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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4602-4611
The Human Poliovirus Receptor Related 2 Protein Is a New
Hematopoietic/Endothelial Homophilic Adhesion Molecule
By
Marc Lopez,
Mustapha Aoubala,
François Jordier,
Daniel Isnardon,
Sophie Gomez, and
Patrice Dubreuil
From INSERM U.119, Unité de Cancérologie et
Thérapeutique Expérimentales, Marseille, France; and the
Institut Paoli-Calmettes, Marseille, France.
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ABSTRACT |
We have recently described Poliovirus Receptor Related 2 (PRR2), a
new cell surface molecule homologous to the poliovirus receptor
(PVR/CD155). Both molecules are transmembrane glycoproteins belonging
to the Ig superfamily (IgSF). They contain 3 Ig domains of V, C2, and
C2 types in their extracellular regions that share 51% aa identity.
The PRR2 gene encodes two mRNA isoforms of 3.0 kb (hPRR2 [short
form]) and 4.4 kb (hPRR2 [long form]), both widely expressed in
human tissues, including hematopoietic cells. To further characterize
PRR2 expression during hematopoiesis and to analyze its function, we
have developed a monoclonal antibody (MoAb) directed against its
extracellular region (R2.477). PRR2 was expressed in 96% of the
CD34+, 88% of the CD33+, and 95% of the
CD14+ hematopoietic lineages and faintly in the CD41
compartment. Ectopic expression of both PRR2 cDNAs induced marked cell
aggregation. A soluble chimeric receptor construct with the Fc fragment
of human IgG1 (PRR2-Fc) as well as a fab fragment of the anti-PRR2 MoAb
(R2.477) inhibit aggregation. PRR2-Fc binds specifically to
PRR2-expressing cells. These results suggest that PRR2 is a homophilic
adhesion receptor. PRR2 was also expressed at the surface of
endothelial cells at the intercellular junctions of adjacent cells but
not at the free cellular edges. Homophilic interactions are associated
with dimerization of isoforms of PRR2 and lead to the tyrosine
phosphorylation of PRR2. Altogether, these results suggest that
homophilic properties of PRR2 could participate to the regulation of
hematopoietic/endothelial cell functions.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ADHESION MOLECULES play a fundamental
role in the regulation of different biological processes such as
embryonic development, neuronal ontogeny and physiology, immune
response, and hematopoietic differentiation. Most adhesion molecules
mediate cell/cell or cell/substratum interactions through heterophilic
mechanisms. In some cases, adhesion molecules interact with themselves
through homophilic mechanisms. Adhesion molecules are classified in
different families and, among them, the Ig superfamily (IgSF) includes
molecules involved in homophilic adhesion processes.1
We have recently cloned two new human genes encoding transmembrane
glycoproteins that contain 3 Ig domains of V, C2, and C2 types in their
extracellular regions. The encoded proteins are homologous to the
poliovirus receptor (PVR; CD155),2 and we named them PRR1
and PRR2 (poliovirus receptor related).3,4 The PVR and PRR2
genes are located in the same human chromosomal region (19q13); they
encode proteins sharing 51% identity in their extracellular regions.
The PRR1 gene is located on chromosome 11q23 and encodes protein
sharing 30% and 32% identity with the PVR and PRR2,
respectively.3 Among the three members of this family, PVR
and PRR2 proteins are more closely related.
At least four PVR transcripts, resulting from alternative splicing,
have been cloned: two transmembrane forms (PVR and PVR) and two
soluble forms (PVR and PVR ) with common Ig domain
sequences.5 Two different PVR monkey cDNAs (AGM1 and
AGM2) have been isolated and encode receptors to
poliovirus.6 Two murine PVR homolog (MPH) cDNAs have been
cloned, but the corresponding proteins do not bind
poliovirus.7,8 By cloning the human PRR2 gene, we showed
that the true human homolog of MPH was hPRR2 and not hPVR. MPH was
renamed mPRR2.4 Two hPRR2 cDNAs (hPRR2 [short form] and hPRR2 [long form]) were identified. These two cDNAs encode alternative forms of the same gene sharing identical extracellular regions but different transmembrane and intracytoplasmic regions. The
mPRR2 gene encodes also two glycoproteins, mPRR2 and mPRR2, sharing 69% and 64% identities with hPRR2 and hPRR2,
respectively, along the whole amino acid sequence, but only 51%
identity with PVR in the extracellular region. Finally, no cDNAs
encoding soluble forms of hPRR2 or mPRR2 have been identified.
