 fragment was
excised and ligated into BamHI/EcoRI digested
pCDEF (Clontech, Palo Alto, CA) expression vector. To generate the
cytoplasmic deletion SHPS-1 mutant construct (CT SHPS-1) the sense
primer "Bam" and the antisense primer "FL2" were used for PCR
to introduce the Flag-epitope tag behind the signal sequence.
The sense primer "H2" was used with the antisense
primer "Eco" within the cytoplasmic region proximal to the
transmembrane domain (5'-CCTGAATTCGATTCGGAGGAGGTAGAG-3')
for PCR to generate the remaining portion of the SHPS-1 lacking
amino acids 400–509. The "Bam/FL2" PCR product digested with
BamHI/HindIII was ligated with the "H2/Eco"
PCR product digested with HindIII/EcoRI into
BamHI/EcoRI digested pCDEF-Myc vector. The
pCDEF-Myc vector was generated by introducing a Myc-epitope tag
followed by a stop codon into the pCDEF polylinker. This was
accomplished by annealing a sense oligonucleotide
(5'-AATTCGAACAGAAGCTGATATCGGAAGAAGACCTCTGACTGACTGACGC-3')
with an antisense oligonucleotide
(5'-GGCCGCGTCAGTCAGTCAGAGGTCTTCTTCCGATATCAGCTTCT
GTTCG-3') and ligating the annealed product into
EcoRI/NotI-digested pCDEF. The sequences of both
Flag-tagged cDNAs were verified to be correct by DNA sequence
analysis.
Transfections and cell selection
A total of 1 x 107 Ba/F3 cells were washed once with PBS and resuspended in 800 µl of ZAP buffer (25 mM HEPES, 0.75 mM Na2HPO4, 140 mM KCl, 5 mM NaCl, 2 mM MgCl2, and 0.5% Ficoll 400) with 12 µg of PvuI-linearized plasmid, and electroporated at 300 mV and 960 µF using a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA). Cells were resuspended in 30 ml of Ba/F3 medium. Cells were allowed to recover for 20 min at room temperature before returning to the 37°C incubator. After 48 h cells were centrifuged, washed with PBS, and resuspended in Ba/F3 medium with 1 mg/ml geneticin (G418) (Life Technologies) selection and plated in 96-well plates. After 3–4 wk of selection, plates were screened using anti-Flag staining and FACS. Wells containing clones showing the highest fluorescence signals were then expanded and isolated by cell sorting (see below). To generate green fluorescent protein (GFP)-expressing Ba/F3 cells, 1 x 107 Ba/F3 cells were transfected with PvuI-linearized pCDEF vector containing the GFP cDNA. After G418 selection, Ba/F3 clones expressing high levels of GFP were isolated by cell sorting.
Flow cytometry and cell sorting
A total of 1 x 106 cells were washed
twice with FACS buffer (2% FBS in PBS) and resuspended in 100 µl of
FACS buffer. Cells were stained with 1 µg Ab (anti-Flag or
anti-CD47) for 1 h on ice then washed twice with FACS buffer
and incubated with goat anti-mouse IgG FITC for anti-Flag
stained cells, or goat anti-rat FITC for anti-CD47 stained
cells, for 30 min on ice. Cells were then washed twice with FACS buffer
and resuspended in 300 µl of FACS buffer before analysis on a FACSort
(Becton Dickinson, Mountain View, CA) flow cytometer equipped with
CellQuest software program (Becton Dickinson). For cell sorting,
1 x 107 cells were stained with
anti-Flag Ab, and cells showing greater than two log-fold shift in
fluorescence were gated for sorting. Approximately 1 x
105 cells were collected and resuspended in Ba/F3
medium with 1 mg/ml G418.
Immunoblotting
Ba/F3 cells transfected with the empty pCDEF vector or with this
vector containing wild-type (WT) SHPS-1 or the CT SHPS-1 deletion
mutant construct were washed once in PBS and lysed in Nonidet P-40
(NP-40) lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
2 mM EDTA, and 10% glycerol), 100 µg/ml PMSF, 1 µg/ml aprotinin, 1
µg/ml pepstatin, 1 µg/ml leupeptin, 40 µg/ml bestatin, 10 µg/ml
soybean trypsin inhibitor (Boehringer Mannheim, Indianapolis, IN), 10
mM sodium fluoride, 1 mM sodium vanadate, and 1 mM sodium molybdate.
