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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2800-2809
CD148 Is a Membrane Protein Tyrosine Phosphatase Present in All
Hematopoietic Lineages and Is Involved in Signal Transduction on
Lymphocytes
By
Miguel Angel de la Fuente-García,
Josep Maria Nicolás,
John H. Freed,
Eduard Palou,
Andrew P. Thomas,
Ramón Vilella,
Jordi Vives, and
Antoni Gayá
From the Servei d'Immunologia, Servei de Medicina Interna, Hospital
Clínic, Barcelona, Spain; the National Jewish Center for
Immunology and Respiratory Medicine, Denver, CO; and the Department of
Pathology, Anatomy and Cell Biology, Thomas Jefferson University,
Philadelphia, PA.
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ABSTRACT |
Evidence is presented showing that a protein tyrosine phosphatase
different from CD45 is present on the membrane of human hematopoietic
cells. The molecule recognized by the monoclonal antibody 143-41, which
has been classified as CD148 in the VI International Workshop on
Leukocyte Differentiation Antigens, was immunopurified and sequenced.
The sequence obtained from N-terminus as well as from two different
CNBr-digested peptides showed a close identity with a previously
described tyrosine phosphatase named HPTP- /DEP-1. CD148 is present
on all hematopoietic lineages, being expressed with higher intensity on
granulocytes than on monocytes and lymphocytes. Interestingly, whereas
it is clearly present on peripheral blood lymphocytes, it is poorly
expressed on different lymphoid cell lines of T and B origin. When this protein tyrosine phosphatase was cocrosslinked with CD3, an inhibition of the normally observed calcium mobilization was observed. This inhibition correlates with a decrease in phospholipase C- (PLC-) phosphorylation and is similar to the one observed with CD45. In
addition, it is shown that the crosslinking of the CD148 alone is also
able to induce an increase in [Ca2+]i. This
increase is abolished in the presence of genistein and by
cocrosslinking with CD45. These data, together with the induction of
tyrosine phosphorylation on several substrates, including PLC-, after CD148 crosslinking, suggest the involvement of a tyrosine kinase-based signaling pathway in this process. In conclusion, the data
presented show that CD148 corresponds to a previously described protein
tyrosine phosphatase HPTP-/DEP-1 and that this molecule is involved
in signal transduction in lymphocytes.
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INTRODUCTION |
THE PHOSPHORYLATION of tyrosine residues
of proteins is a crucial event in the regulation of cellular processes,
including those of proliferation and differentiation. The level of
protein phosphorylation is mainly the result of the antagonistic
functions of protein-tyrosine kinases (PTKs) and protein-tyrosine
phosphatases (PTPs).1,2 Thus, the activation and
inactivation of both enzymes are relevant in determining the functional
state of a great variety of intracellular molecules.
In recent years an increasing number of PTPs have been described.
Currently over 40 PTPs have been reported.3 They have been
subcategorized4 into three groups: (1) receptor-like PTPs, (2) intracellular PTPs, and (3) dual specific PTPs. The common structural features of the receptor-like PTPs include an extracellular domain of variable length and composition, a single membrane-spanning region, and one or two intracellular catalytic domains.
The interaction of T cell receptor (TCR) with the appropriate antigen
or its stimulation with antireceptor antibodies induces a signal
transduction cascade that leads to the expression of a number of genes
and eventually to effector functions. One of the earliest biological
events after lymphocyte stimulation is the activation of PTKs, which
results in tyrosine phosphorylation of various cellular proteins.
Recently it has been observed that the rapid, and generally transient,
tyrosine phosphorylation response is the result of a complex and still
poorly understood kinase cascade involving at least three families of
PTKs: src, syk, and csk (see Zenner et al5 for review).
Although leukocytes express a wide variety of PTPs in the cytoplasm,
their precise role remains unknown in most of the cases.6
Among the PTPs, CD45 is the only membrane PTP that has been described
to be involved in the process of signal transduction,7
modulating the response to antigen receptor engagement in both
T8 and B lymphocytes.9
In this report we present evidence that the recently described
CD14810 corresponds to a previously described membrane PTP, HPTPh/DEP-1.11-13 This molecule is present on all
hematopoietic lineages and, in addition to being able to transduce
signals by itself, it is also able to modulate the signal transduction
through the TCR/CD3 complex in a manner similar to CD45.
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MATERIALS AND METHODS |
Cells.
Blood samples were obtained from healthy adult donors. Peripheral blood
mononuclear cells (PBMCs) were isolated by centrifugation over
Ficoll-Hypaque (Pharmacia LKB, Uppsala, Sweden) density-gradient sedimentation. The following cell lines were grown in RPMI plus 10%
fetal calf serum (FCS): CEM, HPB-ALL, HSB2, JURKAT, MOLT-4, RAJI, KM3,
NAMALWA, RAMOS, NALM-6, K562, U937, and HL-60.
Monoclonal antibodies (MoAbs).
The following MoAbs were produced in our laboratory and ascribed to
their CDs through one of the International Workshop on Human Leukocyte
Differentiation Antigens (WLDA): CRIS-7 (CD3, IgG2a), 72-5D3 (CD45,
IgG2a), and 111-5A1 (CD41, IgG1). MoAbs were purified from ascitic
fluid by protein A affinity chromatography. The 143-41 (IgG1) hybridoma
was produced in accordance with a previously described
method14 after immunization of BALB/c mice with PBMCs that
had previously been stimulated with phytohemagglutinin (PHA) for 3 days. The following phycoerythrin (PE)-labeled MoAbs were used: HD-37
(CD19), Leu 4 (CD3), and mouse IgG2a control (Becton Dickinson, San
Jose, CA).
Immunofluorescence assay.
The 143-41 MoAb was labeled with fluorescein following conventional
techniques. Cells were washed with immunofluorescence buffer
(phosphate-buffered saline [PBS] containing 0.02 mmol/L sodium azide
and 1% bovine serum albumin [BSA]) and incubated with specific MoAb
or isotype-matched control MoAb for 30 minutes on ice in
immunofluorescence (IF) buffer containing 5% rabbit serum. For
two-color analysis the simultaneous combination of 143-41 fluorescein
isothiocyanate (FITC)-conjugated MoAb with a PE-conjugated MoAb, was
used. Samples were run on a FACScan flow cytometer (Becton Dickinson).
