Redox-regulated Rotational Coupling of Receptor Protein-tyrosine Phosphatase alpha  Dimers*

Thea van der Wijk, Christophe Blanchetot, John Overvoorde, and Jeroen den HertogDagger

From the Hubrecht Laboratory, Netherland Institute for Developmental Biology, Uppsalalaan 8, Utrecht, 3584 CT, The Netherlands

Received for publication, January 21, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Receptor protein-tyrosine phosphatase alpha  (RPTPalpha ) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTPalpha revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTPalpha and of constitutively dimeric, active RPTPalpha -F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTPalpha -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 RPTPalpha that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTPalpha -D2). Here we report that H2O2 treatment of cells induced a change in rotational coupling in RPTPalpha dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys723 in RPTPalpha -D2, which was required for the conformational change of RPTPalpha -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 RPTPalpha led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 RPTPbeta , leukocyte common antigen-related (LAR), and PTPsigma 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 RPTPbeta /zeta activity, in that beta -catenin phosphorylation was enhanced in cells expressing RPTPbeta /zeta in response to pleiotrophin (7).

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 RPTPalpha by dimerization. The crystal structure of RPTPalpha -D1 provided structural evidence that dimerization leads to inhibition of the catalytic activity of RPTPalpha 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 RPTPalpha leading to the formation of a stable intermolecular disulfide bridge (12). A disulfide bridge at position 137 (RPTPalpha -P137C) resulted in reduced substrate dephosphorylation, whereas constructs with a disulfide bridge at position 135 (RPTPalpha -F135C) had activities similar to the wild type construct. These data indicate that dimerization of RPTPalpha 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 RPTPalpha (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 RPTPalpha 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.

Recent studies indicate that the second catalytic domain of RPTPalpha , RPTPalpha -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 RPTPalpha -D2, Cys723, suggesting that direct oxidation of this residue underlies the observed effects.

Here we report that antibody binding to the HA tag within the N-terminal part of the ectodomain of plasma membrane localized RPTPalpha reflected the state of rotational coupling. Although the epitope tag was masked in wild type RPTPalpha and constitutively dimeric, active RPTPalpha -F135C with a single cysteine in the extracellular domain, the epitope tag in inactive dimeric RPTPalpha -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 RPTPalpha . Masking of the epitope tag was dependent on residues 20-42 in the extracellular domain and on dimerization of RPTPalpha . RPTPalpha -D2 and more specifically the catalytic site Cys in RPTPalpha -D2, Cys723, were required for H2O2-induced HA tag accessibility. Our results suggest that a conformational change in the cytoplasmic domain of RPTPalpha leads to a change in the conformation of the ectodomain of RPTPalpha and therefore provide evidence that RPTPs have the potential for inside-out signaling.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Constructs-- SV40-driven expression vectors have been described for full-length HA-RPTPalpha (20), HA-RPTPalpha -F135C, HA-RPTPalpha -P137C (12), HA-RPTPalpha -Delta 224-235, HA-RPTPalpha -Delta 207-238 (14), HA-RPTPalpha -C723S, HA-RPTPalpha -Delta D2 (21), and Myc-RPTPalpha (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-RPTPepsilon was constructed by fusing the RPTPalpha signal sequence (residues 1-19) and HA epitope tag to RPTPepsilon (from residue 20 to the end).

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 RPTPalpha -/- mice (22) were a kind gift of Jan Sap. These cells were stably co-transfected using LipofectAMINE with HA-tagged wild type RPTPalpha , RPTPalpha -F135C, or RPTPalpha -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) RPTPalpha .

Immunoprecipitation of Accessible and Nonaccessible HA-RPTPalpha -- 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.

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-RPTPalpha 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.

Biotinylation of Membrane-localized RPTPalpha -- 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.

Total RPTPalpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 RPTPalpha , we studied HA antibody binding on living mouse embryo fibroblasts derived from RPTPalpha -/- mice (22), stably expressing N-terminally HA-tagged wild type RPTPalpha or constitutive dimer mutants with a disulfide bridge in the extracellular domain (RPTPalpha -F135C and RPTPalpha -P137C) as described under "Experimental Procedures" (Fig. 1). In short, we incubated cells stably expressing HA-tagged RPTPalpha 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 RPTPalpha in the lysate is the accessible fraction (a). The rest of the HA-tagged RPTPalpha was precipitated from the supernatant and represents the nonaccessible fraction (n). Surprisingly, almost no wild type HA-RPTPalpha was detected in the accessible fraction (Fig. 2A). Constitutively dimeric HA-RPTPalpha -F135C, which is catalytically active (12), behaved similarly to wild type HA-RPTPalpha with very little signal in the accessible fraction. In contrast, inactive dimeric HA-RPTPalpha -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 RPTPalpha in Triton-soluble fractions.


