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. Author manuscript; available in PMC: 2026 Aug 28.
Published in final edited form as: J Neurochem. 2009 Jul 1;110(5):1635–1647. doi: 10.1111/j.1471-4159.2009.06261.x

Dimerization of tyrosine phosphatase PTPRO decreases its activity and ability to inactivate TrkC*

Amy E Hower 1,2, Pedro J Beltran 2,3,5, John L Bixby 1,2,3,4
PMCID: PMC13521113  NIHMSID: NIHMS2202283  PMID: 19573017

Abstract

Receptor protein tyrosine phosphatases (RPTPs), like receptor tyrosine kinases, regulate neuronal differentiation. While receptor tyrosine kinases are dimerized and activated by extracellular ligands, the extent to which RPTPs dimerize, and the effects of dimerization on phosphatase activity, are poorly understood. We have examined a neuronal type III RPTP, PTPRO; we find that PTPRO can form dimers in living cells, and that disulfide linkages in PTPRO’s intracellular domain likely regulate dimerization. Dimerization of PTPRO’s transmembrane and intracellular domains, achieved by ligand binding to a chimeric fusion protein, decreases activity towards artificial peptides and towards a putative substrate, TrkC. Dephosphorylation of TrkC by PTPRO may be physiologically relevant, as it is efficient, and TrkC and PTPRO can be co-precipitated from transfected cells. Inhibition of PTPRO’s phosphatase activity by dimerization is interesting, as dimerization of a related RPTP, CD148/PTPRJ, increases activity. Thus, our results suggest a complex relationship between dimerization and activity in type III RPTPs.

Keywords: tyrosine phosphorylation, neurotrophin, CD148, cell adhesion, PTP, CRYP-2

INTRODUCTION:

Activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs) regulate many aspects of neuronal differentiation (Bixby 2000, Johnson & Van Vactor 2003, Stoker 2001, Chisholm & Tessier-Lavigne 1999). Receptor tyrosine kinase (RTKs) are controlled by extracellular ligands that dimerize and activate them (Ullrich & Schlessinger 1990, Heldin 1995, Weiss & Schlessinger 1998). However, ligand regulation of transmembrane receptor PTPs (RPTPs), is poorly understood. There are 21 known RPTPs, divided into five structural classes, based primarily on motifs in their extracellular domains (ECDs) (Alonso et al. 2004). In most cases, the ligands for these RPTPs, the regulation of their dimerization state, and the effects of dimerization on activity are unknown. We have focused on the type III RPTP, PTPRO, which is most strongly expressed in the developing nervous system (Bodden & Bixby 1996, Beltran et al. 2003, Thomas et al. 1994, Tagawa et al. 1997) and regulates axon guidance during development (Stepanek et al. 2005, Shintani et al. 2006).

Several studies have examined dimerization of RPTPs. Dimerization in vivo has been demonstrated for CD45/PTPRC, and homodimerization has been shown to occur with several RPTPs overexpressed in tissue culture cells (Jiang et al. 2000, Takeda et al. 1992, Xu & Weiss 2002, Walchli et al. 2005, Lee et al. 2007). For PTP-α/PTPRA, dimerization is mediated in part by the transmembrane domain (TMD) (Tertoolen et al. 2001); this mechanism has also been proposed for PTP-σ/PTPRS and for the transmembrane PTPRR isoforms (Lee et al. 2007). Dimerization through TMD interactions has been suggested as a general mechanism for RPTPs (Chin et al. 2005), but has not been tested for most native RPTPs. In fact, dimerization of the type III RPTP, PTPRH, appears to be mediated through its ECD (Walchli et al. 2005). The dimerization of RPTPs has physiological significance, since dimerization state can be regulated both by extracellular ligands (den Hertog et al. 1999) and by changes in oxidation state (den Hertog et al. 2005).