Where applicable, different cell populations (eg, lymphocytes,
monocytes, and neutrophils) were identified based on 2-dimensional
light scatter characteristics.
Surface biotinylation, immunoprecipitation, specific glycosidases
treatment, and immunoblotting.
Adult human peripheral blood mononuclear cells were isolated from
healthy donors by centrifugation over Ficoll-Hypaque. Cells were
prepared for surface biotinylation by washing twice in PBS and
resuspending at 5 × 107 cells/mL in PBS containing
200 µg/mL Sulfo-NHS-Biotin (Pierce, Rockford, IL). Labeling was
allowed to proceed for 30 minutes at 4°C and was quenched by
incubation for 15 minutes at room temperature with an equal volume of
RPMI 1640 medium supplemented with 10% FCS. Cells were then washed
three times in cold PBS and lysed. After 20 minutes on ice, postnuclear
extracts were added to CNBr-activated Sepharose 4B (Pharmacia LKB) that
had been previously coupled to 143-41 antibody and blocked with 2% BSA
(Sigma Chemical Co, St Louis, MO). After 60 minutes at 4°C,
immunoprecipitates were washed five times in lysis buffer (0.5%
Nonidet P-40, 10 mmol/L Tris-HCL, pH 7.40, 150 mmol/L NaCl, 1 mmol/L
EDTA, 1 mmol/L EGTA, 1 mmol/L NaF, 20 mg/mL egg white trypsin
inhibitor, 1 mg/mL leupeptin, 1 mg/mL pepstatin, 1 IU/mL aprotinin, and
1 mmol/L phenylmethyl sulfonyl fluoride [PMSF]) containing 0.05%
sodium dodecyl sulfate (SDS). For deglycosylation, samples of
immunoprecipitates were washed and, after boiling, resuspended in the
corresponding buffers prepared according to manufacturer's
instructions for Neuraminidase (150 mU/50 mL; Boehringer-Mannheim,
Mannheim, Germany), O-Glycosidase (1.5 mU/50 mL,
Boehringer-Mannheim) and recombinant N-glycosidase F (0.7 mU/100 mL,
Boehringer-Mannheim). Overnight digestions at 37°C were usually
used; however, for Neuraminidase treatment a shorter digestion time (2 hours) was used. The glycosidase-treated proteins were run on 5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to Immobilon-P (Millipore Corp, Bedford, MA) in 39 mmol/L glycine, 48 mmol/L Tris-base, 1.3 mmol/L SDS, and 5% methanol
for 2 hours at 60 V. After incubating the filters with blocking
solution (10% nonfat milk protein in PBS) for 2 hours at 4°C,
blots were incubated with streptavidin-peroxidase (Sigma Chemical Co)
at 1:2,000 in blocking solution for 1 hour at 20°C. Filters were
washed again with 0.1% Tween-20 in PBS, and the Western blots were
developed using substrate solution (0.6 mg/mL diaminobenzidine, 0.1%
hydrogen peroxide (30%), and 0.3% (wt/vol) CoCl2).
Immunoaffinity purification and protein sequencing.
The molecule recognized by 143-41 MoAb was purified following
previously described procedures15 with modifications. Buffy coats from normal healthy donors were obtained and erythrocytes separated by sedimentation in PBS-2% Dextran-500 (Pharmacia LKB). Leukocyte-rich supernatants were centrifuged, and about 20 × 109 white blood cells were obtained and washed twice in
PBS. Pellets were disrupted in lysis buffer for 30 minutes on ice.
Insoluble material was removed by centrifugation at 50,000g for
30 minutes at 4°C and the supernatant was precleared by passing it
sequentially through columns of CNBr-activated Sepharose-4B beads alone
and CNBr-activated Sepharose 4B beads coupled with polyclonal mouse Ig
or irrelevant IgG1 MoAb. The resulting lysate was then applied to a
column of CNBr-activated Sepharose 4B beads (15 mL) derivatized with
MoAb 143.41. The affinity column was washed extensively with modified
lysis buffer containing 0.05% NP40 and then with two column volumes of
PBS 0.5 mol/L MgCl2. The bound material was eluted with 4 mol/L MgCl2 in PBS and the sample concentrated to 150 µL
using a Centriprep 30 membrane (AMICON, Beverly, MA) while the buffer
was changed to PBS. Cyanogen bromide cleavage of the protein was
performed according to a previously described protocol15:
400 mg of protein were resolved with SDS-PAGE (5%) and electroblotted onto nitrocellulose membrane. After transfer, a band visualized by
Ponceau S (Sigma Chemical Co), was excised, transferred to a screwcap
microvial, and incubated in 300 µL of 0.150 mol/L CNBr (Pierce
Chemical Co) in 70% formic acid (vol/vol) (E. Merck, Damstadt, Germany) for 4 hours in the dark, at room temperature. After cleavage, membrane fragment was dried completely with N2 and washed
with 200 µL water and dried again. The protein fragments were
redissolved in Laemmli's sample buffer containing 5% mercaptoethanol,
and the SDS-PAGE for the separation of the peptides was 20% acrylamide (200:1, acrylamyde:bis), 10% (vol/vol) glycerol, 0.75 mol/L Tris pH
9.3, and 0.1% SDS. The gel was aged 2 days, and the running buffer
contained 0.1 mmol/L thioglycolate. After the run, the proteins were
blotted onto polyvinylidine difluoride membrane (ProBlott; Applied
Biosystems, Inc, Foster City, CA) with transfer buffer (48 mmol/L Tris
pH 9, 39 mmol/L Tricine, 1.3 mmol/L SDS, and 20% methanol). SDS-PAGE
molecular weight standards (Bio-Rad Laboratories, Richmond, CA) were
used. The membrane was stained with AmidoBlack (0.1% in 40% methanol,
1% acetic acid), washed thoroughly in H2O, and dried. Two
bands were cut out and stored in microvials filled with N2.
NH2-terminal sequence analysis of the intact protein and
the two protein fragments was performed on an Applied Biosystems
470A/120A microsequencer.
Phosphatase assay.