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Fig. 1.   Separation of accessible and nonaccessible HA-tagged RPTPalpha . Cells expressing HA-tagged (mutant) RPTPalpha 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-RPTPalpha 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 RPTPalpha dimers determines ectodomain epitope tag accessibility. A, accessible (a) and nonaccessible (n) RPTPalpha was isolated as described in Fig. 1 from mouse embryo fibroblasts, derived from RPTPalpha -/- mice, stably transfected with either wild type HA-RPTPalpha (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 RPTPalpha was immunoprecipitated from mouse embryo fibroblasts, stably transfected with either wild type RPTPalpha (WT) or constitutive dimer mutants (F135C and P137C). Biotinylated RPTPalpha was detected by labeling the immunoblots with avidin-biotin complexes. Three independent stably transfected clones expressing either wild type or mutant RPTPalpha were tested in these assays with similar results. Representative examples of these experiments are depicted here.

The variation in accessibility of the HA tag between the constructs might be due to a difference in subcellular localization of the HA-RPTPalpha mutants. To investigate this possibility, we biotinylated the membrane proteins using EZ-linked sulfo-NHS-biotin. Subsequently, we immunoprecipitated RPTPalpha and detected membrane-localized RPTPalpha with an avidin-biotin complex. All three proteins, wild type RPTPalpha 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-RPTPalpha and constitutively dimeric HA-RPTPalpha -F135C, despite the fact that these proteins were expressed on the cell surface as shown by the biotinylation experiments. In contrast, HA-RPTPalpha -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-RPTPalpha and HA-RPTPalpha -F135C but not in HA-RPTPalpha -P137C.

H2O2-induced Change in Ectodomain Accessibility of RPTPalpha -- The accessibility of the HA tag of the forced RPTPalpha 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 RPTPalpha , which was dependent on the catalytic site cysteine in RPTPalpha -D2 (Cys723) and resulted in stabilization of RPTPalpha dimers (19). We wondered whether H2O2 treatment of cells expressing wild type HA-RPTPalpha changed the accessibility of the HA tag. H2O2 induced a remarkable increase in the accessible fraction of RPTPalpha (Fig. 3A). Biotinylation of cell surface proteins and detection of the amount of biotinylated HA-RPTPalpha demonstrated that there is no significant change in membrane localization of RPTPalpha after H2O2 treatment (Fig. 3B). By first biotinylating membrane proteins and subsequently separating the accessible and nonaccessible fraction of HA-RPTPalpha , we found that a large part of the biotinylated HA-RPTPalpha 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-RPTPalpha is present on the cell membrane and that the H2O2-induced increase in accessibility is not due to an increase in membrane localization of RPTPalpha or to an increase in amount of RPTPalpha to more accessible membrane sub-domains (Fig. 3, B and C). Expression of different amounts of RPTPalpha by transfecting variable quantities of DNA in 293HEK cells did not change the ratio of accessible and inaccessible RPTPalpha 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 RPTPalpha -P137C and RPTPalpha -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 RPTPalpha .


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Fig. 3.   Increased accessibility of HA-RPTPalpha in response to H2O2 treatment. Mouse embryo fibroblasts stably transfected with HA-tagged RPTPalpha -WT were treated for 5 min with 1 mM H2O2 (+) or left untreated (-). A, the accessible (a) and nonaccessible (n) fractions of RPTPalpha were isolated. Immunoblots are labeled with anti-HA antibody. B, membrane proteins were covalently biotinylated with sulfo-NHS-LC-biotin, and RPTPalpha was precipitated. The immunoblot was labeled with avidin-biotin complexes. C, after biotinylation of membrane proteins, the accessible (a) and nonaccessible (n) fractions of RPTPalpha were isolated. The immunoblot was labeled with avidin-biotin complexes. D, 293HEK cells were transiently transfected with different amounts of DNA encoding HA-RPTPalpha 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 RPTPalpha were isolated. Immunoblots probed with anti-HA antibody and developed by ECL are depicted.

Masking of the Ectodomain Requires RPTPalpha Dimerization-- RPTPepsilon is highly homologous to RPTPalpha but differs from RPTPalpha in its ectodomain (27 residues for RPTPepsilon and 123 residues for RPTPalpha ). RPTPalpha and RPTPepsilon form stable heterodimers upon H2O2 treatment as tested by a co-immunoprecipitation experiment with cells transfected with HA-tagged RPTPepsilon and Myc-tagged RPTPalpha (Fig. 4A). RPTPalpha -RPTPepsilon heterodimerization is only detected by co-immunoprecipitation after H2O2 treatment, similar to RPTPalpha homodimerization (19) (Fig. 4A). Accessibility studies with HA-RPTPepsilon showed a large accessible fraction in the control situation and no increase after H2O2 treatment (Fig. 4B), presumably because the RPTPepsilon ectodomain is too short to mask the HA tag. Furthermore, as shown in Fig. 4B, co-transfection of HA-RPTPalpha and HA-RPTPepsilon led to an increase in accessibility of HA-RPTPalpha consistent with the inability of the RPTPepsilon ectodomain to mask the HA tag of RPTPalpha in RPTPalpha -RPTPepsilon heterodimers. These results suggest that the extracellular domains are responsible for steric hindrance of antibody binding to the RPTPalpha dimer.