Dimerization of PTPRA or PTPRC results in a decrease in PTPase activity (Jiang et al. 1999, Desai et al. 1993, Xu & Weiss 2002, Takeda et al. 2004). This decrease is thought to be mediated by a helix-loop-helix region on the intracellular domain (ICD), called an inhibitory wedge (Bilwes et al. 1996, Majeti et al. 1998). However this inhibitory wedge region is not highly conserved among RPTPs, and is unlikely to be a general mechanism through which dimerization regulates activity (Hoffmann et al. 1997, Nam et al. 1999, Nam et al. 2005, Barr et al. 2009).

Dimerization and its consequences for PTPase activity are poorly understood for type III RPTPs. The 5 mammalian type III RPTPs are characterized by variable numbers of FN type III repeats in their ECDs, and by a single intracellular catalytic motif. Of this group, only SAP-1/PTPRH has been shown to dimerize in cells, whereas dimerization of another type III RPTP (PTP-β/PTPRB) was not seen using the same methodology (Walchli et al. 2005). The relationship between dimerization and PTPase activity in this group of RPTPs is not resolved. Dimerization of PTPRH has been proposed to decrease its activity (Walchli et al. 2005). However, dimerization of CD148/PTPRJ appears to increase its activity (Takahashi et al. 2006). Whether other type III RPTPs dimerize, and how such dimerization influences activity, are not known.

We have now examined dimerization of the type III RPTP, PTPRO, and its effect on PTPase activity. PTPRO can exist in a dimerized state in living cells, which is regulated, at least in part, by disulfide linkages in the ICD. Using a chimeric protein strategy, we found that dimerization of PTPRO leads to a decrease in PTPase activity. The activity changes resulting from dimerization of PTPRO are capable of regulating the phosphorylation (and therefore activity) of the NT-3 receptor TrkC, which is likely to be a relevant substrate for PTPRO.

MATERIALS AND METHODS:

Expression Constructs

The Trk-RO chimera fused the ECD of human TrkA (nt 1-1305 of M23102) with the TMD and ICD of chick PTPRO (nt 2629-4053 of U65891). This and all other constructs, unless otherwise noted, were placed into the pcDNA3.0 expression vector (Invitrogen). Trk-RO was fused at its C-terminal either to mVenus (a monomeric version of YFP; (Nagai et al. 2002, Zacharias et al. 2002) or to mCherry (Shaner et al. 2004) after a short flexible linker peptide (2x GGGS). The Trk-PTPRD chimera (Trk-δv) fused the ECD of human TrkA (nt 1-1305 of M23102) with the TMD and ICD of human PTPRD (nt 3922 – 5889 of L38929.1). Full length PTPRO (chick) was fused at its C-terminal end to the GGS linker peptide followed by mVenus. Flag-tagged proteins Trk-ROf and flROflag were created by adding restriction sites for insertion of Trk-RO or flRO respectively into the C-terminal p3xFLAG-CMV expression vector (Sigma). The catalytically inactive Trk-RO mutant was made by mutating the catalytic C to S using a site directed mutagenesis kit (Stratagene). The substrate trap mutant of flRO (flRO-QADA) was produced using PCR to mutate the highly conserved D1153 and Q1231 to alanines. The TrkA, TrkC, and TrkCvenus constructs (the mVenus tag was placed C-terminal to human TrkC in pcDNA3) were gifts from Dr. Pantelis Tsoulfas (University of Miami). The PTPRM construct was a gift from Dr. Martijn Gebbink (University of Utrecht). The mVenus and mCherry cDNAs were gifts from Dr. Atsushi Miyawaki (RIKEN, Japan) and Dr. Roger Tsien (University of California, San Diego) respectively. The flag-tagged PTPRJ construct was a gift from Dr. Art Weiss (UCSF), and a CSF-Met chimera was a gift from Dr. Morag Park (McGill). DNA constructs were transfected into COS-7 cells using Nucleofection (Amaxa), or with standard transfection reagents (Fugene, Roche; Lipofectamine, Invitrogen). Transfected cells were analyzed 2 days post transfection for all experiments. Live immunostaining with WGA-594 (Molecular Probes/Invitrogen) was performed according to the manufacturer’s instructions.