Substrate preparation and PTP assay were performed with the Malachite
Green Phosphatase Assay (Upstate Biotechnology Inc, Lake Placid, NY) as
described in the product manual. Affinity purified 143-41 and CD50
molecules (kindly provided by Dr C. Vilardell, H. Clinic, Barcelona,
Spain) were diluted in assay buffer (10 mmol/L Tris-HCl, pH 7.4)
and added to microtiter wells with or without the substrate
phosphopeptide (2 mmol/L, TSTEPQpYQPGENL) allowing
enzyme reaction to proceed for 30 minutes. One hundred microliters per
well Malachite Green solution was added, and after incubation for 15 minutes the absorbance at 620 nm was determined with a Titertek
Multiscan enzyme-linked immunosorbent assay reader (Flow Laboratories,
Rockville, MD). The assay was performed in the presence or absence of
10-mmol/L sodium orthovanadate (Sigma Chemical Co).
Transfection of COS cells.
A full-length cDNA (hp21) encoding the human protein-tyrosine
phosphatase (HPTP-)12 was the kind gift of
Drs H. Honda and H. Hirai, Faculty of Medicine, University of Tokyo,
Japan. To examine whether MoAb 143-41 recognizes HPTP- gene product, COS-7 cells were transfected by lipofection. Briefly, 1 × 106 cells in log phase were washed twice with PBS and
incubated in serum-free Dulbecco's modified Eagle's medium containing
30 µg/mL DOTAP
(N-[1-(2,3-Dioleoyloxy)propyl]-N,n,n-trimethylammonium methylsulfate; Boehringer Mannheim GmbH, Mannheim, Germany) and 5 µg of the pSSRa expression vector with hp21 insert, or 5 µg of the pSSRa plasmid alone (mock transfection) at 37°C for 6 hours. After 2 days, cells were stained with 143-41 MoAb and FITC-labeled goat antimouse Ig and
analyzed on a FACScan flow cytometer (Becton Dickinson).
Analysis of [Ca2+]i.
[Ca2+]i was measured in individual
lymphocytes basically following the method described by Wacholtz and
Lipsky.16 Briefly, peripheral blood lymphocytes (PBL) were
resuspended at a final concentration of 30 × 106 cells/mL in RPMI supplemented with 10% FCS. Cells were
loaded with fura-2 acetoxymethyl ester (fura-2/AM; 2 mmol/L;
Calbiochem, San Diego, CA) by incubation for 25 minutes at 37°C,
with gentle shaking. After fura-2 loading, lymphocytes were incubated
with MoAbs (CRIS-7: 10 mg/mL and 72-5D3, 143-41,111-5A1: 40 mg/mL) for
30 minutes at 4°C. After washing, fura-2-loaded PBL
(106 cells) were plated on the center of a 25-mm glass
coverslip coated with Cell-Tak (Collaborative Biomedical Products,
Bedford, MA) in 50 mL of RPMI medium without FCS. The cells were
incubated for 20 minutes at 37°C under an atmosphere of 5%
CO2/air, and washed with incubation buffer composed of 121 mmol/L NaCl, 10 mmol/L HEPES, 5 mmol/L NaHCO3, 4.7 mmol/L
KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L
MgSO4, 2 mmol/L CaCl2, 10 mmol/L glucose, and
0.01% BSA at pH 7.4 to remove unattached and nonviable cells.
Coverslips with attached lymphocytes were transferred into an open flow
chamber (1 mL, volume) mounted on the heated stage of a Nikon
Diaphot-300 inverted epifluorescence microscope. The stage, 40 × fluor immersion objective (Nikon) and chamber were maintained at
37°C. Ca2+ mobilization was induced by the crosslinking
of cell surface molecules after addition of a second step saturating
amount of polyclonal affinity purified goat-antimouse antiserum (GAM;
Tago Inc, Burlingame, CA). Cells were considered to respond when
[Ca2+]i increased more than 100% of the
basal level. Fluorescence images were obtained by a CCD CH250 camera
(Photometrics, Tucson, AZ) and were digitized, stored, and analyzed in
an Apple-MacIntosh 840AV computer (Apple Computers Inc, Cupertino, CA).
Images were collected alternately at excitation wavelengths of 340 and
380 nm (10 nm bandwidth filters) to excite the Ca2+-bound
and Ca2+-free forms of this ratiometric dye, respectively.
The emission wavelength was 510 nm (120 nm bandwidth filter). The
integration time for each image was 100 ms, and individual pixels were
binned into 2 × 2 superpixels at read out from the charge coupled
device detector to improve signal to noise. To minimize photobleaching, a computer-controlled shutter was used to limit the exposure of the
cells to excitation light. [Ca2+]i values
were calculated on a single-cell basis from the 340- to 380-nm
fluorescence ratios at each time point as described previously.17,18 All images were checked for movement
artifacts, and a reference point was used to obtain true coregistration
of the 340- and 380-nm images. At the end of each experiment, cells were exposed to ionomicyn (10 mmol/L) and MnCl2 for 20 minutes. This treatment quenches the fluorescence of the intracellular Ca2+-sensitive fura-2, leaving the residual fluorescence at
each wavelength because of cell autofluorescence and any
Ca2+-insensitive forms of the dye. The residual
fluorescence was measured over the same region of each cell as the
Ca2+-dependent fluorescence.
Statistics.
Standard statistical methods from SPSS Statistical Analysis System
V4.0+ (SPSS, Chicago, IL) were used. Paired two-tailed t-tests
were used to analyze the differences between conditions in each
experiment. All variables were expressed as mean ± standard error
(SE), and a significance level of P < .05 was used.
Cell stimulation and tyrosine phosphorylation analysis.
Cells (50 × 106) were incubated with the
different MoAbs (10 µg) for 15 minutes at 4°C followed by the
addition of crosslinking rabbit antimouse Igs (5 µg). Incubation was
terminated after different periods of time by the addition of 1 mL of
ice-cold stop buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 100 mmol/L NaF,
10 mmol/L EDTA, 10 mmol/L Na4P2O7,
2 mmol/L sodium pervanadate, 2 mmol/L PMSF, 10 mg/mL aprotinin, 10 mg/mL pepstatin, 1 mg/mL leupeptin, 100 mmol/L PAO). Cells were
pelleted and lysed with stop buffer containing 1% NP-40. Proteins were
separated by SDS-PAGE and transferred to nitrocellulose membranes.
