From the Hubrecht Laboratory, Netherland Institute for Developmental Biology, Uppsalalaan 8, Utrecht, 3584 CT, The Netherlands
Received for publication, January 21, 2003
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ABSTRACT |
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Receptor protein-tyrosine phosphatase Two antagonistically acting families of enzymes regulate
phosphorylation of protein tyrosine residues, an important determinant for many cellular functions: protein-tyrosine kinases
(PTKs)1 and protein-tyrosine
phosphatases (PTPs). PTKs catalyze the phosphorylation of tyrosine
residues, and PTPs catalyze the dephosphorylation. The classical PTPs
can be subdivided into the cytoplasmic PTPs and the transmembrane PTPs
(reviewed in Ref. 1). Transmembrane PTPs, tentatively called
receptor-like PTPs (RPTPs), are interesting because of their potential
to signal across the membrane. Besides one transmembrane domain, most
RPTPs possess two catalytic domains of which the first contains most of
the catalytic activity. The RPTP extracellular domains vary greatly
(2). Clear evidence for ligands that regulate RPTP activity remains
elusive. In some cases, as for the interaction between RPTP Dimerization induced by ligand binding is a well known regulatory
mechanism for receptor PTK activity. In the absence of ligand, RPTK
monomers are in equilibrium with RPTK dimers. A limited population of
the RPTK dimers are in the active configuration, which becomes stabilized upon ligand binding and results in cross-phosphorylation and
stimulation of PTK activity (reviewed in Refs. 8 and 9). The first
evidence for regulation of RPTPs by dimerization came from studies with
the RPTP CD45. Chimeric EGFR-CD45 with the extracellular domain of the
EGFR fused to the cytoplasmic domain of CD45 is functionally
inactivated by dimerization upon ligand binding (10). In line with
this, there is evidence for the regulation of RPTP Recent studies indicate that the second catalytic domain of RPTP Here we report that antibody binding to the HA tag within the
N-terminal part of the ectodomain of plasma membrane localized RPTP Constructs--
SV40-driven expression vectors have been
described for full-length HA-RPTP Cell Culture and Transfection--
Human embryonic kidney (HEK)
293 cells were routinely grown in Dulbecco's modified Eagle's
medium/Ham's F-12 medium supplemented with 7.5% fetal bovine serum.
Transient transfection of HEK293 cells was done by calcium phosphate
precipitation as described previously (20). The medium was refreshed
the day after transfection, and the experiments were performed after
another 16 h. Mouse embryo fibroblasts derived from RPTP Immunoprecipitation of Accessible and Nonaccessible
HA-RPTP
For precipitation of the accessible fraction, the supernatant was
rotated for 2 h at 4 °C with protein A-Sepharose beads (see Fig. 1). The beads were spun down and washed four times with HNTG (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton
X-100, and 10% glycerol), yielding the accessible fraction. The
supernatant was collected for immunoprecipitation of the nonaccessible
fraction by incubation for 2 h with anti-HA antibody linked to
protein A-Sepharose beads. The beads were washed four times with HNTG. The samples were boiled in Laemmli sample buffer and separated on 7.5%
SDS-PAGE gels. The material on the gel was transferred to
polyvinylidene difluoride membrane by semi-dry blotting. The membranes
were stained with Coomassie Blue, blocked for 1 h at room
temperature in TBST (50 mM Tris, pH 8.0, 150 mM
NaCl, 0.05% Tween 20) containing 5% milk, and incubated for another
hour with anti-HA antibody or anti-RPTP Biotinylation of Membrane-localized RPTP
Total RPTP Rotational Coupling Determines Accessibility of the HA Tag in the
Ectodomain--
For RPTKs it is well known that dimerization induced
by ligand binding results in internalization of the complex (reviewed in Ref. 9). To investigate the role of dimerization on subcellular localization of RPTP
The variation in accessibility of the HA tag between the constructs
might be due to a difference in subcellular localization of the
HA-RPTP H2O2-induced Change in Ectodomain
Accessibility of RPTP Masking of the Ectodomain Requires RPTP
HA-RPTP
Constructs of HA-RPTP The Mechanism of the H2O2-induced
Conformational Change in the Ectodomain--
We have previously shown
that treatment of cells with H2O2 leads to a
conformational change in RPTP
We showed that the catalytic site cysteine in RPTP Here we report that the conformation of the extracellular domain
of RPTP We developed an assay to assess accessibility of the epitope tag in the
ectodomain of HA-RPTP Taken together, based on our results, we propose a model for the
effects of H2O2 on the conformation of RPTP (RPTP
) constitutively forms dimers in the membrane, and activity
studies with forced dimer mutants of RPTP
revealed that rotational
coupling of the dimer defines its activity. The hemagglutinin (HA) tag
of wild type RPTP
and of constitutively dimeric, active
RPTP
-F135C with a disulfide bond in the extracellular domain was not
accessible for antibody, whereas the HA tag of constitutively dimeric,
inactive RPTP
-P137C was. All three proteins were expressed on the
plasma membrane to a similar extent, and the accessibility of their
extracellular domains did not differ as determined by biotinylation
studies. Dimerization was required for masking the HA tag, and we
identified a region in the N terminus of RPTP
that was essential for
the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTP
-D2). Here we report that H2O2 treatment of cells induced a
change in rotational coupling in RPTP
dimers as detected using
accessibility of an HA tag in the extracellular domain as a read-out.