In the present report, we demonstrate that hPRR2 is expressed at the
surface of hematopoietic cells of the myelo-monocytic and
megakaryocytic lineages. hPRR2 is also detected at the surface of
endothelial cells; its localization is restricted to intercellular borders. Both hPRR2 isoforms could mediate homophilic intercellular adhesion, confirming another recently published report.9
Homophilic adhesion mediated by PRR2 correlates with dimerization of
the molecule at the cell surface and phosphorylation of the long form on tyrosine residues. Altogether, these results suggest that hPRR2 is a
new homophilic adhesion molecule that could be involved not only in
cell-cell interactions, but also in the regulation of intracellular
functions through signal transduction.
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MATERIALS AND METHODS |
Cells.
The murine hematopoietic cell line Da-1 and the human erythroleukemic
cell line TF110 were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Murine interleukin-3 (IL-3; supernatant from transfected X63Ag8-653 myeloma) was added to
the Da-1 culture and 5 ng/mL of recombinant human
granulocyte-macrophage colony-stimulating factor (huGM-CSF; Sandoz,
Rueil Malmaison, France) was added to the TF1 culture. The
murine cell line expressing the CD28 antigen (DWT6.11) was cultivated
in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FCS.11 The recombinant CHO DG44 cell
line (kindly provided by Dr L. Chasin, Columbia University, New York,
NY) was cultivated in CHOSFMII serum-free medium (LifeTechnologies,
Grand Island, NY).
Adult bone marrow was aspirated from the posterior iliac crest of
healthy volunteers after informed consent had been obtained. ECV304
endothelial cell line (umbilical cord origin; ATCC, Rockville, MD) was cultivated in DMEM supplemented with 10% FCS.
Eahy924 cell line (kindly provided by Dr C.J. Edgell, University of
North Carolina, Chapel Hill, NC) was cultivated in DMEM
supplemented with 10% FCS and hypoxanthine aminopterin thymidine
(HAT) medium.12 Endothelial cells were
harvested from human umbilical cord vein (HUVEC) as previously
described.13 The cells were maintained in RPMI containing
20% FCS.
Antibodies.
Flow cytometric analyses were performed using fluorescein
isothiocyanate (FITC)-labeled monoclonal antibodies (MoAbs). CD34 (HPCA-2) was purchased from Becton Dickinson (Mountain View,
CA). CD33 (D3HL60.251), CD14 (RMO52), CD41 (P2),
glycophorin A (11E4B-7-6), and CD19 (J4.119) were purchased from
Immunotech (Marseille, France).
The MoAbs directed against PRR2 (R2.477 and R2.525) and PVR (PV.404)
were obtained in the laboratory and submitted at the VIth International
Workshop on Human Leukocyte Differentiation Antigens.14 Fab
fragments were obtained after papain digestion according to the
manufacturer's protocol (Pierce, Rockford, IL). The MoAb
against PRR2 (BC-12) was kindly provided by Dr Claude Clement
(Diaclone, Berançon, France).
Immunesera directed against the intracellular domain of PRR2
(anti-PRR2) and (anti-PRR2) were obtained after rabbit
immunization with C-terminal peptides SQLDGSLISRRAVYV and
SYQGKGFVMSRAMYV, respectively.
The PECAM-1/CD31 MoAb (clone JC/70A) was purchased from Dako (Glostrup,
Denmark).
The 4G10 MoAb (UBI; Euromedex, Souffelweyersheim, France)
was used in Western blotting to analyze the tyrosine phosphorylation status of PRR2.
Immunohistochemistry.
Immunodetection of PRR2 and PECAM-1/CD31 were performed on frozen
sections (5 µm) of human placenta using the R2.477 and the PECAM-1/CD31 MoAbs. Specimen were processed with the Universal Dako Kit
ChemMate according to the supplier's recommendations and
counterstained for 5 minutes in Harris hematoxylin and mounted in Dako
glycergel mounting medium.
Cell transfection.
The PRR2 and cDNAs were cloned in the LXSN vector and then
transfected in Da-1 cells. The extracellular region of PRR2 (aa 1 to
347) was cloned in frame with the Fc fragment of the human IgG1
sequence using the Cos Fc Link vector (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). CHO DG44 cells
were then transfected with PRR2-Fc plasmid to produce an Fc-tagged PRR2 soluble receptor.