Phosphotyrosine-containing proteins were probed with
antiphosphotyrosine MoAb PY-20 from Santa Cruz Biotechnology (Santa
Cruz, CA) and horseradish peroxidase-conjugated rabbit antimouse and
visualized by fluorography with enhanced chemiluminescence reagent
(Amersham, Buckinghamshire, UK).
Immunoprecipitation of phospholipase C-1.
For immunoprecipitation experiments, cell lysates of 25 × 105 stimulated cells were prepared as described previously.
Precleared lysates were incubated overnight with 1 µg of anti-PLC-g1
MoAb (UBI Inc, Lake Placid, NY). Immunoprecipitates were recovered by
incubation with 20 µL of Protein A-Sepharose beads for 120 minutes at
4°C and washed three times in lysis buffer. The proteins were then
eluted and dissolved by boiling for 5 minutes in Laemmli sample buffer
and subsequently resolved by SDS-PAGE. Western blot analysis was then
performed using PY20 antiphosphotyrosine antibody (UBI Inc),
biotinylated goat antimouse (Sigma Chemical Co), and avidin-peroxidase
(Sigma Chemical Co) as described above. Next, membranes were stripped
of primary antibody with stripping buffer (100 mmol/L 2-ME, 2% SDS,
Tris-Cl 65 mmol/L, pH = 7.5) washed and reprobed with anti-PLC-1
MoAb (UBI Inc).
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RESULTS |
Phenotypic and immunochemical characterization of CD148.
With the aim of producing MoAbs defining new membrane proteins, several
MoAbs were obtained in our laboratory. One of them, MoAb 143-41, defined a molecule present on peripheral blood cells that shows its
highest expression on granulocytes, being present at intermediate
intensity on monocytes and lymphocytes (Fig
1A, left). Its expression on red blood cells and platelets was even lower than the one observed on those cells (Fig 1A, right). This molecule was detected on both T and B lymphocytes as determined by
double immunofluorescence with FITC-labeled 143-41 and PE-labeled CD3
and CD19 (Fig 1B). The reaction of 143-41 MoAb with different hematopoietic cell lines was also tested
(Fig 2). Thus, CD148 showed a clear
reaction with cell lines of myeloid origin (K562, U937, and HL-60) and
with some B-cell lines (Raji, KM3, Nalm-6), whereas this was weakly
expressed on Namalwa and Ramos, also of B-cell origin. Surprisingly,
and in contrast with the clear expression observed on CD3+
lymphocytes, it was not detected on T-cell lines (CEM, HPB-ALL, Jurkat,
and MOLT-4) with the exception of HSB-2.
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| Fig 1.
Flow cytometric analysis of human hematopoietic cell
lineages. Whole blood was stained with fluoresceinated-CD148 as
described in Materials and Methods and analyzed on a FACScan flow
cytometer. (A) Erythrocytes and platelets were analyzed after selecting
cell populations by side scatter and forward size. In parallel, after washing, erythrocytes were lysed by incubating with lysis buffer and
the different leukocyte populations were selected on basis of cell
scatter and forward size characteristics. Histograms for fluorescence
of simultaneously stained lymphocytes, monocytes, and granulocytes have
been superimposed. (B) PBL were obtained from normal healthy donors by
Ficoll-Hypaque gradient density centrifugation and lymphocyte
populations were analyzed by two color fluorescence by using
fluoresceinated 143-41 MoAb and comercially available PE-labeled CD3
and CD19 MoAb. The appropiate negative control FITC- and PE-labeled
MoAbs were used to establish the marker position. The staining
intensity of PE-labeled cells is shown in the vertical axis with
143-41-FITC staining on the horizontal axis.
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| Fig 2.
Expression patterns of CD148 on different cell lines of
myeloid and lymphoid origin. The 143-41 MoAb was assayed for their binding to cell lines by first incubating the cells with a saturating amount of antibody and, after washing, cells were incubated with a
FITC-labeled goat antimouse antibody ( ). Cells were also stained with
a control isotype-matched antibody. The staining intensity of
FITC-labeled cells is shown on the horizontal axis with the number of
cells on the vertical axis.
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Although this MoAb was analyzed during the IV WLDA it could not be
clustered. During the work of the VI WLDA held recently in Kobe,
another MoAb (A3) with a similar pattern of reaction was detected. In
basis of the comparative analysis of this antibody with our MoAb
143-41, a new cluster of differentiation could be defined:
CD148.10
To further characterize this molecule a thoroughly biochemical
characterization was performed. The molecule was immunoprecipitated, and before and after digestion with neuraminidase, O-glycanase, and
N-glycanase it was subjected to electrophoresis in reducing conditions
and analyzed by immunoblotting, as described in Materials and Methods.
As can be observed in Fig 3, CD148 appeared
before treatment as a broad band with an apparent molecular weight of 240 kD. Treatment of purified CD148 with the various glycosidases led
to alterations in the electrophoretic mobility of the protein. Thus,
after treatment with N-glycosidase F, the molecular weight of CD148 was
shifted to a much smaller size in Western blot analysis, indicating
that the molecule recognized by 143-41 was a glycoprotein containing
Asn-linked carbohydrate and that the molecular weight of this molecule
was, for the most part, modified by the N-glycosylation. O-Glycanase
treatment also affected the electrophoretic mobility of the CD148 by
decreasing its apparent molecular weight under reducing conditions. In
addition, a slight decrease in the electrophoretic mobility was
observed after treatment with neuraminidase suggesting the existence of
sialic acid residues. The reaction of CD148 with Maackia
amurensis and Sambucus nigra L biotinylated lectins,
confirmed this presence and showed that they are linked in both
 (2-3) and (2-6) to galactose (data not shown).
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| Fig 3.