The catalytic site Cys723 in RPTP
-D2, which was required
for the conformational change of RPTP
-D2 upon
H2O2 treatment, was essential for the
H2O2-induced increase in accessibility. These
results show for the first time that a conformational change in the
intracellular domain of RPTP
led to a change in conformation of the
extracellular domains, indicating that RPTPs have the capacity for
inside-out signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
leukocyte common antigen-related (LAR), and PTP
and their
ligands contactin, laminin/nidogen, and heparan sulfate proteoglycans,
respectively, the binding compound is known, but the effect of the
ligand on PTP activity needs to be established (3-5). Sorby et
al. (6) reported an effect of Matrigel on DEP1 activity; however,
the exact responsible compound still needs to be established. Only
pleiotrophin, a heparin-binding cytokine, was reported to reduce
RPTP
/
activity, in that
-catenin phosphorylation was enhanced
in cells expressing RPTP
/
in response to pleiotrophin (7).
by dimerization.
The crystal structure of RPTP
-D1 provided structural evidence that
dimerization leads to inhibition of the catalytic activity of RPTP
because a wedge-like structure of one monomer inserts into the
catalytic cleft of the other, thereby occluding the catalytic site
(11). In vivo activity studies with constitutively dimeric
mutants confirm this hypothesis. In these mutants a single cysteine
residue was introduced in the extracellular domain of RPTP
leading
to the formation of a stable intermolecular disulfide bridge (12). A
disulfide bridge at position 137 (RPTP
-P137C) resulted in reduced
substrate dephosphorylation, whereas constructs with a disulfide bridge
at position 135 (RPTP
-F135C) had activities similar to the wild type
construct. These data indicate that dimerization of RPTP
only leads
to inactivation of the catalytic activity if the rotational coupling of
the dimer is such that the wedge of one monomer inserts into the
catalytic cleft of the other (12). Point mutations in the wedge of
EGFR-CD45 chimeras (13) and constitutive dimer mutants of RPTP
(12)
indeed abolished the dimerization-induced inhibition of PTP activity.
Taken together, RPTPs can be regulated by dimerization. Evidence is
accumulating that RPTPs indeed dimerize in vivo.
Cross-linking experiments (14) and fluorescence resonance energy
transfer analysis (15) indicate that RPTP
constitutively forms
dimers in living cells. Similarly, CD45 forms dimers in living cells,
as demonstrated by chemical cross-linking and fluorescence resonance
energy transfer analysis (16-18). Interestingly, dimerization of
distinct alternatively spliced isoforms of CD45 is different (18),
suggesting regulation of dimerization at the level of alternative
splicing. Rotational coupling appears to be an important determinant
for RPTP activity, and it will therefore be interesting to see how
rotational coupling in RPTP dimers is regulated.
,
RPTP
-D2, may have a regulatory role in dimer conformation. Using
fluorescence resonance energy transfer and co-immunoprecipitation techniques, we found that oxidative stress leads to opening up of D2,
resulting in the formation of more stable, inactive dimers (19).
Importantly, as shown by bis[sulfosuccinimidyl]suberate cross-linking
experiments, H2O2 does not induce de
novo dimerization (19). The H2O2-induced
conformational change is dependent on the catalytic site cysteine in
RPTP
-D2, Cys723, suggesting that direct oxidation of
this residue underlies the observed effects.