Brain lysates

The original PTPRO−/− mouse strain has been described (Wharram et al. 2000); these animals were a donation from Dr. Roger Wiggins (University of Michigan). We backcrossed PTPRO−/− mice onto a 100% 129P3/J background using the MaxBax service (Charles River Laboratories, Wilmington, MA). Brain lysates from postnatal day 0 (P0) PTPRO knockout mice and wt littermates were prepared by homogenization in lysis buffer (20mM Tris, pH 8.0; 120mM NaCl, 10% glycerol, 1mM Na3VO4, 1X Roche protease inhibitor cocktail). The homogenate was centrifuged for 30 min at 14,000 rpm in a microfuge; the pellet was dissolved in lysis buffer containing 1 % Triton X-100. Before loading, samples were boiled 5 minutes in reducing or non-reducing sample buffers. Reducing buffer contained 100mMTris-HCL, 4% SDS, 30% glycerol, and fresh 10mM DTT, and DTT was omitted for non-reducing buffer.

Trk-expressing cell lines

COS cells were transfected using Lipofectamine (Invitrogen) and selected with 600 μg/ml G418, which was replaced every 3 days. Colonies were lifted by aspiration to establish independent clones; the rest of the plate was pooled for Western blotting and immunohistochemistry for TrkC. Stable TrkC-expressing cells were transiently transfected with PTPRO, PTPRD, or a control vector, and cultured for 18 hrs prior to 24 hrs of serum starvation. Cells were treated with 75 ng/ml NT-3 for 20 mins prior to lysis and examination by phosphotyrosine Western blotting. Blots were stripped and re-probed to analyze total TrkC levels.

Stimulation with neurotrophins or antibody

NGF and NT-3 were from Preprotech. Neurotrophins or vehicle (PBS + 0.1%BSA for NGF or H2O + 0.1%BSA for NT-3) were diluted into serum-free media, and incubated with cells for 30 min in at 37°C. Cells were rinsed twice with cold PBS and lysed on ice by scraping in 0.02M Imidazole/0.002M EDTA/0.002M EGTA/0.5% CHAPS/protease inhibitors (1x Roche Complete Tablets). Crude cell lysates were spun in a benchtop centrifuge for 10 min at 13,000rpm to remove debris.

Immunoprecipitation and Western Blot Analysis

For immunoprecipitation experiments, anti-flag agarose beads (E-Z view, Sigma) or GammaBind Sepharose beads (GE Healthcare Bio-Sciences/Amersham Biosciences) were used according to the manufacturer’s protocols. Briefly, lysates were incubated overnight with beads at 4°C with rotation. Beads were pelleted by centrifugation, followed by 3 washes in TBS (50mM Tris HCl, 150mM NaCl, pH 7.4) for 5 minutes at 4°C. Bound proteins were eluted with sample buffer and subjected to SDS-PAGE and Western blotting essentially as described (Stepanek et al. 2005). Normalization was performed using internal controls in the same lanes. Primary antibodies included rabbit anti-PTPRO (Beltran et al., 2003), a mouse monoclonal antibody to the ECD of Trk (Zymed Laboratories), polyclonal anti-GFP (Invitrogen), monoclonal anti-flag (M2) (Sigma), anti-phosphotyrosine (4G10) (Upstate), anti-phosphotyrosine (PY-20) (BD Bioscience), HRP-conjugated anti-phosphotyrosine (RC20) (BD Bioscience), anti-pan-Trk (c-14) (Santa Cruz), and a goat anti-TrkC (Huang et al. 1999) which was a gift from Dr. Louis Reichardt (UCSF). Secondary antibodies included Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes) and multiple IRDye 800-conjugated IgG antibodies (Rockland).