Immunochemical characterization of CD148 molecule. Adult
human PBMCs were surface biotinylated and CD148 molecule was
immunoprecipitated by using 143-41-coupled CNBr-activated Sepharose
4B. Sample aliquots were subjected to treatment with
N-endoglycosidase F (lane 3), O-endoglycosidase (lane 2), and
neuraminidase (lane 1) as described in the Materials and Methods. Next,
samples were analyzed on a 5% SDS-polyacrylamide gel under reducing
conditions before (lane 4) and after glycanase treatment (lanes 1, 2, and 3), followed by electrophoretic transfer of proteins onto
Immobilon-P. After blocking and incubating with
streptavidin-peroxidase, the Western blots were developed using
diaminobenzidine with cobalt enhancement.
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Protein sequencing and identification as HPTP-/DEP-1.
CD148 was purified by immunoaffinity chromatography from leukocyte
membranes and subjected to N-terminal sequencing. After digestion of
CD148 with CNBr, several peptides were obtained, two of them being
subjected to N-terminal sequencing. The sequences obtained are shown in
Fig 4. A complete homology of these
sequences with the recently described PTP HPTP-12 or
DEP-113 was detected. As can be seen in Fig 4, in all the positions in which a clear sequence was obtained, an identity of
sequence was observed. In both CNBr-derived peptides it was observed
that the sequence started after a methionine residue in the sequence of
the PTP, in agreement with the existence of a point of cleavage for
CNBr. From the N-terminal sequence data we obtained, it should be noted
that the mature N terminus corresponds to Ala 36 and not to Thr 39 (HPTP-)12 or Gly 37 (DEP-1).13
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| Fig 4.
Identity between CD148 N terminal and peptide sequence
and deduced HPTP-h/DEP-1 protein sequence from the cDNA. Numbering of
HPTP-/DEP-1 amino acid positions are from Honda et al12 and Östman et al.13 Assignment of X in the CD148
sequence represents nonidentifiable signal in the sequence analysis.
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To confirm that the molecule recognized by 143-41 MoAb corresponds to
the previously described protein tyrosine phosphatase HPTP-/DEP-1
two different approaches were undertaken. First, hp21, a full length
HPTP- cDNA was used to transfect COS-7 cells. When stained with
143-41 MoAb, whereas mock-transfected COS-7 cells were negative
(Fig 5A), the COS-7 cells transfected with the HPTP- cDNA showed a clear positive reaction (Fig 5B). In addition, the phosphatase activity of CD148 was analyzed by testing its
ability to release phosphate groups from a tyrosine phosphorylated peptide. As can be observed in Fig 6 a
clear tyrosine phosphatase activity was observed when immunopurified
CD148 was tested. This activity was clearly diminished in the presence
of sodium orthovanadate, a PTP inhibitor. No PTP activity was detected
when a similarly immunopurified molecule, CD50, was tested in the same
assay.
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| Fig 5.
Immunofluorescence analysis of mock-transfected COS-7
cells (A) or COS-7 cells transfected with HPTP- cDNA (B). COS-7
cells were transfected with hp21 clone encoding HPTP- or plasmid
only and stained with 143-41-FITC as described in the Materials and Methods.
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| Fig 6.
PTP activity of purified CD148 molecule. Affinity
purified CD148 molecule was incubated with a tyrosine phosphorylated
syntethic peptide (TSTEPQpYQPGENL). The amount of free phosphate
released as inorganic phosphate in the absence or presence of vanadate was measured by the Malachite Green Phosphatase Assay (UBI) and it is
shown as nmol/L concentration. Affinity purified CD50 molecule was used
as a negative control.
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Ca2+ mobilization after CD3, CD148, and CD45
crosslinking.
Previous studies had shown that CD3-induced Ca2+
mobilization is modulated by the tyrosine phosphatase activity of CD45
when both molecules were cocrosslinked.7 Based on the fact
that CD148 was identified as a PTP expressed on the membrane of
lymphocytes, we evaluated whether CD148 could modify the activation
induced by CD3 crosslinking. Therefore, PBL were loaded with fura-2/AM, and Ca2+ mobilization after cell stimulation was analyzed
by computer-aided fluorescence imaging.
Figure 7 depicts the mean changes of
[Ca2+]i including all PBL populations after
crosslinking the CD3 molecule. These data are normalized to the basal
values before the addition of affinity purified GAM, and
Ca2+ changes were measured in individual cells. Mean
[Ca2+]i increased from a baseline of 71 ± 4 nmol/L to 163 ± 15 nmol/L after addition of the CD3
crosslinking agent. In agreement with previous results,7 we
observed a significant decrease in the CD3-induced response because of
CD45 cocrosslinking. Moreover, when CD3 was cocrosslinked with CD148,
we also observed a clear reduction in the mean peak
[Ca2+]i response (P < .05; n = 5 experiments; Fig 7), suggesting that the PTP activity of CD148 could
modulate signals transduced through the CD3 complex, similarly to CD45.
Interestingly enough, when CD148 alone was crosslinked, we observed an
increase in the mean [Ca2+]i, reaching a mean
peak of 130 ± 8 nmol/L, which was delayed and significantly lower
(P < .05, n = 6 in both experiments; Fig 7) when compared
with CD3 crosslinking stimulation. In addition, to determine the role
of tyrosine phosphorylation in [Ca2+]i
increase induced by CD148 stimulation we tested whether the phosphatase
activity of CD45 could abolish this effect when both molecules were
cocrosslinked, as has been described in other tyrosine kinase-mediated
activations.19 As can be observed in
Fig 8, the
[Ca2+]i increase induced by CD148
crosslinking was inhibited when CD45 was cocrosslinked with CD148.
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| Fig 7.
Calcium mobilization induced after CD3, CD148, and
CD3+CD148 crosslinking. Peripheral blood lymphocytes were loaded with
fura-2, and [Ca2+]i was measured as
described in the Materials and Methods. Cells were incubated 30 minutes
at 4°C with the different MoAbs (CD3, 10 µg/mL; 143-41, 50 µg/mL; CD45, 50 µg/mL) and adhered to Cell-Tak coated coverslips.
After establishing baseline values, a saturating amount of GAM was
added to prewarmed samples (arrow). [Ca2+]i
was measured every 5 seconds for 15 minutes on a single-cell basis in a
computer-aided fluorescence imaging. The average curve of a minimum of
200 individual cells for each condition of at least four independent
experiments are shown. Calcium changes are expressed as the percentage
change from basal.
|
|
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| Fig 8.