reflected the state of rotational coupling. Although the epitope tag
was masked in wild type RPTP
and constitutively dimeric, active
RPTP
-F135C with a single cysteine in the extracellular domain, the
epitope tag in inactive dimeric RPTP
-P137C was accessible, suggesting that accessibility of the HA tag was dependent on rotational coupling. H2O2 induced accessibility of the HA
tag in wild type RPTP
. Masking of the epitope tag was dependent on
residues 20-42 in the extracellular domain and on dimerization of
RPTP
. RPTP
-D2 and more specifically the catalytic site Cys in
RPTP
-D2, Cys723, were required for
H2O2-induced HA tag accessibility. Our results suggest that a conformational change in the cytoplasmic domain of
RPTP
leads to a change in the conformation of the ectodomain of
RPTP
and therefore provide evidence that RPTPs have the
potential for inside-out signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(20), HA-RPTP
-F135C,
HA-RPTP
-P137C (12), HA-RPTP
-
224-235, HA-RPTP
-
207-238
(14), HA-RPTP
-C723S, HA-RPTP
-
D2 (21), and Myc-RPTP
(19).
The deletion mutants in the juxtamembrane domain (see Fig. 5) and
ectodomain (see Fig. 6) were made by site-directed mutagenesis and
checked by sequencing. HA-RPTP
was constructed by fusing the RPTP
signal sequence (residues 1-19) and HA epitope tag to RPTP
(from
residue 20 to the end).
/
mice (22) were a kind gift of Jan Sap. These cells were stably
co-transfected using LipofectAMINE with HA-tagged wild type
RPTP
, RPTP
-F135C, or RPTP
-P137C and pSV2neo, conferring
resistance to G418. Following selection for 2 weeks, individual
colonies were picked, and these clones were tested for expression of
(mutant) RPTP
.
--
Subconfluent (stimulated) cells were washed with
ice-cold phosphate-buffered saline. The cells were incubated for 1 h on ice with anti-HA antibody in Dulbecco's modified Eagle's
medium/Ham's F-12 medium. Subsequently, the cells were washed three
times with ice-cold phosphate-buffered saline, lysed in cell lysis
buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 1% Triton X-100,
aprotinin, and leupeptin), and centrifuged at 14,000 × g for 15 min.
antibody (AP 5478) (23) in
TBST with 5% milk. The blots were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody for
1 h. After washing with TBST, the immunoreactivity on the membranes was visualized using ECL according to standard protocols.
--
Subconfluent
(stimulated) cells were washed with ice-cold phosphate-buffered saline
and incubated for 45 min with 0.5 mg/ml EZ-linked sulfo-NHS-LC-biotin
(Pierce) in borate buffer (10 mM boric acid, pH 8.0, 150 mM NaCl). The cells were washed three times with ice-cold
15 mM glycine in phosphate-buffered saline, lysed in cell
lysis buffer, and centrifuged at 14,000 × g for 15 min.
was precipitated by incubation of the lysate for 2 h
with anti-HA antibody linked to protein A-Sepharose beads. The beads
were washed five times with HNTG and boiled in Laemmli sample buffer.
The immunoprecipitates were separated on 7.5% SDS-PAGE gels and
transferred to polyvinylidene difluoride membrane after separation.
Immunoblotting was done as described above using horseradish peroxidase-conjugated avidin-biotin complexes and ECL to detect biotinylated proteins on the blots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, we studied HA antibody binding on living mouse
embryo fibroblasts derived from RPTP
/
mice (22), stably expressing N-terminally HA-tagged wild type RPTP
or constitutive dimer mutants with a disulfide bridge in the extracellular domain (RPTP
-F135C and RPTP
-P137C) as described under "Experimental Procedures" (Fig. 1). In short, we
incubated cells stably expressing HA-tagged RPTP
with anti-HA
antibody for 1 h on ice. After extensive washing we lysed the
cells and precipitated the protein antibody complexes. This part of the
total RPTP
in the lysate is the accessible fraction (a).
The rest of the HA-tagged RPTP
was precipitated from the supernatant
and represents the nonaccessible fraction (n). Surprisingly,
almost no wild type HA-RPTP
was detected in the accessible fraction
(Fig. 2A). Constitutively
dimeric HA-RPTP
-F135C, which is catalytically active (12), behaved
similarly to wild type HA-RPTP
with very little signal in the
accessible fraction. In contrast, inactive dimeric HA-RPTP
-P137C was
readily detectable in the accessible fraction (Fig. 2A).
Similar results were found when cells were lysed in RIPA buffer (data
not shown), indicating that a difference in accessibility of the HA tag
is not due to a difference in amount of RPTP
in Triton-soluble
fractions.
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Fig. 1.
Separation of accessible and nonaccessible
HA-tagged RPTP . Cells expressing
HA-tagged (mutant) RPTP
were incubated with anti-HA antibody (12CA5)
for 1 h at 0 °C. Excess antibody was washed away, and the cells
were lysed. The lysates were incubated with protein A-Sepharose beads
to precipitate the accessible fraction. The remaining HA-RPTP
was
immunoprecipitated from the supernatant, which is the nonaccessible
fraction. For details, see under "Experimental Procedures."