Transfection of Da-1 and CHO DG44 cells was performed with Lipofectin
according to the manufacturer's protocol (Life Technologies, Inc).
Production and purification of soluble chimeric PRR2-Fc protein.
CHOSFMII medium was harvested every 4 days and stored at
 20°C. Approximately 1 L of supernatant was filtered and
loaded on a 5-mL Affigel protein A column according to the
manufacturer's protocol (Bio-Rad, Hercules, CA). After
washing, the PRR2-Fc protein was eluted with a 0.1 mol/L citrate
buffer, pH 3.5, concentrated, and dialyzed against phosphate-buffered
saline (PBS). Purification steps were monitored by enzyme-linked
immunosorbent assay (ELISA) using a sandwich revelation system composed
with coated antibody against human Fc (Sigma, St Louis,
MO) and biotinylated R2.477 antibody.
Purity and quality of the protein were controlled by gel
electrophoresis followed by silver gel staining (Bio-Rad). As shown in
Fig 6B, Western blot analysis of purified PRR2-Fc molecule showed a
molecular weight of 80 kD.
The B7.1-Fc protein, used as an irrelevant control, was kindly provided
by Dr R. Sweet (SmithKline Beecham Pharmaceuticals) and was produced
similar to PRR2-Fc.
Immunoprecipitation and Western blot analyses.
Cells (5 × 106) were lysed in ice-cold lysis buffer
containing 1% Triton X100, 10% glycerol, 0.1% sodium dodecyl sulfate
(SDS), 50 mmol/L HEPES buffer, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L
MgCl2, 1 mmol/L EGTA, 10 µg/mL aprotinin, 10 µg/mL
leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Orthovanadate (1 mmol/L) was added in the case of phosphotyrosine analysis. Cell lysates
were clarified by centrifugation at 13,000 rpm for 15 minutes at
4°C. They were incubated for 1 hour at 4°C with 5 µg of
R2.477 antibody and 50 µL of protein A Sepharose (Pharmacia, Uppsala,
Sweden). Immune complexes were washed three times with
cold lysis buffer, heated in SDS-sample buffer, separated by gel
electrophoresis, semi-dry transferred to polyvinylidene difluoride
membranes (Immobilon-P; Millipore, Boston, MA), and
immunoblotted with a 1:10,000 dilution anti-PRR2 immunesera.
PRR2-Fc protein was shown by immunoblotting using a
peroxidase-conjugated antibody directed against human Fc fragment
(Jackson ImmunoResearch, West Grove, PA).
Chemical cross-linking.
Before cross-linking, 5 × 106 Da-1/PRR2 aggregated
cells were dissociated by agitation and then incubated with 1 mmol/L of Bis (sulfosuccinimidyl) suberate (BS3) cross-linker (Pierce). This
concentration gives maximum cross-linking efficiency. After 15 minutes
at 14°C, the reaction was quenched by the addition of 10 mmol/L
ammonium acetate. Cells were washed twice with PBS and counted to
confirm that no aggregation of suspended cells had occurred. Cells
lysis, immunoprecipitation, and Western blot analyses were performed as
described in the previous paragraph.
Aggregation assays.
Experiments were performed as previously described.15
Briefly, Da-1/PRR2 cells were suspended in complete medium at 1 × 106 cells/mL and transferred to polystyrene tubes.
Incubation was performed at 37°C and aliquots were taken every 15 minutes after gentle mixing. The number of aggregates (>3 cells) were
counted in a hemocytometer. Mixed-cell aggregation experiments were
performed with Da-1 cells stained with 0.5 µmol/L of CM-DiI dye as
recommended by the manufacturer (Molecular Probes, Eugene,
OR) and Da-1/PRR2 cells at a 1:1 ratio.
PRR2-Fc binding assays.
Three different populations of Da-1/PRR2 cells were sorted according to
the membrane levels of PRR2 and further put into culture. After 10 days, cells were controlled for the expression of PRR2 and the three
populations were named Da-1/PRR2 lo (low), Da-1/PRR2 me (medium), and
Da-1/PRR2 hi (high).
Each population of Da-1/PRR2 cells was analyzed in a test of PRR2
soluble receptor binding. Briefly, Da-1/PRR2 cells (1 × 105) were washed three times with cold PBS/bovine serum
albumin (BSA) and incubated for 1 hour at 4°C with 40 µg/mL of
PRR2-Fc protein. After two washes with the PBS/BSA buffer, cells were
incubated for 30 minutes at 4°C with a 1:60 dilution of
phycoerythrin-labeled goat antihuman IgG (Immunotech). Cells were
washed three times in the PBS/BSA buffer, and pellets were resuspended
in 200 µL of PBS. PRR2-Fc binding was analyzed using a FacScan
cytometer (Becton Dickinson).