Biotinylation Assay

Biotinylation experiments were performed using the Cell Surface Biotinylation Kit (Pierce). Trk-ROv transfected cells were rinsed twice with ice-cold PBS prior to addition of either Sulfo-NHS-SS-Biotin in cold PBS or PBS alone for control cells. Cells were incubated for 30 min at 4°C with shaking, prior to quenching (3X). Cells were scraped, rinsed with ice-cold TBS (2X), and lysed. Lysates were sonicated on ice 3X. Lysates were cleared, aliquots removed for Western blots, and supernatants incubated with Immobilized NeutrAvidin (1 hr at RT). Flow-through from the columns was collected for Western blots. Columns were washed 4X and bound proteins eluted with Sample Buffer + 50 mM DTT. Eluates were analyzed on Western blots together with the “pre-avidin” and “flow-through” samples. Quantification of each was performed and used to estimate surface and internal levels as a percentage of the total.

PTPase Assay

Protein content was quantified using an Amido Black assay (Schaffner & Weissmann 1973). PTPase activity was measured by dephosphorylation of a synthetic peptide using the PTP Assay Kit 2 (Upstate), according to the manufacturer’s instructions. Standards provided in the kit and controls were run on each plate with every experiment. Background release was defined by samples with buffer replacing the lysate. Background-subtracted measurements were fitted to a standard curve to yield data in the form of nmol/min of phosphate released.

RESULTS:

PTPRO forms oligomers in living cells

Dimerization is important for Type I transmembrane proteins (Ullrich & Schlessinger 1990, Heldin 1995, Weiss & Schlessinger 1998); whether PTPRO forms dimers is unknown. We examined this question in native brain, using Western blots in reducing and non-reducing conditions. In reducing conditions, PTPRO formed a smear from 180-220 kD (Fig. 1A; see also Beltran et al., 2003). In non-reducing conditions, a major immunoreactive band appeared at ca. 340 kD, with a smear above it (Fig. 1A, arrow). All bands were specific, as they did not appear in brain lysates from PTPRO null mice. Thus PTPRO forms disulfide-linked oligomers, at approximately the size of PTPRO dimers, in native brain. To examine whether these were homomeric, or might reflect specific associations with other brain proteins, we transfected COS-7 cells with a full-length flag-tagged version of PTPRO (flROflag). On blots run under reducing conditions, immunoreactive bands were seen near 170kD and 190kD, with a smear near 230 kD (Fig. 1B). A similar pattern has previously been seen for PTPRO in transfected cells (Beltran et al. 2003). Under non-reducing conditions, a band appeared around 350kD, which likely represents the dimeric form of full length PTPRO (Fig. 1B, arrow). Taken together, these data strongly suggest that native PTPRO can form dimers, and that at least some such dimers are maintained by disulfide linkages.

Figure 1. Full length PTPRO forms disulfide-linked dimers.

Figure 1.

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A. P0 brain lysates from wt mice and PTPRO−/− littermates (KO) were analyzed on anti-PTPRO Western blots in reducing (right) and non-reducing (left) conditions. In reducing conditions PTPRO appeared as a smear between 180-220 kD; in most cases a band at 180 kD was clear (data not shown; see Beltran et al., 2003). In non-reducing conditions a prominent band appeared at ca. 340 kD (small arrow), smearing to higher molecular weights. No immunoreactive bands were present in the PTPRO−/− lysates. Similar results were seen in 2 other independent experiments. B. Lysates from untransfected (un) COS-7 cells and cells transfected with increasing amounts of flag tagged full length PTPRO (flROflag), were analyzed on anti-flag Western blots in both reducing (left) and non-reducing (right) conditions. Reduced bands appear at 170kD, 190kD, and 235kD, with some smearing, and a putative dimeric form (ca 350kD; small arrow) of PTPRO can be seen in non-reducing conditions.