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Fig. 2.
Rotational coupling of
RPTP dimers determines ectodomain epitope tag
accessibility. A, accessible (a) and
nonaccessible (n) RPTP
was isolated as described in Fig.
1 from mouse embryo fibroblasts, derived from RPTP
/
mice, stably
transfected with either wild type HA-RPTP
(WT), or
HA-tagged constitutive dimer mutants (F135C and
P137C). Immunoblots probed with anti-HA antibody are
depicted here. B, membrane proteins were covalently
biotinylated using sulfo-NHS-LC-biotin, and RPTP
was
immunoprecipitated from mouse embryo fibroblasts, stably transfected
with either wild type RPTP
(WT) or constitutive dimer
mutants (F135C and P137C). Biotinylated
RPTP
was detected by labeling the immunoblots with avidin-biotin
complexes. Three independent stably transfected clones expressing
either wild type or mutant RPTP
were tested in these assays with
similar results. Representative examples of these experiments are
depicted here.
mutants. To investigate this possibility, we biotinylated the membrane proteins using EZ-linked sulfo-NHS-biotin. Subsequently, we immunoprecipitated RPTP
and detected membrane-localized RPTP
with an avidin-biotin complex. All three proteins, wild type RPTP
and the two constitutively dimeric mutants, were readily detectable, and there was no increase in membrane localization of the P137C mutant
compared with the wild type or the F135C mutant (Fig. 2B). These results indicate that the anti-HA antibody hardly bound to wild
type HA-RPTP
and constitutively dimeric HA-RPTP
-F135C, despite
the fact that these proteins were expressed on the cell surface as
shown by the biotinylation experiments. In contrast, HA-RPTP
-P137C
was readily detected in the HA-accessibility assay. Taken together,
these results suggest that the HA epitope tag is masked in wild type
HA-RPTP
and HA-RPTP
-F135C but not in HA-RPTP
-P137C.
--
The accessibility of the HA tag of the
forced RPTP
dimers seemed to depend on the position of the cysteine
bridge and therefore on the rotational coupling in the dimer.
Previously, we found that H2O2 treatment of
cells induced a conformational change in the cytoplasmic domain of
RPTP
, which was dependent on the catalytic site cysteine in
RPTP
-D2 (Cys723) and resulted in stabilization of
RPTP
dimers (19). We wondered whether H2O2
treatment of cells expressing wild type HA-RPTP
changed the
accessibility of the HA tag. H2O2 induced a
remarkable increase in the accessible fraction of RPTP
(Fig.
3A). Biotinylation of cell
surface proteins and detection of the amount of biotinylated HA-RPTP
demonstrated that there is no significant change in membrane localization of RPTP
after H2O2 treatment
(Fig. 3B). By first biotinylating membrane proteins and
subsequently separating the accessible and nonaccessible fraction of
HA-RPTP
, we found that a large part of the biotinylated HA-RPTP
ends up in the nonaccessible fraction. H2O2
treatment led to an increase in the biotinylated accessible fraction
(Fig. 3C). These results show that a large fraction of the
inaccessible HA-RPTP
is present on the cell membrane and that the
H2O2-induced increase in accessibility is not
due to an increase in membrane localization of RPTP
or to an
increase in amount of RPTP
to more accessible membrane sub-domains
(Fig. 3, B and C). Expression of different
amounts of RPTP
by transfecting variable quantities of DNA in 293HEK
cells did not change the ratio of accessible and inaccessible RPTP
significantly (Fig. 3D), indicating that the effect of
H2O2 treatment is not determined by the amount
of overexpression of the protein. Similar results were found if media
containing higher concentrations of anti-HA-antibodies were used for
the purification of the accessible fraction (data not shown). These
results, together with the finding that the two constitutively dimeric
mutants RPTP
-P137C and RPTP
-F135C show differences in accessible
fractions (Fig. 2A), make it unlikely that the increase in
accessible fraction after H2O2 treatment is
merely due to co-immunoprecipitation of oligomeric complexes. The
results indicate that the difference in accessibility is due to a
change in conformation of the ectodomains of RPTP
.
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Fig. 3.
Increased accessibility of
HA-RPTP in response to
H2O2 treatment. Mouse embryo fibroblasts
stably transfected with HA-tagged RPTP
-WT were treated for 5 min
with 1 mM H2O2 (+) or left
untreated (
). A, the accessible (a) and
nonaccessible (n) fractions of RPTP
were isolated.