After 30 min on ice, cell lysates were centrifuged at 10,000 x
g for 15 min. Lysates were then separated by SDS-PAGE, then
transferred to nitrocellulose membranes by electroblotting. Membranes
were blocked for 1 h in 5% BSA in Tris-buffered saline (TBS),
washed in TBS with 1% Tween 20 (TBST), and incubated with
anti-Flag M2 (10 µg/ml) for 1 h in TBS. The membrane was
washed with TBST and incubated with a HRP-conjugated secondary
anti-mouse Ab (Dako, Carpinteria, CA) for 1 h and washed with
TBST. Enhanced chemiluminescence (Amersham, Arlington Heights, IL)
detection was performed according to manufacturers instructions.
Ba/F3-RBC adhesion assay
Poly-L-lysine at 100 µg/ml (500 µl) was added to
wells of 24-well plates and incubated at room temperature for 1 h,
then washed twice with dH2O. Ba/F3 cells
transfected with vector alone, WT SHPS-1, or CT SHPS-1 were washed
twice with PBS and resuspended in PBS at 2 x
105 cells/ml; 500 µl of cells was then added to
the poly-L-lysine-coated wells. After a 15 min incubation
plates were centrifuged at 400 x g for 4 min, and
nonadherent cells were aspirated and the surface washed twice with PBS.
Blood obtained either from control or CD47-deficient (23)
C57BL/6 mice was collected in heparinized tubes and washed three times
with 1% BSA in PBS. RBCs were counted and resuspended in 1% BSA in
PBS at 3 x 106 cells/ml, and 500 µl of
3 x 106 cells/ml RBCs were added to the
immobilized Ba/F3 cells. Plates were incubated at room temperature for
15 min, then centrifuged at 400 x g for 5 min. RBCs
not bound were aspirated and the surface washed three times with PBS.
Immobilized cells were imaged using a CCD camera and Northern Eclipse
5.0 software (Empix Imaging, Mississauga, Ontario, Canada).
Generation of recombinant GST-V1SHPS-1 and cell adhesion assay
The amino-terminal V domain of rat SHPS-1 was tagged with Flag-epitope at the carboxyl terminus and fused to GST by PCR using the sense primer "G1" (5'-GGTGCGGATCCAAAGAACTGAAGGTGACT-3') and the antisense primer "G2" (5'-GTCGGAATTCTTGTCATCGTCGTCCTTGTAGTCGAGTACATAGAGCGT-3'). The amplified product was digested with BamHI/EcoRI and cloned in-frame into BamHI/EcoRI digested pGEX-2T vector (Pharmacia, Piscataway, NJ) to generate GST-tagged V1SHPS-1. The construct thus had GST fused in-frame with amino acids 32–147 of SHPS-1. The GST fusion construct was sequenced before protein isolation. Bacterial expression and fusion protein purification was performed as previously described (25). For the cell adhesion assay, 100 µg GST or GST-V1SHPS-1 protein was spotted on a 1 x 1 cm nitrocellulose filter under vacuum aspiration. The filter was washed with PBS and placed in wells of a 24-well plate with 4% BSA in PBS blocking solution for 30 min. Blood from control and CD47-deficient mice was collected in a heparinized tube and washed three times with PBS. Erythrocytes were resuspended at 1 x 107 cells/ml, and 500 µl of these washed cells was added to the filters. For the blocking experiments RBCs were preincubated with anti-CD47, or anti-CD8 at 20 µg/ml for 10 min. RBCs were then incubated with the filters for 15 min and then centrifuged 5 min at 400 x g. The supernatant was removed by aspiration and the filters washed three times with 1% BSA in PBS. Cells that remained bound to the filters were lysed by adding 100 µl of mouse red cell removal buffer (140 mM NH4Cl and 17 mM Tris (pH 7.2)). The lysis solution was transferred to a microtiter plate, and OD405 measured on a Bio-Kinetics microplate reader (Bio-Tek, Burlington, VT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the effects of SHPS-1 expression within a
hematopoietic cell line, we expressed SHPS-1 in the pro-B cell line
Ba/F3. This cell line lacks detectable endogenous SHPS-1 protein (Ref.