To examine dimerization of PTPRO without requiring disulfide bonding, we co-expressed flROflag with a second construct consisting of full length PTPRO fused to a monomeric Venus (mVenus) tag (flROvenus). mVenus is designed to prevent artifactual dimerization of fusion proteins (Nagai et al. 2002, Zacharias et al. 2002). Precipitation of flROflag led to co-precipitation of flROvenus, indicating the presence of PTPRO-PTPRO oligomers, presumably dimers (Fig. 2A). Co-precipitation did not occur in the presence of excess flag peptide, or if GFP was co-expressed instead of the tagged PTPRO construct. Further, flROvenus did not co-precipitate with a flag-tagged version of a related Type III RPTP, CD148/PTPRJ (Fig. 2B). Together with the earlier results, these data indicate that PTPRO can form dimers in living cells.

Figure 2. Full length PTPRO self-associates in cells.

Figure 2.

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A. COS-7 cells were co-transfected with flROflag (RO-fl) and an mVenus tagged version of full-length PTPRO (RO-v). Lysates were precipitated with anti-flag beads and Western blots were probed for both the flag and the Venus (GFP) epitopes. Precipitation of flROflag co-precipitated flROvenus (2nd lane), indicating the presence of oligomers; specific precipitation was blocked with excess flag peptide (1st lane). flROvenus was not substantially precipitated directly in the absence of flROflag (3rd lane), and no bands corresponding to flROvenus were seen if GFP was co-transfected instead (4th lane). Un= control untransfected cells. B. flROflag specifically associates with flROvenus. flROvenus was co-expressed with either flRO-fl (RO-fl) or full length CD148/PTPRJ (RJ-fl); the left 2 lanes show that expression levels of flROvenus were similar in the 2 conditions. Immunoprecipitation with anti-flag brought down more RJ-fl than RO-fl (middle 2 lanes), yet flROvenus was co-precipitated only with flRO-fl (right 2 lanes).

A TrkA-PTPRO fusion protein is expressed on the surface of transfected cells

The ability of PTPRO to form dimers in cells raises the question of the effect of dimerization on enzymatic activity. Because native ligands for the PTPRO ECD are unknown, we produced a chimeric cDNA encoding the ECD of the nerve growth factor (NGF) receptor, TrkA, fused to the TMD and ICD of PTPRO (Trk-RO) (Fig. 3A). Application of NGF to cells expressing this chimeric protein should dimerize it, leading to dimerization of the intracellular catalytic domains. The Trk-RO chimera was tagged with mVenus (Trk-ROv).

Figure 3. A fluorescent Trk-RO chimera is expressed on cell membranes and the cell surface.

Figure 3.

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(A) A Trk-RO chimera was produced by fusing the ECD of the high affinity NGF receptor, TrkA, to the TMD and ICD of PTPRO. Addition of NGF is expected to dimerize the TMD and ICD of PTPRO, potentially leading to changes in PTPase activity. (B) Western blot analysis of cell lysates transfected with either Trk-ROv (a) or cytoplasmic GFP (b) were run under reducing conditions and probed with anti-GFP (upper panels), and anti-GAPDH (lower panel) as a loading control. The chimera is seen as a band near the expected molecular size of 135kD. A 26kD band present in the Trk-ROv lysate is likely a fragment containing the mVenus tag. (C) The Trk-RO chimera was tagged on the intracellular C-terminus with mVenus (Trk-ROv). Confocal imaging of COS-7 cells transfected with Trk-ROv (green), stained live with an impermeant fluorescent wheat germ agglutinin (WGA; red) to mark the cell surface. A single Z-slice through two cells (a) shows that both the Trk-ROv and WGA can be visualized at the cell periphery. Both are absent from the cell center (likely the nucleus), and Trk-ROv can be seen in an epi-nuclear location, apparently in cell trafficking intermediates. The entire Z-stack is shown in b; the top and side windows are orthogonal views at the locations corresponding to the x (green) and y (red) lines that cross the center image, and show that Trk-ROv is localized together with WGA on the cell surface.