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Fig. 4.   The ectodomain of RPTPalpha is responsible for masking the epitope tag. A, 293HEK cells were transiently transfected with HA-RPTPalpha , Myc-RPTPalpha , HA-RPTPepsilon , and Myc-RPTPalpha or HA-RPTPalpha and Myc-RPTPalpha . The cells were treated for 5 min with 1 mM H2O2 (+) or left untreated (-) lysed, and HA-RPTPalpha or HA-RPTPepsilon 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 RPTPalpha , 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-RPTPalpha , HA-RPTPepsilon , or both HA-RPTPalpha and HA-RPTPepsilon 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 RPTPalpha and RPTPepsilon were isolated. The immunoblots were labeled with anti-HA antibody. The positions of HA-RPTPalpha (~130 kDa) and HA-RPTPepsilon (~100 kDa) are indicated on the right.

HA-RPTPalpha 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-RPTPalpha 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 RPTPalpha . 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 RPTPalpha were isolated from 293HEK cells, transiently transfected with HA-tagged RPTPalpha -WT or mutants of RPTPalpha with deletions of the entire wedge (RPTPalpha -Delta 207-238), of the tip of the wedge (RPTPalpha -Delta 224-235), or of small deletions of the juxtamembrane domain (RPTPalpha -Delta 175-182, RPTPalpha -Delta 183-194, and RPTPalpha -Delta 195-206). The cells were treated for 5 min with 1 mM H2O2 (+) or left untreated (-). HA-RPTPalpha was detected in the different fractions by labeling the immunoblot with anti-HA antibody.

Constructs of HA-RPTPalpha 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 RPTPalpha -Delta 20-130 mutant was insensitive to H2O2 treatment and showed a large accessible fraction, whereas all other deletions, including Delta 43-142 behaved similar to full-length HA-RPTPalpha 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 RPTPalpha . B, the accessible (a, top panels) and nonaccessible (n, bottom panels) fractions of RPTPalpha were isolated from 293HEK cells, transiently transfected with HA-tagged RPTPalpha -WT and RPTPalpha mutants with deletion of different parts of the extracellular domain (RPTPalpha -Delta 20-130, RPTPalpha -Delta 43-142, RPTPalpha -Delta 63-142, RPTPalpha -Delta 93-142, and RPTPalpha -Delta 118-142). The cells were treated for 5 min with 1 mM H2O2 (+) or left untreated (-). Immunoblots probed with affinity purified anti-RPTPalpha antibody show the amount of RPTPalpha in the different fractions. The molecular masses of the deletion mutants range from ~85 to ~120 kDa. WT, wild type.

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 RPTPalpha -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-RPTPalpha -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 RPTPalpha (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 RPTPalpha 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 RPTPalpha were isolated. The amount of RPTPalpha in the different fractions was determined by labeling the immunoblot with anti-RPTPalpha antibody. B, 293HEK cells were transiently transfected with both HA- and Myc-tagged RPTPalpha (lanes 2-8) or only Myc-tagged RPTPalpha 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-RPTPalpha was immunoprecipitated using anti-HA antibody. Co-immunoprecipitating Myc-tagged RPTPalpha was detected by immunoblotting using anti-Myc antibody (MT, top panel). The amount of transfected MT-RPTPalpha and HA-RPTPalpha was monitored by immunoblotting of whole cell lysates (WCL) with anti-Myc antibody (middle panel) or anti-HA antibody (bottom panel).

We showed that the catalytic site cysteine in RPTPalpha -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 RPTPalpha -C723S and RPTPalpha -Delta 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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Fig. 8.   The catalytic site Cys723 in RPTPalpha -D2 is required for the H2O2-induced increase in accessibility. 293HEK cells were transiently transfected with either wild type HA-RPTPalpha , HA-RPTPalpha -C723S (A), or RPTPalpha -Delta D2, which lacks RPTPalpha -D2 (B). The cells were treated for 5 min with 1 mM H2O2 (+) or left untreated (-). The accessible (a) and nonaccessible (n) fractions of RPTPalpha were isolated. The immunoblots depicted here were probed with anti-HA antibody and developed using ECL. The position of full-length RPTPalpha (WT) and of RPTPalpha -Delta D2 (Delta D2) are indicated in B. C, HEK293 cells were transiently transfected with RPTPalpha , RPTPalpha -C723S, or RPTPalpha -Delta D2. The membrane proteins were covalently biotinylated using sulfo-NHS-LC-biotin, and RPTPalpha was immunoprecipitated using anti-HA antibody. Biotinylated RPTPalpha was detected by labeling the immunoblots with avidin-biotin complexes. The positions of wild type RPTPalpha /RPTPalpha -C723S (WT/C723S) and RPTPalpha -Delta D2 (Delta D2) are indicated on the immunoblot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we report that the conformation of the extracellular domain of RPTPalpha dimers changed in response to H2O2 treatment, reflecting the catalytic state of RPTPalpha , active or inactive. We provide evidence that the catalytic site cysteine in the membrane-distal PTP domain, RPTPalpha -D2, is required for this effect. Our results suggest that redox signaling regulates rotational coupling of RPTPalpha dimers, thereby reversibly switching RPTPalpha from an active to an inactive dimeric state.