Fluorescence imaging of
[Ca2+]i responses from individual cells
after CD3, CD148, and CD3+CD148 crosslinking. Cells previously loaded
with fura-2 (2 µmol/L), were incubated with the corresponding antibody (CD3, 10 µg/mL; 14341, 40 µg/mL; CD45, 40 µg/mL) for 30 minutes at 4°C. After washing, cells were plated on
Cell-Tak-coated glass coverslips. After establishing baseline values,
saturating amount of polyclonal GAM was added to prewarmed samples.
[Ca2+]i was measured every 5 seconds for 15 minutes in every cell. Calcium changes are expressed as the percentile
change of basal. A total of 20 cells representative of each condition
are shown. Each line represents an individual cell.
|
|
Because the mean [Ca2+]i kinetics may be
influenced either by the percentage of responding cells, appearance of
unsynchronized responses, or variable intensity response,20
individual cell measurements of the [Ca2+]i
changes after CD3, CD45, and CD148 crosslinking were also evaluated (Fig 8). For the analysis of the single cell responses, we considered a
response significant when the Ca2+ mobilization induced by
crosslinking was at least twice the [Ca2+]i
of the basal level. In this sense, anti-CD3 induced a rapid and
significant (within 1 minute) [Ca2+]i
increase in 65% of cells, with these cells exhibiting a mean peak
[Ca2+]i of 414 ± 10 nmol/L. This was
followed by smaller [Ca2+]i oscillations,
especially in those cells with a higher
[Ca2+]i peak (polytopic response). The
asynchronous [Ca2+]i oscillations observed at
the single cell level are responsible for the sustained phase of the
[Ca2+]i changes identified in cell suspension
experiments. The reduction in the mean
[Ca2+]i response observed after CD3+CD45
cocrosslinking was basically caused by a decrease in the immediate
Ca2+ mobilization, affecting an average of 69% of the
responding cells. As may also be observed in Fig 8, crosslinking of
CD45 alone did not induce any significant modification of the
[Ca2+]i levels. With respect to the
inhibitory effect of CD148 on CD3-induced stimulation, we observed a
decrease in the immediate response in 35% of the cells.
Individual responses to CD148 crosslinking exhibited a synchronized,
more than twofold increase in [Ca2+]i in 39%
of the cells (Fig 8). This increase, which was delayed (1 minute) if it
was compared with the rapid response observed after CD3 crosslinking,
was significantly different from the levels observed in the presence of
an isotype matched control MoAb (111-5A1, CD41; data not shown).
Likewise, no response was observed after adding GAM to the sample. The
inhibitory effect of CD45 cocrosslinking was observed to affect 80% of
CD148 responding cells, affecting especially the early phase of the
response (Fig 8). These data suggest that the crosslinking of CD148
could induce Ca2+ mobilization through tyrosine
phosphorylation processes. To better characterize this point, the
response to CD148 crosslinking in the presence of a known protein
tyrosine kinase inhibitor, genistein, was analyzed. The data presented
in Fig 9A show that genistein clearly
inhibits the Ca2+ mobilization induced by CD148
croslinking. When individual cell measurements of the
[Ca2+]i changes were analyzed after CD148
crosslinking in the absence (Fig 9B) or presence of genistein 75 µmol/L (Fig 9C) it was confirmed that the inhibition observed in the
presence of genistein was real and not caused by the induction of
unsynchronized responses. The inhibitory effect of genistein affected
70% of CD148 responding cells, mainly during the early phase of the
response (Fig 9C).
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| Fig 9.
Effect of Genistein on calcium mobilization induced by
CD148 crosslinking. Peripheral blood lymphocytes were loaded with
fura-2, and [Ca2+]i was measured as
described in the Materials and Methods. Cells were incubated 30 minutes
at 4°C with 50 mg/mL of CD148 and adhered to Cell-Tak-coated
coverslips. After establishing baseline values, a saturating amount of
GAM was added in the absence or presence 0f 75 mmol/L Genistein to
prewarmed samples (arrow). [Ca2+]i was
measured every 5 seconds for 15 minutes on a single-cell basis in a
computer-aided fluorescence imaging. (A) The average curve of a minimum
of 200 individual cells for each condition of at least four independent
experiments are shown. Calcium changes are expressed as the percentage
change from basal. Fluorescence imaging of
[Ca2+]i responses from 20 individual cells,
after CD148 crosslinking, in the absence (B) or the presence (C) of
genistein (75 µmol/L). Each line represents an individual cell.
|
|
Induction of tyrosine phosphorylation by CD148 crosslinking.
The results of the CD45 cocrosslinking and genistein experiments led us
to investigate whether specific tyrosine phosphorylation events were
associated with anti-CD148-triggered Ca2+ mobilization.
Thus, cells previously coated with CD3, CD148, or both were lysed in
SDS sample buffer at various times after addition of rabbit antimouse
antiserum and tyrosine-phosphorylated proteins were detected by
immunoblotting. As shown in Fig 10, and as it has been exhaustively described, a rapid increase in the phosphotyrosine content of several proteins was observed after CD3
crosslinking (Fig 10A, lanes 2 and 3). In addition, phosphorylation of
several substrates after CD148 crosslinking was also evident (Fig 10A,
lanes 4 and 5). These results are in agreement with those that have
been presented previously and suggest that the Ca2+
mobilization induced by CD148 crosslinking could be mediated by
tyrosine phosphorylation events.
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| Fig 10.