Western blot analysis of COS cells transfected with Trk-ROv revealed an immunoreactive doublet at around 140kD, similar to the predicted size of the fusion protein (Fig. 3B). The relationship between the bands in the doublet is unclear, though PTPRO can also appear as a doublet. Confocal microscopy demonstrated cell surface localization, as assessed by co-staining with a fluorescent wheat germ agglutinin conjugate (Fig. 3C). Trk-ROv was also seen in intracellular puncta, likely representing trafficking intermediates. Surface expression was also demonstrated by staining of live cells with antibodies to the ECD of TrkA, and by imaging the fluorescent protein using TIRF microscopy (data not shown). In cell surface biotinylation experiments, we found that the steady-state percentage of total Trk-RO chimera present at the cell surface ranged from 9-23% (16 ± 4%; N= 3 experiments). Because Trk-RO is likely to traffic between cell surface and intracellular compartments, these levels represent a minimum cell surface expression level. Nevertheless, they indicate that only a minority of the chimeric molecules would be available to bind NGF administered to the cells.

The Trk-RO chimera is dimerized by NGF

To determine whether Trk-ROv, like full-length PTPRO, can dimerize in transfected cells, we examined the chimeric protein on Western blots under non-reducing conditions. Similar to full length PTPRO, both monomeric and oligomeric (presumed dimeric) Trk-ROv were present under non-reducing conditions (Fig. 4A), but not reducing conditions (Fig. 4B). To test whether NGF can increase dimerization of the chimera, we incubated Trk-ROv-transfected cells in the presence and absence of NGF. Indeed, the relative amount of oligomerized, presumably dimerized, Trk-ROv increased upon treatment with NGF (Fig. 4A). Quantification revealed nearly a two-fold increase in the relative amount of oligomerized Trk-ROv (1.83 ± 0.3 fold; mean ± SEM; p < 0.02; N=11).

Figure 4. NGF increases disulfide linkage of the Trk-ROv chimera.

Figure 4.

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Lysates of control cells (expressing GFP alone) and cells expressing Trk-ROv (± NGF) were analyzed on Western blots under non-reducing (A) and reducing (B) conditions. Both a 135/145 kDa monomeric form (doublet) and an oligomerized form (arrows) of the Trk-ROv chimera can be seen in non-reducing conditions. NGF addition increases the relative amount of the oligomer. Quantification revealed that the oligomer represented 4% of the total in the absence of NGF, and 10% of the total in the presence of NGF. Oligomers are fully reduced by βME. No signals are seen in the GFP control lanes. Similar results were obtained in 2 other experiments.

We also measured self-association of Trk-ROv using a co-precipitation strategy similar to that used for full-length PTPRO. Cells were co-transfected with Trk-ROv and a flag-tagged version of the Trk-RO chimera (Trk-ROf). Precipitation of Trk-ROf co-precipitated Trk-ROv, and this association was increased more than two-fold when cells were incubated with NGF (2.39 ± 0.39; mean ± SEM; p < 0.02; N =4). Because most of the Trk-RO is intracellular and not available for NGF binding, this magnitude of increase in association with NGF addition likely reflects relatively efficient dimerization. Co-precipitation of Trk-ROf and Trk-ROv was specific because it did not occur in the presence of excess flag peptide, or if GFP was co-expressed instead of Trk-ROv (Fig. 5A). Further, a Venus-tagged version of PTP-δ/PTPRD failed to co-precipitate with Trk-ROf (Fig. 5B).

Figure 5. NGF increases self-association of the Trk-RO chimera.

Figure 5.