22 and our observations) and therefore provided a model
system where different SHPS-1 mutants could be evaluated. Both the
full-length SHPS-1 (WT SHPS-1) and a cytoplasmic deletion mutant with
amino acids 400–509 deleted (CT SHPS-1) were introduced into Ba/F3
cells and oligoclonal populations with high level expression
identified. Out of these populations, cells showing a two-log shift by
FACS analysis were sorted to obtain cell subpopulations having similar
levels of both proteins (Fig. 1
A). Cell surface expression
was confirmed by anti-Flag Ab staining and FACS analysis of the
transfected cells (Fig. 1B). During their growth, Ba/F3
cells expressing the full-length SHPS-1 displayed a spontaneous
aggregation phenotype that resulted in the formation of large cell
clusters (Fig. 1B). Cells transfected with a SHPS-1 cDNA
that lacked the Flag epitope tag at the amino terminus were also able
to form aggregates, demonstrating that this phenotype was not
Flag-dependent (data not shown). Aggregate formation occurred rapidly.
Thus, when aggregates were disrupted to yield a single cell suspension,
and then replated, SHPS-1-expressing cells, but not control Ba/F3
cells, were able to begin reaggregating within 1–2 h. Cells
transfected with empty pCDEF vector failed to aggregate, growing as a
single cell suspension like the parental Ba/F3 cells (Fig. 1B). The ability to form aggregates varied with SHPS-1
expression level, with cells expressing relatively low levels of SHPS-1
showing only a weak tendency to cluster (data not shown). The
expression of CT SHPS-1, the mutant lacking the intracellular domain
of this molecule, at levels similar to those for SHPS-1, also produced
Ba/F3 cell aggregation (Fig. 1B), indicating that this
process was likely independent of the SHPS-1 intracellular domain.
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Cell cluster formation could have been due either to a
SHPS-1-SHPS-1 (homophilic) interaction, or to a heterophilic
interaction between SHPS-1 and another molecule. To evaluate these
alternative possibilities, CT SHPS-1-expressing Ba/F3 cells were
co-cultured at a 1:1 ratio with Ba/F3 cells expressing the fluorescent
marker GFP for 24 h. As seen in Fig. 2A, the SHPS-1-transfected
cells produced clusters. When these were viewed under fluorescence,
GFP-expressing Ba/F3 cells were also evident within the clusters (Fig. 2B). The ability of SHPS-1-expressing Ba/F3 cell to include
GFP expressing cells within the aggregates, while not excluding the
possibility of a homophilic interaction, suggested that SHPS-1 might be
able to bind to another cell surface molecule on Ba/F3 cells.
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Jiang et al. (17) reported that SHPS-1 (P84) was one
of the ligands for CD47 in neural cells; therefore we examined Ba/F3
cells for CD47 expression, verifying that these cells express CD47 and
that expression levels of this molecule were not altered by SHPS-1
expression (data not shown). To investigate whether
SHPS-1-expressing Ba/F3 cell aggregation was due to an interaction
between SHPS-1 and CD47, the Ab mIAP301, known to block CD47-mediated
cell migration (23, 24), was added to the cells. At 50
µg/ml, mIAP301, but not an isotype control, was able to block
aggregation of SHPS-1-expressing cells (Fig. 3). The anti-CD47 Ab was effective at
preventing aggregation at the lowest concentration tested (1 µg/ml),
whereas the isotype control was ineffective, even at concentrations of
100 µg/ml (data not shown). As a further specificity control, the
anti-Flag mAb was unable to inhibit cell-cell aggregation of cells
expressing Flag epitope-tagged SHPS-1 (data not shown). This result
suggested that a heterophilic interaction was likely responsible for
aggregate formation of SHPS-1-transfected cells, and the result of a
specific SHPS-1-CD47 interaction.
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We evaluated whether SHPS-1 expression on the surface of Ba/F3
cells could mediate cell-cell adhesion with another hematopoietic cell
type, RBCs. The latter are known to express CD47 (20, 21).
RBCs from control mice were incubated with vector-alone transfected
Ba/F3 cells, or with cells expressing the CT SHPS-1 protein. In this
experiment, RBCs were able to form rosettes only with CT SHPS-1
expressing Ba/F3 cells (Fig. 4). RBCs
from control mice were also able to form rosettes with Ba/F3 cells that
expressed full-length SHPS-1 (data not shown). To determine whether
rosetting was due primarily to a SHPS-1-CD47 interaction, the assay was
also performed employing RBCs obtained from CD47-deficient mice. These
RBCs were unable to associate with CT SHPS-1-expressing Ba/F3 cells
(Fig. 4), indicating that RBC-Ba/F3 cell adhesion required the presence
of both SHPS-1 and CD47. Furthermore, as RBCs do not express integrins
(20), the results suggested that SHPS-1-CD47-dependent
cell-cell binding occurs independently of this class of cell adhesion
molecule.