Immunoblots are labeled with anti-HA antibody. B, membrane
proteins were covalently biotinylated with sulfo-NHS-LC-biotin, and
RPTP
was precipitated. The immunoblot was labeled with avidin-biotin
complexes. C, after biotinylation of membrane proteins, the
accessible (a) and nonaccessible (n) fractions of
RPTP
were isolated. The immunoblot was labeled with avidin-biotin
complexes. D, 293HEK cells were transiently transfected with
different amounts of DNA encoding HA-RPTP
as indicated. The cells
were treated for 5 min with 1 mM
H2O2 (+) or left untreated (
), and the
accessible (a) and nonaccessible (n) fractions of
RPTP
were isolated. Immunoblots probed with anti-HA antibody and
developed by ECL are depicted.
Dimerization--
RPTP
is highly homologous to RPTP
but differs
from RPTP
in its ectodomain (27 residues for RPTP
and 123 residues for RPTP
). RPTP
and RPTP
form stable heterodimers
upon H2O2 treatment as tested by a
co-immunoprecipitation experiment with cells transfected with HA-tagged
RPTP
and Myc-tagged RPTP
(Fig.
4A). RPTP
-RPTP
heterodimerization is only detected by co-immunoprecipitation after
H2O2 treatment, similar to RPTP
homodimerization (19) (Fig. 4A). Accessibility studies with
HA-RPTP
showed a large accessible fraction in the control situation
and no increase after H2O2 treatment (Fig.
4B), presumably because the RPTP
ectodomain is too short
to mask the HA tag. Furthermore, as shown in Fig. 4B,
co-transfection of HA-RPTP
and HA-RPTP
led to an increase in
accessibility of HA-RPTP
consistent with the inability of the
RPTP
ectodomain to mask the HA tag of RPTP
in RPTP
-RPTP
heterodimers. These results suggest that the extracellular domains are
responsible for steric hindrance of antibody binding to the RPTP
dimer.
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Fig. 4.
The ectodomain of RPTP
is responsible for masking the epitope tag. A, 293HEK
cells were transiently transfected with HA-RPTP
, Myc-RPTP
,
HA-RPTP
, and Myc-RPTP
or HA-RPTP
and Myc-RPTP
. The cells
were treated for 5 min with 1 mM
H2O2 (+) or left untreated (
) lysed, and
HA-RPTP
or HA-RPTP
was immunoprecipitated (IP) from
the lysate, using anti-HA antibody (anti-HA, 12CA5). The
samples were run on SDS-PAGE gels, and the immunoblots were labeled
with anti-Myc antibody (anti-MT, 9E10) (top
panel). The blots were stripped and reprobed with anti-HA antibody
to monitor immunoprecipitation of equal amounts of the HA-tagged
proteins (middle panel). To check for expression of
Myc-tagged RPTP
, samples of the whole cell lysate (WCL)
were run on SDS-PAGE gels, and the immunoblots were labeled with
anti-Myc antibody (bottom panel). B, 293HEK cells
transiently transfected with HA-RPTP
, HA-RPTP
, or both HA-RPTP
and HA-RPTP
were treated for 5 min with 1 mM
H2O2 (+) or left untreated (
), and the
accessible (a, top panels) and nonaccessible
(n, bottom panels) fractions of RPTP
and
RPTP
were isolated. The immunoblots were labeled with anti-HA
antibody. The positions of HA-RPTP
(~130 kDa) and HA-RPTP
(~100 kDa) are indicated on the right.
constructs with a deletion of the entire wedge (31 residues)
or the tip of the wedge (11 residues) (Fig.
5A), which is known to abolish
dimer formation (14), were found to be insensitive to
H2O2 treatment and showed a large accessible
fraction in the control situation (Fig. 5B). Deletion of
8-11-residue sequences elsewhere in the juxtamembrane domain did not
affect accessibility (Fig. 5B), indicating that the effects
of the wedge deletions were specific. These results indicate that dimer
formation of HA-RPTP
is required for an interaction of the
ectodomains in the dimer, leading to steric hindrance of antibody
binding to the HA tag.
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Fig. 5.
Mutation of the wedge increased ectodomain
accessibility in RPTP . A,
schematic representation of the deletion constructs in the wedge
(residues 208-240) and juxtamembrane region (residues 167-207).