Adhesion assays.
Experiments were performed as previously described.16
Briefly, 3.5-mm tissue culture Petri dishes were first coated with methanol-solubilized nitrocellulose and then with polyornithin (1.5 µg/mL; Sigma). Three-microliter spots of different soluble proteins
(PRR2-Fc or B7-1-Fc) at concentrations of 200 µg/mL were applied in
triplicate to the nitrocellulose/polyornithin-coated surfaces and
incubated for 12 hours at 37°C in a humidified atmosphere. Spots
were washed three times with Ca2+- and
Mg2+-free PBS. Cells were then incubated for 15 minutes at
37°C, washed three times with PBS, and then fixed with PBS
containing 2% formaldehyde (Fluka, St Quentin Fallavier,
France). Cell adhesion was analyzed by microscopy and
photomicrographs were taken at the edges of the spots of soluble
proteins.
Confocal immunofluorescence analysis of PRR2 expression.
HUVEC were cultured on coverslips, fixed with 3%
paraformaldehyde for 20 minutes, and indirectly stained with the R2.477
MoAb and goat antimouse Ig conjugated to FITC (Immunotech). Serial optical sections were collected using a TCS 4D Leica laser scanning confocal microscope (Heidelburg, Germany). Microscope
settings were adjusted so that black level values were obtained with a mouse IgG1 isotypic control.
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RESULTS |
PRR2 expression in hematopoiesis.
Northern blot analysis has shown that PRR2 is expressed in various
tissues, including bone marrow cells.4 This approach does
not allow us to define precisely which hematopoietic cells express
PRR2. We therefore developed MoAbs against PRR2 molecules. FACS
analysis of bone marrow cells demonstrated that PRR2 was expressed in
96% of the progenitor compartment defined by the expression of the
CD34 antigen (Fig 1A). Similar results were obtained with CD34+ cells purified from mobilized
peripheral blood (data not shown). PRR2 expression was also detected in
the myelo-monocytic compartment, ie, in 88% of CD33+ and
in 95% of CD14+ cells. PRR2 was also expressed at a low
level in the megakaryocytic lineage (50%). Expression of PRR2 on
CD19-expressing cells was found to be negative or low. No expression
was detected in the glycophorin A-expressing cells. Among peripheral
blood cells, PRR2 expression was predominantly detected on monocytes
(89%) and slightly on polymorphonuclear cells (data not shown).
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| Fig 1.
Expression of PRR2 in human bone marrow cells and
endothelial cells. (A) Bone marrow cells were doubled stained with the
indicated MoAbs and the MoAb R2.477 as described in Materials and
Methods. Fluorescence density plots were gated on specific regions
defined on scatter plot. The bottom-left quadrant was defined according
to an isotypic-matched control antibodies. The percentage represents
the number of PRR2-expressing cells in each lineage. (B) FACS analysis
of PRR2 and PECAM-1 expression in HUVEC. HUVEC were stained either with
the MoAb R2.477 or the MoAb anti-PECAM-1. Each fluorescence
distribution was superposed and compared with the background
fluorescence distribution of an isotypic-matched control antibody.
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PRR2 was expressed in greater than 98% of HUVEC. FACS analysis showed
that PRR2 expression is homogenous and weaker than PECAM-1 expression
(Fig 1B). PRR2 was equally expressed in HUVEC, EaHy926, and ECV304
cells (data not shown). The R2.477 MoAb was used in immunohistochemistry (IHC) on frozen sections of normal human placenta
to confirm that PRR2 was actually expressed in endothelial cells.
PECAM-1 expression was analyzed, as a control, by IHC in parallel on
serial sections. PRR2 immunostaining was detected in endothelial cells
from vessels located inside the placental villi
(Fig 2B) and was weaker than PECAM-1
immunostaining in the same vessels (Fig 2C). These differences in
expression levels are in agreement with the profiles of expression
detected by FACS analysis on cultured endothelial cells (Fig 1B). A
faint PRR2 immunostaining was also detected by IHC in trophoblastic
cells lining the placental villi (Fig 2B), indicating that PRR2
expression is probably not restricted to endothelial cells.
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| Fig 2.