We developed an assay to assess accessibility of the epitope tag in the ectodomain of HA-RPTPalpha on living cells. Surprisingly, this assay allowed us to discriminate between active and inactive constitutively dimeric conformations of RPTPalpha (Fig. 2). Moreover, H2O2 treatment of cells significantly increased the accessibility of the HA tag in HA-RPTPalpha (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 RPTPalpha -D2 (Fig. 8), suggesting that the conformational change in RPTPalpha -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 RPTPepsilon , masked the epitope tag in RPTPalpha -Delta 43-142, whereas the ectodomain of RPTPepsilon 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 RPTPalpha 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 RPTPalpha (14) specifically abolished masking of the epitope tag (Fig. 5). Moreover, the short extracellular domain of RPTPepsilon did not mask the RPTPalpha ectodomain in RPTPalpha -RPTPepsilon heterodimers. Therefore, we conclude that intermolecular interactions, likely mediated by direct interactions of the ectodomains in RPTPalpha dimers, mask the epitope tag in the extracellular domain of RPTPalpha .

Taken together, based on our results, we propose a model for the effects of H2O2 on the conformation of RPTPalpha dimers (Fig. 9). In the prestimulation state RPTPalpha exists as a preformed, active dimer. Using fluorescence resonance energy transfer and chemical cross-linkers, we have demonstrated that RPTPalpha 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 RPTPalpha is dimeric. In addition to a conformational change in RPTPalpha -D2 leading to stabilization of RPTPalpha dimers (19), we demonstrate here that H2O2 treatment alters the conformation of the extracellular domain from a state resembling active RPTPalpha -F135C to a state resembling inactive RPTPalpha -P137C. Our results suggest that oxidative stress induces a rapid change in rotational coupling, which slowly reverts to the prestimulation state upon reduction of RPTPalpha .


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Fig. 9.   Rotational coupling of RPTPalpha is regulated by redox signaling. The epitope tag in active RPTPalpha is masked under normal conditions (left situation). Oxidation of RPTPalpha leads to a conformational change of the intracellular D2 domain of RPTPalpha (right situation), leading to stabilization of RPTPalpha 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 RPTPalpha allows the complex to return to its prestimulation conformation. The two rotational coupling states are mimicked by the two constitutively dimeric RPTPalpha mutants with a disulfide bond in the extracellular domain. Active RPTPalpha -F135C resembles the active, reduced state (left), and inactive RPTPalpha -P137C resembles the inactive, oxidized form (right).

The function of the extracellular domain of RPTPalpha remains to be determined. Both RPTPalpha and RPTPepsilon 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 RPTPalpha and RPTPepsilon and their corresponding cytoplasmic forms (24) and in studies addressing the effect of calpain-induced cleavage of RPTPalpha and RPTPepsilon that occurs in the intracellular juxtamembrane domain of the RPTPs (25). However, previous studies with EGFR-RPTPalpha chimeras suggest that the RPTPalpha ectodomain does not solely serve as a membrane localizer but may also confer ligand-dependent regulatory mechanisms to RPTPalpha (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 RPTPalpha to its putative ligand. Conversely, ligands may bind to the extracellular domain of RPTPalpha , 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 RPTPalpha activity. However, bona fide ligands have not been reported yet for RPTPalpha . Only the GPI-linked protein contactin was reported to bind to the ectodomain of RPTPalpha in cis (27). However, co-transfection of contactin did not affect accessibility of RPTPalpha in our hands (data not shown). Our results suggest that RPTPalpha 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 RPTPalpha 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 RPTPalpha dimerization, which is responsible for complete inactivation of RPTPalpha . Here, we demonstrate a change in the ectodomain of RPTPalpha 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 RPTPalpha in an inactive conformation.

Not only RPTPalpha 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 RPTPalpha implicating that RPTPalpha 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 RPTPalpha -/- mouse embryo fibroblasts and Ari Elson for the RPTPepsilon 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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