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When RBCs from control mice were incubated with Ba/F3 cells
expressing a SHPS-1 mutant lacking the amino-terminal IgV domain, no
rosettes were obtained (data not shown). This suggested that the IgV
domain was responsible for mediating the association between SHPS-1 and
CD47. To investigate this further, we generated a Flag-epitope-tagged
SHPS-1 mutant that lacked the two membrane proximal IgC domains.
Interestingly, despite repeated attempts, we were unable to isolate
cells expressing this protein at levels (by FACS or by immunoblotting
of cell lysates) comparable to those of the other SHPS-1 proteins.
Thus, the ability of a GST-fusion protein (GST-V1SHPS-1) containing the
IgV domain of SHPS-1 (amino acids 32–147) to bind CD47-expressing RBCs
was evaluated. The GST or GST-V1SHPS-1 proteins were immobilized on
nitrocellulose filters and incubated with the RBCs obtained either from
control or CD47-deficient mice. Filters with immobilized GST-V1SHPS-1
incubated with control RBCs were visibly covered with these cells,
whereas GST control filters, and GST-V1SHPS-1 filters incubated with
cells from Cd47-/- mice
failed to retain as many RBCs and appeared blank on visual inspection.
Cells remaining associated with the filters were lysed to enable the
quantitation of RBC-derived hemoglobin by spectrophotometry (Fig. 5A). As further evidence of
the role of CD47 in the RBC adhesion process, GST-V1SHPS-1 binding of
RBCs was inhibited by preincubation of these cells with the
anti-CD47 Ab, but not with the isotype control (Fig. 5B). It is also notable that Ba/F3 cells transfected with a
SHPS-1 mutant lacking the amino-terminal IgV domain failed to aggregate
(data not shown), further suggesting that this domain is both necessary
and sufficient for the interaction between SHPS-1 and CD47.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, CD47 (IAP) was identified as a ligand for neural cell-expressed P84 (SHPS-1) (17). In this paper we have shown that CD47 is also capable of being a ligand for SHPS-1 in the hematopoietic cell line, Ba/F3. Interestingly, Ba/F3 cell aggregation was also observed in cells expressing a mutant of SHPS-1 that lacked the intracellular region of this molecule. This result suggested that SHPS-1 was unlikely to be acting via the induction of intracellular events, such as stimulation of an "inside-out" effect on integrins, to bring about the aggregate formation. However, given that CD47 has been shown to be closely associated with specific integrins (19, 28), it remained possible that the Ba/F3 cell aggregation might in part be due to an integrin-cell adhesion molecule interaction. Although this was not formally excluded in our study, the ability of SHPS-1-expressing Ba/F3 cells to bind CD47-expressing RBCs argues against this possibility. As RBCs do not express detectable integrins (20), we propose that a CD47-integrin complex is not required for CD47 recognition by SHPS-1, or even for the observed cell-cell adhesion that resulted in SHPS-1-induced Ba/F3 cell aggregation. The ability of the GST-V1SHPS-1 fusion protein to bind CD47-positive (but not CD47-negative) RBCs further supports the notion that SHPS-1-CD47-mediated aggregation can occur in the absence of integrins. Interestingly, our finding that the bacterially expressed amino-terminal SHPS-1 Ig-like domain was able to bind CD47-expressing RBCs also suggests that the recognition of CD47 by SHPS-1 may not be dependent on the glycosylation of this domain. Given the large number of SIRP isoforms that appear to be encoded by the human genome (1), it will be of considerable interest to determine whether CD47 is a universal ligand for this family, or alternatively, whether the sequence variations in the extracellular domains of the various isoforms specify interactions with ligands other than CD47.
Arguing that the SHPS-1-CD47 interaction leading to Ba/F3 cell aggregation was mediated by the amino-terminal IgV domain of SHPS-1, we found that a bacterially expressed GST-fusion protein containing this domain was capable of binding RBCs obtained from Cd47+/+, but not from Cd47-/- mice. Interestingly, Veillette et al. (22) identified an isoform of SHPS-1 in macrophages that lacked the two membrane-proximal Ig domains, raising the possibility that expression of this single Ig-like domain in this cell type might have a specific physiological role. Although the function of this particular isoform is as yet unknown, our results suggest it may indeed be capable of binding CD47. Another protein structurally related to SHPS-1, PECAM (platelet-endothelial cell adhesion molecule), also mediates both homophilic and heterophilic interactions via the amino-terminal Ig loop, a region that is required for the transendothelial migration of hemopoietic cells (29). Likewise, the finding that the amino-terminal Ig domains of both killer cell Ig-like receptor p58 and CD22 inhibitory receptors are responsible for binding to HLA-C allotypes and sialic acid ligands, respectively (30, 31), raises questions about the precise function of the membrane proximal IgC domains in these various molecules.