Immunohistochemical detection of PRR2 in endothelial
cells. Frozen sections of human placenta were stained with either an
isotypic-matched control antibody (A), the R2.477 MoAb (B), or the
PECAM-1/CD31 MoAb (C) as described in Materials and Methods. Slides
were observed by light microscopy at 40× magnification.
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Biochemical characterization of the two PRR2 isoforms (PRR2 and
).
We previously identified two transcripts of 3.0 and 4.4 kb
corresponding to PRR2 and cDNAs. Cells of a murine hematopoietic cell line Da-1 were transfected with the PRR2 and cDNAs to analyze the proteins encoded by the two transcripts. Western blot analyses of Da-1/PRR2 and Da-1/PRR2 cells are represented in Fig 3A and B, respectively. After
immunoprecipitation of PRR2 with the R2.477 MoAb, a unique band of 64 kD was detected with the immuneserum directed against the C terminal
region of PRR2 in Da-1/PRR2 cells (Fig 3A, lane 2), but not in
control cells or Da-1/PRR2 cells. The immuneserum directed against
the C terminal region of PRR2 detected a 72-kD band in Da-1/PRR2
cells only (Fig 3B, lane 3). Identical results were obtained with the
R2.525 and BC-12 MoAbs (data not shown). Tunicamycin treatment of both transfectants reduced the molecular weight of both proteins,
suggesting that PRR2 is N-glycosylated (data not shown).
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| Fig 3.
Biochemical characterization of huPRR2 and PRR2.
Da-1 (1), Da-1/PRR2 (2), and Da-1/PRR2 (3) cell lines were lysed
and immunoprecipitated with MoAb R2.477 as described in Materials and
Methods. Blots were then incubated with a 1/10,000 rabbit immune serum
directed against PRR2 (A), PRR2 (B), or both (C). and isoforms of PRR2 were indicated and the calculated molecular weights
were 64 and 72 kD, respectively. The band at 50 kD corresponds to the
heavy chain of MoAb R2.477. Molecular weight markers were from Biolabs.
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PRR2 mediates intercellular homophilic adhesion.
Both Da-1/PRR2 and cells formed aggregates with a similar
phenotype, whereas control Da-1 cells did not
(Fig 4). To link cell aggregation with PRR2
expression, aggregation experiments were performed with Da-1/PRR2
and Da-1/PRR2 and compared with Da-1 cells
(Fig 5). For both transfectants, an
increase in the number of aggregates was observed as a function of
time, with a maximum reached between 30 and 45 minutes. Da-1 control
cells do not aggregate in the same conditions (Fig 5A). To precisely measure the contribution of both forms of PRR2,
aggregation experiments were performed in the presence of either the
soluble PRR2-Fc protein or the fab fragment of the MoAb R2.477. In both
cases, aggregation of transfectants was strongly decreased,
demonstrating that PRR2 mediates cell aggregation (Fig 5B). Mixed-cell
aggregation experiments between transfectants and CM-diI-stained Da-1
cells demonstrated that aggregates were exclusively formed with
unstained cells (data not shown). These results strongly suggest that
PRR2 mediates aggregation via a homophilic mechanism.
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| Fig 4.
Aggregation of PRR2 transfectants. Da-1 cells transfected
either with the LXSN vector (A), PRR2 (B), or PRR2 (C)
constructions were then plated on to 96 mutliwell plates. After 2 hours, aggregation was observed by light microscopy. Control cells did
not aggregate in these conditions.
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| Fig 5.
Aggregation assays of PRR2 transfectants. (A) Short-term
kinetic. Values are the mean ± SE for three independant experiments
and correspond to the number of aggregates (n > 3 cells) per
microliter. ( ) Da1/PRR2 cells; ( ) Da1/PRR2 cells; (×) Da1
cells. (B) PRR2-Fc (20 µg/mL) or fab fragment of R2.477 MoAb (20 µg/mL) inhibits aggregation. The number of aggregates was counted
after 60 minutes of incubation. ( ) Da1; () Da1/PRR2; ()
Da1/PRR2. The mock control corresponds to a murine irrelevant IgG1
(20 µg/mL).