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A. Cells were co-transfected with a flag-tagged (RO-fl) and a Venus-tagged (RO-v) Trk-RO chimera, and incubated in the absence (first lane) or presence (second lane) of NGF before precipitation of lysates with anti-flag. More Trk-ROvenus was co-precipitated in the presence of NGF (compare first and second “GFP” lanes; see text for quantification). Excess flag peptide abrogated precipitation (third lane). Little to no Trk-ROvenus was precipitated in the absence of Trk-RO-fl (fourth lane), and no band was seen if GFP was co-transfected instead of Trk-ROvenus (last lane). B. Trk-ROfl specifically associates with Trk-ROvenus. Cells were co-transfected with a flag-tagged Trk-RO chimera (RO-fl) and either a Venus-tagged Trk-RO chimera (RO-v) or a Venus-tagged Trk-δ chimera (δ-v). Proteins were expressed at similar levels in the 2 conditions (left lanes). Immunoprecipitation with anti-flag brought down similar levels of Trk-RO-fl in the 2 conditions, but Trk-ROvenus was co-precipitated only with Trk-RO-fl (right lanes). This experiment was repeated 3 times with similar results.

NGF-induced Dimerization of Trk-RO is accompanied by a decrease in PTPase activity

To examine the effect of dimerization of PTPRO’s catalytic domain on PTPase activity, we measured activity in lysates of COS cells transfected with Trk-ROv. Basal PTPase levels in lysates from untransfected COS cells were 5-15 nmol/min/mg protein (e.g., Fig. 6A), consistent with levels found in most tissues in vivo (Maher 1991). In cells transfected with Trk-ROv, PTPase activity increased 4-fold, when cells were compared at similar levels of confluence (Fig. 6B). Both basal PTPase activity and that conferred by Trk-ROv were inhibited by the PTPase inhibitor, vanadate. Further, the increase in PTPase activity conferred by Trk-ROv was not seen with an inactive mutant version of the chimera (Trk-ROv-CS) (Fig. 6B). Thus most of the PTPase activity measured in Trk-ROv transfected cells was attributable to the activity of the PTPRO catalytic domain.

Figure 6. NGF binding to the Trk-ROv chimera decreases PTPase activity.

Figure 6.

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Phosphatase activity was measured in lysates from untransfected COS-7 cells, and cells transfected with Trk-ROv or GFP as a control. (A) Representative experiment plotting phosphatase activity against protein levels in transfected and untransfected lysates. (B) Mean PTPase activity (+/− SEM) of transfected and untransfected cells, incubated with or without 100ng/ml NGF prior to lysis, normalized to the unstimulated GFP control. Expression of the Trk-ROv chimera increased total cellular phosphatase activity almost 4-fold. This increase can be attributed to the activity of the Trk-RO chimera since it was not seen with expression of a catalytically dead mutant, in which the catalytic cysteine was mutated to a serine (Trk-RO-CS). Nearly all activity was abolished in the presence of the tyrosine PTPase inhibitor vanadate. **significant difference from GFP; p<0.0001; N=16 independent experiments. NGF treatment of cells expressing Trk-ROv led to a significant decrease in PTPase activity, while it did not change PTPase activity in cells expressing GFP or the inactive C-S mutant. *significant difference from Trk-ROv no NGF; p<0.005; N=14 independent experiments. N=3 and N=9 for the Trk-ROCS and GFP experiments with NGF, respectively.

To test whether dimerization of Trk-ROv affects its PTPase activity, we treated Trk-ROv–transfected cells with NGF (100ng/ml). NGF treatment of cells expressing Trk-ROv decreased PTPase activity by 33% (Fig. 6B). In control experiments, NGF treatment did not affect PTPase activity in cells transfected with GFP, nor in cells expressing the Trk-ROv-CS mutant. Since basal PTPase activity is not responsive to NGF, the 33% decrease in total activity suggests a >40% decrease in the activity conferred by Trk-ROv. As a relatively small percentage of Trk-RO is likely to be available to the added NGF during the course of 30 min incubation, this decrease is substantial. Our data thus suggest that dimerization of the PTPRO catalytic domain leads to a strong decrease in intrinsic PTPase activity. This result is opposite to that reported for another type III RPTP, PTPRJ, for which activity was reported to increase upon dimerization(Takahashi et al. 2006).