Protein tyrosine phosphorylation induced by CD148
crosslinking. (A) Cells were incubated without (lane 1) or with (lanes
2 to 7) 10 µg of the different monoclonal antibodies for 15 minutes at 4°C followed by the addition of crosslinking rabbit antimouse Igs. Incubation was terminated after 1 minute (lanes 2, 4, and 6) and 5 minutes (lanes 3, 5, and 7). Proteins were resolved by SDS-PAGE
followed by antiphosphotyrosine immunoblotting. (B) PLC-1 was
immunoprecipitated from cells stimulated during 1 and 3 minutes and
analyzed by Western blot with an antiphosphotyrosine antibody and,
after stripping, with an anti-PLC-1.
|
|
On the other hand, and taking into account the tyrosine phosphatase
activity of CD148, we were interested to know whether the CD148-induced
inhibition of CD3-mediated Ca2+ mobilization was caused by
specific dephosphorylation event(s). The pattern of protein tyrosine
phosphorylation after activation of human lymphocytes with CD3
crosslinking was modified when CD148 was cocrosslinked to this receptor
(Fig 10A, lanes 6 and 7). Thus, a selective inhibition of some
substrate was detected, which could be related with the phosphatase
activity of CD148. Taking into account the previously established
relationship between calcium mobilization and tyrosine phosphorylation
of phospholipase C (PLC-) we were interested in analyzing the
phosphorylation status of this enzyme after cell stimulation. Cells
coated with CD3, CD148, or both were stimulated with rabbit antimouse
Ig and after cell lysis at different times, PLC- was
immunoprecipitated. The immunoblotting with an antiphosphotyrosine
antibody (Fig 10B) showed that, whereas after stimulating with CD3 and
CD148 an increase in tyrosine phosphorylation of PLC- was observed,
a clear decrease was detected when both CD3 and CD148 were
cocrosslinked. By reprobing with an anti-PLC- it was observed that
the same quantity of PLC- was loaded on each lane. These results are
in agreement with the data provided by Ca2+ mobilization
analysis.
In addition, it was observed that cotriggering of CD3 with CD148
resulted in an increase in protein tyrosine phosphorylation in some
other proteins. This increase in the phosphorylation pattern was
particularly evident on a 56-kD substrate.
| |
DISCUSSION |
During recent years there has been an increase in the description of
new PTPs,3 although the majority of these PTPs have been
mainly related to activities of the central nervous system. In
contrast, although the number of PTKs involved in lymphocyte signal
transduction has also grown, this has not been the case for the number
of PTPs involved in this process. In addition to the cytoplasmic PTPs
involved in lymphocyte signaling, just one membrane PTP, CD45, is known
to influence the signaling process after antigen receptor engagement
(see Streuli21 for review). In this paper, we have
presented evidences showing that in addition to CD45, there is another
PTP on the lymphocyte membrane that could be able to modulate the
signaling process after CD3 crosslinking. Thus, we have observed that
the cocrosslinking of the molecule recognized by the MoAb 143-41, together with CD3 inhibits the subsequent
[Ca2+]i increase. Our results show that this
molecule is a membrane protein tyrosine phosphatase identical to a
recently described HPTP called HPTP-12 or
DEP-1.13 This phosphatase is present on the membrane of all
the hematopoietic cells and it has been classified as CD148 during the
last VI WLDA.10 Within the lymphoid cells, it is preferentially expressed on B cells, memory T lymphocytes, and mature
thymocytes (A. Gayá, unpublished observations). In contrast, its
expression on hematopoietic cell lines is heterogeneous. Thus, whereas
it is clearly expressed on myeloid cell lines and on the majority of
B-cell lines tested it is absent from the majority of T-cell lines
analyzed. Although this pattern of distribution could seem
contradictory there are other molecules that display a similar pattern
of distribution. Thus, CD26, which is clearly present on all mature T
lymphocytes is absent from T-cell lines with the exception of
HSB-2.22
We have presented several pieces of evidence showing that CD148 is
identical to HPTP-12 and DEP-1.13 First, the
sequences we obtained by protein sequencing and the previously
published cDNA sequences of HPTP-12 and
DEP-113 are identical. These sequences include an
N-terminal fragment of 18 amino acids as well as two different
CNBr-derived peptides of 18 aa. We consider those sequences as
identical at all of the resolved positions, and the nonresolved
positions can be explained by technical problems inherent in the
sequencing process. The most interesting aspect of these data is the
determination that the N-terminal residue of the mature protein
corresponds to an Ala and not to the previously proposed
Thr12 or Gly.13 Second, when COS-7 cells were
transfected with a plasmid (hp21) containing a HPTP- encoding cDNA
clone,12 a clear reaction with 143-41 MoAb was observed.
Third, by using an immunopurified preparation of CD148 molecule,
obtained from leukocyte membranes, it was possible to determine its
capacity to release free phosphate groups from a tyrosine
phosphorylated peptide, thus, confirming its tyrosine phosphatase
activity. The immunochemical characterization of CD148 molecule
confirmed that this molecule contains both O- and N-linked
carbohydrates. From the analysis with glycosidases it could be deduced
that the major part of carbohydrates are N-linked. This is in agreement
with the 34 potential sites for N-linked glycosilation determined from
the cDNA sequence10,11 and the data presented by Honda et
al12 on N-glycosidase F treatment of HPTP-. Concerning
the apparent molecular weight of HPTP-, differences have been
described among several cell lines11,12 varying from 250 kD
(HL60) to 230 kD (F-36P) or 220 kD (F-36E). Taking into account that
CD148 molecule was obtained from a heterogeneous population of cells,
the broad band around 240 kD we have observed both by
immunoprecipitation and immunoafinity purification could include all
the forms previously mentioned, suggesting the existence of a certain
level of heterogeneity in the expression of this phosphatase among
several cellular lineages.