WT, wild type. B, the accessible (a,
top panels) and nonaccessible (n, bottom
panels) fractions of RPTP
were isolated from 293HEK cells,
transiently transfected with HA-tagged RPTP
-WT or mutants of RPTP
with deletions of the entire wedge (RPTP
-
207-238), of the tip of
the wedge (RPTP
-
224-235), or of small deletions of the
juxtamembrane domain (RPTP
-
175-182, RPTP
-
183-194, and
RPTP
-
195-206). The cells were treated for 5 min with 1 mM H2O2 (+) or left untreated (
).
HA-RPTP
was detected in the different fractions by labeling the
immunoblot with anti-HA antibody.
with deletions of various lengths in the
extracellular domain (Fig. 6A)
were used to map the site that is responsible for masking the HA tag.
Only the RPTP
-
20-130 mutant was insensitive to
H2O2 treatment and showed a large accessible fraction, whereas all other deletions, including
43-142 behaved similar to full-length HA-RPTP
in that H2O2
induced accessibility of the HA tag (Fig. 6B). Therefore,
residues 20-42, close to the HA tag, which was inserted at
position 19, were required for steric hindrance of antibody
binding.
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Fig. 6.
Mapping of the region responsible for masking
the epitope tag. A, schematic representation of the deletion
constructs in the ectodomain of RPTP . B, the accessible
(a, top panels) and nonaccessible (n,
bottom panels) fractions of RPTP
were isolated from
293HEK cells, transiently transfected with HA-tagged RPTP
-WT and
RPTP
mutants with deletion of different parts of the extracellular
domain (RPTP
-
20-130, RPTP
-
43-142, RPTP
-
63-142,
RPTP
-
93-142, and RPTP
-
118-142). The cells were treated
for 5 min with 1 mM H2O2 (+) or
left untreated (
). Immunoblots probed with affinity purified
anti-RPTP
antibody show the amount of RPTP
in the different
fractions. The molecular masses of the deletion mutants range from
~85 to ~120 kDa. WT, wild type.
-D2, resulting in the formation of more
stable dimers, which is reversible (19). To investigate the
reversibility of the H2O2-induced change in
ectodomain accessibility, HA-RPTP
-transfected cells were stimulated
with H2O2. After 10 min the medium was changed,
and the cells were left to recover for up to 2 h. As shown in Fig.
7A,
H2O2 treatment induced a reversible change in
HA tag accessibility, which is restored within 2 h after removing
the H2O2 from the medium. Similar recovery
times were found for stabilization of dimers as detected in
co-immunoprecipitation experiments using cells transfected with both
Myc-tagged and HA-tagged RPTP
(Fig. 7B). These results suggest that both processes may be due to similar mechanisms.
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Fig. 7.
H2O2-induced epitope
accessibility is transient. A, 293HEK cells transiently
transfected with HA-tagged RPTP were treated for 10 min with
H2O2 (+) or left untreated (
). The medium was
replaced by medium without H2O2, and the cells
were left to recover for the times indicated (recovery). The
accessible (a, top panel) and nonaccessible
(n, bottom panel) fractions of RPTP
were
isolated. The amount of RPTP
in the different fractions was
determined by labeling the immunoblot with anti-RPTP
antibody.
B, 293HEK cells were transiently transfected with both HA-
and Myc-tagged RPTP
(lanes 2-8) or only Myc-tagged
RPTP
as a control (lane 1). The cells were treated with 1 mM H2O2 for the times indicated,
and the cells were harvested, or the medium was replaced by medium
without H2O2 to allow the cells to recover for
the times indicated (recovery). The cells were lysed, and
HA-RPTP
was immunoprecipitated using anti-HA antibody.
Co-immunoprecipitating Myc-tagged RPTP
was detected by
immunoblotting using anti-Myc antibody (MT, top
panel). The amount of transfected MT-RPTP
and HA-RPTP
was
monitored by immunoblotting of whole cell lysates (WCL) with
anti-Myc antibody (middle panel) or anti-HA antibody
(bottom panel).
-D2
(Cys723) was responsible for the
H2O2-induced change in conformation (19). In
line with this, we found no H2O2-induced
increase in epitope accessibility after mutation of Cys723
(Fig. 8A) or deletion of the
entire D2 (Fig. 8B). Both RPTP
-C723S and RPTP
-
D2
were expressed on the plasma membrane, because both proteins were
readily biotinylated using sulfo-NHS-biotin (Fig. 8C). In
conclusion, these results suggest that a change in conformation of D2
by H2O2 treatment leads to a rotation in the
dimer, thereby changing the antibody binding characteristics of the HA
tag in the N-terminal part of the ectodomain.
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[in a new window]
Fig. 8.
The catalytic site Cys723 in
RPTP -D2 is required for the
H2O2-induced increase in accessibility.