CD47 has been implicated in the regulation of both cell adhesion and migration; for example, it is required for postadhesive events in the transepithelial or transendothelial migration of neutrophils (32, 33, 34). In keeping with these results, mice rendered deficient in CD47 by gene targeting demonstrated an impaired ability of leukocytes to migrate into sites of infection (23). Because macrophages have also been shown to express SHPS-1, and SHPS-1-CD47 associations can lead to cell-cell association, it is plausible that SHPS-1 will be found to play an important role in adhesion-mediated migration of macrophages, or other hemopoietic cell types, across epithelial or endothelial cell layers. Furthermore, as CD47 was identified as a receptor for the extracellular matrix molecule thrombospondin (35, 36, 37), it would be of interest to determine whether SHPS-1-CD47 binding prevents CD47 binding to this angiogenesis inhibitor.
CD47 is able to function as a costimulatory molecule in the responses of T-lymphocytes to TCR stimulation (38, 39, 40). This appears to occur by at least two mechanisms. First, CD47 promotes the adhesion of T cells to APC. In support of this, COS-7 cells expressing SHPS-1 were able to bind CD4+ T cells (5). Second, CD47 also appears capable of regulating T cell function via intracellular signaling pathways initiated by CD47 interaction with ligands. Interestingly, like CD47, SHPS-1 has been shown to play a costimulatory role in Ag presentation. Thus, not only were dendritic cells expressing SHPS-1 more effective in stimulating T cell responses than were their SHPS-1-deficient counterparts, but SHPS-1-blocking Abs were capable of blocking APC-induced T cell activation (5). Thus, it is conceivable that direct interactions between SHPS-1 and CD47 may be critical to the normal T cell-dependent immune responses. As many CD47-dependent processes have been defined via the use of monoclonal anti-CD47 Abs capable of blocking or stimulating various cell responses (23, 24, 32, 33, 34, 38), it is plausible that binding of CD47 by SHPS-1 will be able to alter signaling pathways lying downstream of CD47. The converse is also possible; namely, that CD47 binding to SHPS-1 at regions of cell-cell contact will be found to regulate both the subcellular localization and tyrosine-phosphorylation of SHPS-1.
Note. While this manuscript was under review, Seiffert
et al. (41) demonstrated that hematopoietic cells were
able to bind recombinant SIRP protein, and that this was likely
mediated by a SIRP:CD47 interaction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frank R. Jirik, Centre for Molecular Medicine and Therapeutics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC, Canada V5Z 4H4. E-mail address:
3 Abbreviations used in this paper: SIRP, signal-regulatory protein; ITIM, immunoreceptor tyrosine-based inhibition motif; SHP, SH2 domain-bearing protein tyrosine phosphatase; SHPS-1, SHP substrate-1; CT SHPS-1, deleted cytoplasmic tail mutant of SHPS-1; IAP, integrin-associated protein; GFP, green fluorescent protein; WT, wild type.
Received for publication October 14, 1999. Accepted for publication January 12, 2000.