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To demonstrate further the homophilic properties of PRR2, two
additional experiments were performed. First, PRR2-Fc binding was
analyzed by FACS analysis on three populations derived on the basis of
their level of PRR2 expression (Da-1/PRR2 lo, me, and hi and
Da-1/PRR2 lo, me, and hi). Expression analyses of Da-1/PRR2 lo,
me, and hi populations are represented in
Fig 6A. Their respective mean fluorescence
intensities (MFI) were 180, 395, and 750. As shown in Fig
6B, the binding of PRR2-Fc was proportional to the expression of
PRR2 at the cell surface, whereas the binding to the control LXSN
cells was lower than in transfected cells. Analysis of PRR2-Fc binding
as a function of PRR2 expression showed a linear relationship between
the respective MFI signals (Fig 6B). The differences of fluorescence
signal intensity between PRR2 expression and PRR2 binding could result
from a difference in affinities between the MoAb R2.477 and the PRR2-Fc
protein. Similar results were obtained with PRR2 transfectants.
Second, adhesion experiments of transfectants to coated PRR2-Fc plates were performed. Da-1/PRR2 hi and Da-1/PRR2 hi cells adhered specifically to PRR2-Fc but not to B7.1-Fc proteins used as a negative
control (Fig 7C through F). This binding
was independent of the presence of divalent cations (data
not shown). The CD28-transfected cells (DWT6.11) were used as a
positive control for B7.1-Fc-coated plates (Fig 7G and H).
Preincubation of Da-1/PRR2 hi cells with the fab fragment of the
MoAb R2.477 totally inhibited adhesion to PRR2-Fc-coated plates (data
not shown).
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| Fig 6.
PRR2-Fc soluble receptor binds to PRR2 transfectants. (A)
FACS analysis of Da-1 (continuous black) and Da-1/PRR2
lo (continuous gray), me (dashed black), and hi (dashed
gray) stained with R2.477 MoAb. (B) FACS analysis of these
transfectants stained with 2 µg of PRR2-Fc. Inserts show Western blot
analysis of the PRR2-Fc soluble receptor using a peroxidase-conjugated
goat antihuman Fc antibody and a plot corresponding to the MFI of
PRR2-Fc binding as a function of the MFI of PRR2 expression.
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| Fig 7.
Adhesion assays of PRR2 transfectants. Adhesion assays of
Da-1/PRR2 hi and Da1/PRR2 hi on either PRR2-Fc (A, C, E, and G)
or B7.1-Fc (B, D, F, and H) -coated petri dishes as described in
Materials and Methods. Adhesion was observed by light microscopy. No or
slight binding was observed with control Da-1 cells (A and B), whereas
Da-1/PRR2 hi adhered specifically on PRR2-Fc-coated petri dish (C
and D), similar to Da1/PRR2 hi (E and F). The CD28-expressing cells
adhere specifically on B7.1-Fc protein (G and H).
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PRR2 is expressed on endothelial cells.
Because PRR2 is a homophilic adhesion molecule expressed on endothelial
cells, the distribution of the molecule was investigated by confocal
immunofluorescence microscopy. The localization of PRR2 on cultured
HUVEC was compared with that of PECAM-1/CD31. Analysis of the
distribution in nonconfluent HUVEC showed that PRR2 expression
(Fig 8B), like that of PECAM-1/CD31 (Fig
8C), was concentrated at the intercellular borders of cultured cells but was absent at the free cellular edges. These observations suggest
that PRR2 could be involved in the physiological functions of vascular
endothelium as described for other homophilic adhesion molecules (for
review, see Dejana17).
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| Fig 8.
PRR2 expression on HUVEC by confocal microscopy. After
trypsinization, HUVEC were plated on coverslips for 6 hours.
Nonconfluent HUVEC were stained with an isotypic-matched control
antibody (A), the MoAb R2.477 (B), and the MoAb against PECAM-1/CD31
(C) and then with goat antimouse FITC antibody. Confocal acquisitions
were then performed at a 63× magnification.
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Dimerization and phosphorylation of PRR2.
As shown in Fig 3A and B, the 64- and 72-kD bands correspond to
monomeric forms of PRR2 and PRR2, respectively. To detect multimeric forms of PRR2, cell surface proteins were chemically cross-linked with BS3 before immunoprecipitation. As shown in Fig 9A, cell surface cross-linking of PRR2
transfectants showed additional bands at approximately 118 and 136 kD
that probably correspond to the dimeric forms of PRR2 and PRR2,
respectively. Because cross-linking was performed after dissociation of
aggregated cells, this suggests that the observed PRR2 homodimerization
results from cis-interactions within the membranes of
individual cells rather than from trans-interactions.
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| Fig 9.