293HEK cells were transiently transfected with either wild type
HA-RPTP
, HA-RPTP
-C723S (A), or RPTP
-
D2, which
lacks RPTP
-D2 (B). The cells were treated for 5 min with
1 mM H2O2 (+) or left untreated
(
). The accessible (a) and nonaccessible (n)
fractions of RPTP
were isolated. The immunoblots depicted here were
probed with anti-HA antibody and developed using ECL. The position of
full-length RPTP
(WT) and of RPTP
-
D2 (
D2) are
indicated in B. C, HEK293 cells were transiently
transfected with RPTP
, RPTP
-C723S, or RPTP
-
D2. The membrane
proteins were covalently biotinylated using sulfo-NHS-LC-biotin, and
RPTP
was immunoprecipitated using anti-HA antibody. Biotinylated
RPTP
was detected by labeling the immunoblots with avidin-biotin
complexes. The positions of wild type RPTP
/RPTP
-C723S
(WT/C723S) and RPTP
-
D2 (
D2) are
indicated on the immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dimers changed in response to H2O2
treatment, reflecting the catalytic state of RPTP
, active or
inactive. We provide evidence that the catalytic site cysteine in the
membrane-distal PTP domain, RPTP
-D2, is required for this effect.
Our results suggest that redox signaling regulates rotational coupling
of RPTP
dimers, thereby reversibly switching RPTP
from an active
to an inactive dimeric state.
on living cells. Surprisingly, this assay
allowed us to discriminate between active and inactive constitutively
dimeric conformations of RPTP
(Fig. 2). Moreover, H2O2 treatment of cells significantly increased
the accessibility of the HA tag in HA-RPTP
(Fig. 3), concomitant
with a loss of catalytic activity in response to
H2O2 treatment. The effect of H2O2 treatment on the antibody binding
characteristics of the HA-tagged ectodomain is dependent on
Cys723 in RPTP
-D2 (Fig. 8), suggesting that the
conformational change in RPTP
-D2 that we reported previously (19) is
involved. The N-terminal part of the extracellular domain was required
for steric hindrance of antibody binding to the epitope tag, as shown
using mutants with deletions in the extracellular domain. It is
noteworthy that deletion of residues 43-142, rendering an
extracellular domain of only 23 residues, which is of a size similar to
that of the ectodomain of RPTP
, masked the epitope tag in
RPTP
-
43-142, whereas the ectodomain of RPTP
did not.
Especially residues immediately to the C-terminal side of the epitope
tag are involved in masking, because deletion of residues 20-130, but
not residues 43-142, rendered the HA tag accessible (Fig. 6), raising
the possibility that intramolecular interactions are involved. However,
H2O2-induced accessibility of the epitope tag
is reversible, and the kinetics are highly similar to
H2O2-induced stabilization of RPTP
dimers (Fig. 7), suggesting that dimerization is required for steric hindrance
of antibody binding. In addition, deletions in the wedge structure that
annihilate dimerization of RPTP
(14) specifically abolished masking
of the epitope tag (Fig. 5). Moreover, the short extracellular domain
of RPTP
did not mask the RPTP
ectodomain in RPTP
-RPTP
heterodimers. Therefore, we conclude that intermolecular interactions,
likely mediated by direct interactions of the ectodomains in RPTP
dimers, mask the epitope tag in the extracellular domain of
RPTP
.
dimers (Fig. 9). In the prestimulation
state RPTP
exists as a preformed, active dimer. Using fluorescence
resonance energy transfer and chemical cross-linkers, we have
demonstrated that RPTP
dimerization is extensive in the
prestimulation state (15). The fact that the epitope tag is not
accessible in the prestimulation state again suggests that most RPTP
is dimeric. In addition to a conformational change in RPTP
-D2
leading to stabilization of RPTP
dimers (19), we demonstrate here
that H2O2 treatment alters the conformation of
the extracellular domain from a state resembling active RPTP
-F135C to a state resembling inactive RPTP
-P137C. Our results suggest that
oxidative stress induces a rapid change in rotational coupling, which
slowly reverts to the prestimulation state upon reduction of
RPTP
.
View larger version (34K):
[in a new window]
Fig. 9.
Rotational coupling of
RPTP is regulated by redox signaling. The
epitope tag in active RPTP
is masked under normal conditions
(left situation). Oxidation of RPTP
leads to a
conformational change of the intracellular D2 domain of RPTP
(right situation), leading to stabilization of RPTP
dimers, which are inactive. The change in conformation of D2 results in
a change in binding characteristics of the epitope tag in the
ectodomain. This process is reversible, in that reduction of RPTP
allows the complex to return to its prestimulation conformation. The
two rotational coupling states are mimicked by the two constitutively
dimeric RPTP
mutants with a disulfide bond in the extracellular
domain. Active RPTP
-F135C resembles the active, reduced state
(left), and inactive RPTP
-P137C resembles the inactive,
oxidized form (right).