HPTP- or DEP-1 is a recently described receptor PTP, the
extracellular portion of which is composed of 813 or
1012 FNIII domains, whereas the intracellular segment
contains a single PTP domain spanning amino acids 1060 to 1296. Therefore, it joins an expanding group of such receptors classified as
type III PTPs3 that includes PTP- ,23
PTP-U2,24 GLEPP1,25 and SAP-126 from humans and DPTP10D,27 DPTP99A,28 and
DPTP4E29 from Drosophila, with HPTP- being the unique
type III PTP expressed on hematopoietic cells. These enzymes are
characterized by a similar organization of their extracellular
segments, which consist of a repeated array of FNIII motifs, and a
single intracytoplasmic phosphatase domain. The FNIII repeats, in
addition to being involved in adhesion processes,29 are
also found in the extracellular regions of the receptors for
interleukin-2 (IL-2), IL-4, IL-6, granulocyte-macrophage
colony-stimulating factor, prolactin, erythropoietin, and growth
hormone.30 The DEP-1 molecule has been implicated in
contact inhibition of cell growth because it is upregulated in dense
cell cultures although its upregulation is initiated before saturation
density is reached. In addition, it has been described that the
expression level of HPTP- was altered when the HL-60 cells were
exposed to differentiating compounds such as dimethyl sulfoxide and
12-O-tetra decanoyl phorbol 13-acetate, suggesting that this gene might
be involved in the differentiating processes for granulocyte or
monocyte/macrophage lineages in these cells.11
The CD45 molecule, the principal protein tyrosine phosphatase present
on the membrane of hematopoietic cells, is capable of regulating signal
transduction and functional responses,7-9 because in T
lymphocytes, CD45 crosslinking inhibits inositol phosphate production,
calcium flux, and proliferation.31 The ability of CD45 to
modulate signals transduced by CD3 correlates with its ability to
inhibit the tyrosine phosphorylation of some intracellular protein
substrates.31,32 Once we showed that the molecule
recognized by the 143-41 MoAb is a membrane protein tyrosine
phosphatase, we were interested to know whether this molecule could
influence the signal transduction through the antigen receptor. The
most striking fact of this analysis was the observation that the
crosslinking of CD148 alone was able to induce a clear increase in
[Ca2+]i. This phenomenon was not observed
after CD45 crosslinking. The kinetic of the process was similar to the
Ca2+ mobilization induced after CD3 crosslinking, although
the lag time was clearly more prolonged in the case of CD148. Another difference was based on the intensity of the response. The CD148 crosslinking produced both a lower level of
[Ca2+]i mobilization and a lower number of
responding cells (39% v 69%). In fact, CD148 crosslinking was
able to decrease the Ca2+ mobilization in 35% of the cells
responding to CD3 (69%), a percentage similar to the percentage of
cells responding to CD148 crosslinking (39%).
Concerning the mechanisms involved in this process, we consider it
plausible that tyrosine phosphorylation could be involved because the
cocrosslinking with CD45 or the presence of genistein were able to
abolish the response induced by CD148. This assumption was proven to be
correct, because after CD148 crosslinking a clear pattern of tyrosine
phosphorylation could be observed. Especially interesting was the
observation that CD148 crosslinking was able to induce tyrosine
phosphorylation of PLC-1. Although it may seem contradictory that a
protein tyrosine phosphatase is able to induce tyrosine
phosphorylation, a similar situation has been described for the
molecule CD45.33-35 In this case, the association of p56lck
with the cytoplasmic tail of CD4535,36 results in an
increase in the catalytic activity of the PTK after CD45
crosslinking.37 In a similar way, the existence of an
association of CD148 with some PTK able to mediate the observed
increase in Ca2+, could be considered. This PTK could be
activated through dephosphorylation by the tyrosine phosphatase domain
of CD148, similar to the way in which CD45 dephosphorylates Y505 on
p56lck, turning the inactive protein into an active PTK.38
In addition, we have also observed that CD148 was able to inhibit the
Ca2+ mobilization induced by CD3 MoAb when both molecules
were cocrosslinked. This could have occurred by bringing the PTP domain
of CD148 into close proximity with the cytoplasmic domain of a
signaling molecule. In this way its activity could be triggered by
changing the phosphorylation status of critical tyrosine residues used
in signal transduction, as has been described with
CD45.31,32 Although Shivnan et al39 have
suggested that some of the results of coclustering experiments with
CD45 may have to do with interfering with CD3 clustering, we have
observed no interference in our experimental model when CD3 was
cocrosslinked with lymphocyte function-associated antigen (data not
shown), thus discarding an unspecific effect. However, whether the
inhibition of CD148 on CD3 signaling is based on the phosphorylation or
the dephosphorylation of some substrate(s) remains to be determined. In
spite of the tyrosine phosphatase activity of CD148 no general
dephosphorylation was observed after CD3 and CD148 cocrosslinking. In
fact, only a very selective inhibition of a few substrates was
detected, the most significant being the dephosphorylation of PLC-.
This dephosphorylation could account for the inhibition of
Ca2+ mobilization, in a way similar to
CD45.31,32 In this case also few very selective
dephosphorylation events have been related by CD3 signaling
inhibition.33,34 It could seem contradictory that the
cocrosslinking of CD148 and CD3 results in no signal whereas engagement
of either molecule by itself causes significant signal transduction
events to occur. Although we have no definitive data to explain
these facts, a possible explanation could be that the kinase activity
activated through CD148 crosslinking would be modified after
cocrosslinking CD148 with CD3, whereas the own phosphatase activity of
CD148 would remain intact. If this were the case, the activation of
PLC- by CD3 would be decreased by the phosphatase activity of CD148
and it would not be compensated by the activation through CD148. The
net result of this process would be a diminished activation of PLC-
and consequently the inhibition of Ca2+ mobilization. On
the other hand, our results also show an increase in the
phosphorylation of some substrates after CD3 and CD148 cocrosslinking.
This fact was particularly evident in a 56-kD substrate.
Corvaïa et al40 have also described an increase in
protein tyrosine phosphorylation of some substrates after cotriggering human monocytes with FcRI or FcRII with CD45. Thus, the
possibility that a specific phosphorylation of some substrate could be
responsible for the inhibition of the signaling process through CD3
could not be ruled out.
In conclusion, in the present report we have shown a functional role
for a recently described membrane protein tyrosine phosphatase, which
is involved in the signaling pathways of T lymphocytes, opening a new
avenue of research into the signal transduction pathways of these
cells.
| |
FOOTNOTES |
Submitted April 14, 1997;
accepted December 9, 1997.
Supported by Grant No. 96/0788 from Fondo de Investigación
Sanitaria. M.A.d.l.F.-G. is a recipient of a predoctoral fellowship from Hospital Clínic i Provincial.
Address reprint requests to Antoni Gayà, MD, Servei
d'Immunologia, Hospital Clínic, Villarroel 170, Barcelona
08036, Spain.
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.
| |
ACKNOWLEDGMENT |
We thank Drs Honda and Hirai for kindly providing the hp21 cDNA clone,
Dr P. Engel for his collaboration in the transfection assays and Dr M. Simarro for her help in the tyrosine phosphorylation analysis.
| |
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Abstract
Introduction
Abstract