PRR2 dimerization and phosphorylation. (A) Da-1/PRR2
me and Da-1/PRR2 me transfectants were immunoprecipitated with
PRR2 and PRR2 antisera, respectively. The ECV304 (B) and the TF-1
(C) cell lines were immunoprecipitated with the MoAb R2.477.
Immunoprecipitation was performed in the absence () or presence
(+) of BS3 cross-linker. Blots were then hybridized with a mix of
PRR2 and PRR2 immunesera. (D) Da-1/PRR2 hi cells were
immunoprecipitated with the PRR2 immuneserum and blot was first
analyzed with the 4G10 MoAb, stripped, and incubated with PRR2
immuneserum. Molecular weight markers were from Biolabs. / and
/ represent PRR2 and PRR2 homodimeric forms, respectively.
/ represents the PRR2 and PRR2 heterodimeric form.
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Dimerization was further investigated with ECV304 endothelial cells
that constitutively express PRR2 (Fig 1B). Two bands could be
identified with molecular weights similar to those found in Da-1/PRR2 and Da-1/PRR2 cells. They correspond probably to the two PRR2 isoforms (Fig 9B). After cell surface cross-linking, three
additional bands at 118, 128, and 136 kD were shown and are likely
homodimers and heterodimers. The 118- and 128-kD bands were
specifically shown with the anti-PRR2 serum, and the 128-and 136-kD
bands were shown with the anti-PRR2 serum (data not shown). Thus,
the intermediate 128-kD band corresponds to a heterodimeric PRR2/PRR2 form as it is recognized by both sera, whereas the 118- and 136-kD bands represent the homodimeric forms of PRR2 and
PRR2, respectively. Although these results were in agreement with
those presented in Fig 9A, it could be noted that, even after a
prolonged trypsinization (10 minutes), ECV304 cells were not completely
dissociated. Thus, in ECV304 cell line, we cannot exclude that PRR2
dimerization occurs exclusively through cis-interactions. Dimeric forms of PRR2 in ECV304 were only shown after a long-time exposure of Western blots, suggesting that they were less abundant than
in PRR2 transfectants. A relationship between PRR2 dimerization and
homophilic aggregation was further established by cross-linking experiments performed on the hematopoietic TF-1 cell line that also
expressed PRR2 but did not form aggregates during cell culture. As for
the ECV304 cell line, the two PRR2 isoforms were detected at similar
molecular weights, but no dimeric forms of PRR2 could be detected after
cross-linking, even after a long exposure time (Fig 9C).
Intracytoplasmic phosphorylation of adhesion receptors has been
frequently described in heterophilic and homophilic aggregation processes. Because the cytoplasmic regions of the two hPRR2 forms contain tyrosine residues (2 for hPRR2 and 8 for hPRR2), we tested a possible tyrosine phosphorylation in Da-1/PRR2hi and PRR2hi. Western blot analysis performed with an antiphosphotyrosine MoAb showed that PRR2 was strongly phosphorylated on tyrosine residues in both monomeric and dimeric forms (Fig 9D). No
phosphorylation was detected for PRR2 (data not shown).
| |
DISCUSSION |
We previously described PRR2, a new cell surface molecule homologous to
the poliovirus receptor (PVR/CD155). This molecule belongs to the IgSF
and is composed of three Ig domains of V, C2, and C2 types. Two cDNA
isoforms were cloned and both transcripts were ubiquitously found among
the various human tissues tested,4 including bone marrow
cells and HUVEC.
The present study demonstrates that, in hematopoiesis, PRR2 expression
is restricted to the myelo-monocytic and megakaryocytic lineages and
detected at the surface of the hematopoietic progenitor cells
expressing the CD34 antigen (Fig 1A). However, Aoki et al9 reported that the mouse homologue of PRR2 (mPRR2) was expressed in
greater than 90% of B cells purified from the spleen, and no expression was detected on monocytes, T cells, and NK cells. The discrepancy between our results and those reported by Aoki et al9 could be explained by the fact that analyses were
performed on cells isolated from different tissues. Alternatively, as
shown for other cell surface receptors, the human and murine PRR2
molecules could have different expression patterns. Recently, PRR2
expression was described on human monocytes.18
Western blot analysis of immunoprecipitated PRR2 isoforms showed two
bands of 64 and 72 kD corresponding to PRR2 and PRR2, respectively (Fig 3). The same molecular weights were observed with
TF-1 and ECV304 cells (Fig 9).