The function of the extracellular domain of RPTP remains to be
determined. Both RPTP
and RPTP
are characterized by their short,
highly glycosylated ectodomains. The extracellular domain may serve to
stabilize the RPTP in the plasma membrane. Subcellular localization may
be a mechanism to provide substrate selectivity for PTPs. This has been
established by localization-function studies with RPTP
and RPTP
and their corresponding cytoplasmic forms (24) and in studies
addressing the effect of calpain-induced cleavage of RPTP
and
RPTP
that occurs in the intracellular juxtamembrane domain of the
RPTPs (25). However, previous studies with EGFR-RPTP
chimeras
suggest that the RPTP
ectodomain does not solely serve as a membrane
localizer but may also confer ligand-dependent regulatory mechanisms to RPTP
(26).
A conformational change of the ectodomain as the result of a change in
conformation of the intracellular C-terminal domain may change binding
characteristics of RPTP to its putative ligand. Conversely, ligands
may bind to the extracellular domain of RPTP
, thereby changing the
conformation of the extracellular domain and shifting rotational
coupling, leading to a state resembling the stabilized dimer
conformation in the cytoplasmic domain and thus to inactivation of
RPTP
activity. However, bona fide ligands have not been
reported yet for RPTP
. Only the GPI-linked protein contactin was
reported to bind to the ectodomain of RPTP
in cis (27).
However, co-transfection of contactin did not affect accessibility of
RPTP
in our hands (data not shown). Our results suggest that RPTP
is capable of conferring a signal from inside cells outwards. Inside-out signaling is a new concept for RPTPs; however, this phenomenon is well known for signal transduction by integrins (reviewed
in Refs. 28 and 29). Integrins not only transduce signals in response
to extracellular matrix interactions from the outside of the cells
inwards, but there is also information flowing from the inside of the
cells outwards. Different cellular conditions affect the
conformation of the extracellular domain of integrins, thereby
affecting their affinity for their ligands. Our results suggest that
RPTPs may be regulated in a similar fashion.
Redox signaling is emerging as an important regulator of PTP activity
(30). Many PTPs have been demonstrated to be inactivated by oxidation
of their catalytic site cysteines (31-34). Moreover, reactive oxygen
species (ROS) are produced in response to physiological stimuli, such
as growth factors, and the levels of ROS are sufficient to inactivate
PTPs (35-38). Regulation of PTPs by ROS is rapid and reversible. It
appears that regulation of RPTP and perhaps other RPTPs by ROS is
more complex, involving not only direct oxidation of the catalytic site
cysteine. Previously, we demonstrated that H2O2
induced stabilization of RPTP
dimerization, which is responsible for
complete inactivation of RPTP
. Here, we demonstrate a change in the
ectodomain of RPTP
in response to H2O2.
Presumably, reduction of the catalytic site cysteine after oxidation is
rapid in cells because of the highly reducing intracellular milieu. The
H2O2-induced conformational changes revert
slowly to the prestimulation state and may help to sustain RPTP
in
an inactive conformation.
Not only RPTP but also other RPTPs may be regulated by
conformational changes in the cytoplasmic domain. We have recently shown that H2O2 induced a conformational change
in LAR-D2 as well (39). It will be interesting to see whether the
conformational change in LAR-D2 is reflected by a conformational change
of the ectodomain. Taken together, our results suggest that redox
signaling regulates rotational coupling of RPTP
implicating that
RPTP
has the capacity for inside-out signaling. It will be
interesting to see whether redox signaling regulates rotational
coupling of other RPTPs as well.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jan Sap for the RPTP/
mouse
embryo fibroblasts and Ari Elson for the RPTP
cDNA.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Research Council for Earth and Life sciences with financial aid from the Netherlands Organization for scientific research (to C. B.) and by the Dutch Cancer Society/Koningin Wilhelmina Fonds (to T. v. d. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
31-302121800; Fax: 31-302516464; E-mail: hertog@niob.knaw.nl.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300632200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PTK, protein-tyrosine kinase; HA, hemagglutinin; PTP, protein-tyrosine phosphatase; EGFR, epidermal growth factor receptor; HEK, human embryonic kidney; LAR, leukocyte common antigen-related; RPTK, receptor-like PTK; RPTP, receptor-like PTP; ROS, reactive oxygen species.
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