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 U. Gurok, C. Steinhoff, B. Lipkowitz, H.-H. Ropers, C. Scharff, and U. A. Nuber Gene Expression Changes in the Course of Neural Progenitor Cell Differentiation J. Neurosci., June 30, 2004; 24(26): 5982 - 6002. [Abstract] [Full Text] [PDF]
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 T. T. Chen, E. J. Brown, E. J. Huang, and W. E. Seaman Expression and Activation of Signal Regulatory Protein {alpha} on Astrocytomas Cancer Res., January 1, 2004; 64(1): 117 - 127. [Abstract] [Full Text] [PDF]
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 K. Tada, M. Tanaka, R. Hanayama, K. Miwa, A. Shinohara, A. Iwamatsu, and S. Nagata Tethering of Apoptotic Cells to Phagocytes through Binding of CD47 to Src Homology 2 Domain-Bearing Protein Tyrosine Phosphatase Substrate-1 J. Immunol., December 1, 2003; 171(11): 5718 - 5726. [Abstract] [Full Text] [PDF]
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 L. A. Maile, J. Badley-Clarke, and D. R. Clemmons The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells Mol. Biol. Cell, September 1, 2003; 14(9): 3519 - 3528. [Abstract] [Full Text] [PDF]
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M. E. Bamberger, M. E. Harris, D. R. McDonald, J. Husemann, and G. E. Landreth A Cell Surface Receptor Complex for Fibrillar beta -Amyloid Mediates Microglial Activation J. Neurosci., April 1, 2003; 23(7): 2665 - 2674. [Abstract] [Full Text] [PDF]
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H. O. Barazi, Z. Li, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Regulation of Integrin Function by CD47 Ligands. DIFFERENTIAL EFFECTS ON alpha vbeta 3 AND alpha 4beta 1 INTEGRIN-MEDIATED ADHESION J. Biol. Chem., November 1, 2002; 277(45): 42859 - 42866. [Abstract] [Full Text] [PDF]
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T. Yamao, T. Noguchi, O. Takeuchi, U. Nishiyama, H. Morita, T. Hagiwara, H. Akahori, T. Kato, K. Inagaki, H. Okazawa, et al. Negative Regulation of Platelet Clearance and of the Macrophage Phagocytic Response by the Transmembrane Glycoprotein SHPS-1 J. Biol. Chem., October 11, 2002; 277(42): 39833 - 39839. [Abstract] [Full Text] [PDF]
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K. Oshima, K. Machida, Y. Ichigotani, Y. Nimura, N. Shirafuji, M. Hamaguchi, and S. Matsuda SHPS-1: A Budding Molecule against Cancer Dissemination Cancer Res., July 15, 2002; 62(14): 3929 - 3933. [Abstract] [Full Text] [PDF]
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Y. Liu, H.-J. Buhring, K. Zen, S. L. Burst, F. J. Schnell, I. R. Williams, and C. A. Parkos Signal Regulatory Protein (SIRPalpha ), a Cellular Ligand for CD47, Regulates Neutrophil Transmigration J. Biol. Chem., March 15, 2002; 277(12): 10028 - 10036. [Abstract] [Full Text] [PDF]
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 H. Park, S. G. Park, J.-W. Lee, T. Kim, G. Kim, Y.-G. Ko, and S. Kim Monocyte cell adhesion induced by a human aminoacyl-tRNA synthetase-associated factor, p43: identification of the related adhesion molecules and signal pathways J. Leukoc. Biol., February 1, 2002; 71(2): 223 - 230. [Abstract] [Full Text] [PDF]
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Y. Liu, D. Merlin, S. L. Burst, M. Pochet, J. L. Madara, and C. A. Parkos The Role of CD47 in Neutrophil Transmigration. INCREASED RATE OF MIGRATION CORRELATES WITH INCREASED CELL SURFACE EXPRESSION OF CD47 J. Biol. Chem., October 19, 2001; 276(43): 40156 - 40166. [Abstract] [Full Text] [PDF]
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S. Latour, H. Tanaka, C. Demeure, V. Mateo, M. Rubio, E. J. Brown, C. Maliszewski, F. P. Lindberg, A. Oldenborg, A. Ullrich, et al. Bidirectional Negative Regulation of Human T and Dendritic Cells by CD47 and Its Cognate Receptor Signal-Regulator Protein-{alpha}: Down-Regulation of IL-12 Responsiveness and Inhibition of Dendritic Cell Activation J. Immunol., September 1, 2001; 167(5): 2547 - 2554. [Abstract] [Full Text] [PDF]
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 M. TICCHIONI, V. RAIMONDI, L. LAMY, J. WIJDENES, F. P. LINDBERG, E. J. BROWN, and A. BERNARD Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow FASEB J, February 1, 2001; 15(2): 341 - 350. [Abstract] [Full Text] [PDF]
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X. Han, H. Sterling, Y. Chen, C. Saginario, E. J. Brown, W. A. Frazier, F. P. Lindberg, and A. Vignery CD47, a Ligand for the Macrophage Fusion Receptor, Participates in Macrophage Multinucleation J. Biol. Chem., November 22, 2000; 275(48): 37984 - 37992. [Abstract] [Full Text] [PDF]
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R. A. Rebres, L. E. Vaz, J. M. Green, and E. J. Brown Normal Ligand Binding and Signaling by CD47 (Integrin-associated Protein) Requires a Long Range Disulfide Bond between the Extracellular and Membrane-spanning Domains J. Biol. Chem., September 7, 2001; 276(37): 34607 - 34616. [Abstract] [Full Text] [PDF]
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