Transfection of either the short or the long form of PRR2 in the murine
hematopoietic cell line Da-1 induced aggregation of the cells in
culture (Fig 4). We demonstrate that both isoforms of PRR2 could
mediate adhesion by an homophilic mechanism (Figs 5, 6, and 7).
PRR2 isoforms form dimers at the surface of Da1/PRR2 cells, probably by
cis-interactions (Fig 9A). Interestingly, no dimeric forms of
PRR2 were detected in the TF-1 cell line (Fig 9C). This could explain
the failure of hematopoietic cells that constitutively express PRR2 to
form aggregates. Because the expression level of PRR2 in TF-1 is lower
than that in the transfectants and because the aggregation of Da1/PRR2
cells was directly correlated with the PRR2 expression level, we can
speculate that aggregation could be proportional to the cell surface
expression level of dimeric PRR2. The functional significance of this
dimerization is unknown. One can speculate that
cis-dimerization is correlated with cell surface expression
levels and that it contributes to strengthen adhesion, as suggested for
other adhesion molecules.19-21
Preliminary long-term culture experiments using a human bone marrow
stromal layer showed no differences in colony formation in the presence
or absence of the blocking MoAbs R2.477 (results not shown). The
contribution of PRR2 could be masked by the presence of many other
adhesion molecules expressed at the cell surface of the
CD34+ progenitors. Other homophilic receptors, such as the
CD66, which belongs to the carcinoembryonic family, and the ALCAM/HCA
antigen, expressed on hematopoietic cells, could mediate homophilic
interactions with no clear associated function.22,23 Only
the PECAM-1/CD31 molecule mediates homotypic homophilic interactions
between endothelial cells and heterotypic homophilic interactions
between leukocytes and endothelial cells leading to leukocyte
transendothelial migration.24 We showed that PRR2 is also
expressed at the surface of endothelial cells isolated from umbilical
cord (HUVEC; Fig 1B), on endothelial cell lines (ECV304 and EAHy924),
and on placental endothelial cells (Fig 2). Confocal microscopy showed
that PRR2 was concentrated at the intercellular borders of adjacent
endothelial cells (Fig 8B). No PRR2 concentration was observed at the
free edges of the cell layer. Because both isoforms of PRR2 could
homodimerize or heterodimerize at the cell surface of endothelial cells
(Fig 9B), we speculate that dimers are only formed at the cellular
junction, thus mediating homophilic interactions. Altogether, these
observations could suggest that PRR2 is involved in leukocyte
transendothelial migration.
Most of the adhesion receptors may also reflect interactions that are
important for signal transduction as described for the CD66 and
PECAM-1/CD31 antigens. These molecules can be tyrosine phosphorylated
in their cytoplasmic region.25,26 We observed that the long
form of PRR2 was phosphorylated on tyrosine residues when cells are
aggregated in culture (Fig 9D). At the present time, we do not know
whether phosphorylation is directly linked to the homophilic engagement
of the molecules either in an outside-in mechanism or, indirectly, via
other conjugated molecules in an inside-out mechanism.
As for CD66 and CD31, hPRR2 contains a YxxL sequence
in its cytoplasmic region. This sequence is conserved between hPRR2 and mPRR2 and could serve as recognition sequence for SH2 domains of
cytoplasmic kinases and phosphatases.
Whereas PRR2 belongs to a new family of at least three receptors
related to the PVR/CD155 molecule (PVR, PRR1, and PRR2) and expressed
in hematopoiesis,27 recent reports demonstrated that these
molecules were also receptors for herpes simplex
viruses.28,29
The identification of the role of this new antigen family is currently
under investigation and should contribute to the understanding of
cellular interactions in normal and pathological hematopoiesis.
| |
ACKNOWLEDGMENT |
The authors are grateful to Elisabeth Devilard, Dr Nathalie Bardin, and
Dr Véronique Francès for expert technical assistance and
helpful discussions. We acknowledge Drs Luc Xerri, Françoise Birg, and Claude Mawas for critically reviewing the manuscript.
| |
FOOTNOTES |
Submitted May 8, 1998;
accepted July 28, 1998.
Supported by INSERM, the Association pour la Recherche Contre le Cancer
(ARC), and the Ligue Nationale Française Contre le Cancer
(LNFCC).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Marc Lopez, PhD, INSERM U.119, 27, Bd
Leï Roure, 13009 Marseille, France; e-mail:
lopez{at}marseille.inserm.fr.
| |
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Abstract
Introduction