PTPRO binds to and dephosphorylates TrkC

The previous assay measured the ability of Trk-ROv to dephosphorylate a synthetic peptide in cell lysates. To obtain a more physiologically relevant measure of PTPase activity, we examined the ability of the chimeric protein to dephosphorylate a biological substrate in an intact cell. One potential PTPRO substrate is the receptor for NT-3, TrkC. PTPRO is co-expressed with TrkC in neurons in several locations, including sensory ganglia, cranial ganglia, spinal cord, and cortex (Beltran et al. 2003).

To test whether PTPRO could interact with TrkC in cells, we used a substrate trapping strategy. Catalytically inactive mutants of various PTPs have been shown to exhibit increased affinity for substrates, and therefore to be useful as “substrate traps”, for substrate identification using immunoprecipitation (Flint et al. 1997, Herbst et al. 1996, Xie et al. 2002). In particular, a double mutant of PTP1B in which two conserved residues are mutated to alanine exhibits very high affinity for substrates (Xie et al. 2002). We therefore co-expressed TrkC with either flROflag or the substrate trapping mutant version of this construct (flRO-QADA). flRO-QADA has no detectable PTPase activity (data not shown). Precipitation of flRO-QADA co-precipitated a phosphotyrosine-containing band at around 140kD, the predicted size for TrkC; this band was also recognized by a pan-Trk antibody (Fig. 7A). The 140 kD band was not co-precipitated with the wild type version of PTPRO, suggesting that interaction of TrkC with the catalytic domain of PTPRO is involved. This TrkC-PTPRO association was selective as numerous tyrosine phosphorylated proteins in the lysate failed to co-precipitate with flRO-QADA (Fig. 7A).

Fig. 7. TrkC interacts with and is dephosphorylated efficiently by PTPRO.

Fig. 7.

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(A) COS cells were transfected with TrkC and with flag-tagged PTPRO (flROflag) or the substrate-trapping mutant (flRO-QADA). The cell lysate and the anti-flag immunoprecipitates were subjected to Western blotting with anti-PY (upper panel) or anti-pan-Trk (lower panel) antibodies. The lysate from cells co-transfected with flRO-QADA showed numerous strong PY bands. Tyrosine-phosphorylated proteins at 185 kD, 140 kD, and 125 KD were immunoprecipitated with flRO-QADA; the band at 125 kD was not consistently seen. The precipitated 140 kD band was recognized by the pan-Trk antibody. (B) COS cells were untransfected (Un), transfected with TrkC alone (Trk), or co-transfected with TrkC plus full-length PTPRO (Trk/RO), SHP-2 (Trk/SHP), PTPRM (Trk/RM), or PTPRD (Trk/RD). Lysates were subjected to SDS-PAGE and Western blotting with antibodies to phosphotyrosine (pY) and to TrkC (Trk) (2 different gels with the same lysates). 140 kD marker (arrowheads) points to TrkC band. Untransfected COS cells show a tyrosine phosphorylated protein at 140 kD that is not immunoreactive for Trk. Expression of TrkC led to an autophosphorylated Trk band that was substantially dephosphorylated by full-length PTPRO. SHP-2 also efficiently dephosphorylated TrkC. PTPRM was about half as effective as PTPRO, and PTPRD was almost completely ineffective. This experiment was repeated once with similar results. Expression of PTPRO, PTPRM and PTPRD was independently confirmed by Western blots with specific antibodies (data not shown). (C) TrkC-expressing cell lines mock transfected (no PTP) or transiently transfected with two different amounts of PTPRO plasmid (RO) or PTPRD (RD), were unstimulated (Co), or stimulated with NT-3 for 20 minutes prior to cell lysis. Lysates were separated on SDS-PAGE for Western blotting with anti-phosphotyrosine antibodies (pY). NT-3 led to a robust activation of TrkC (first 2 lanes), and this activation was strongly suppressed by PTPRO expression (2 different examples are shown). In contrast, PTPRD expression had almost no effect on TrkC activation (last lane). Numbers in parentheses below B and C are ratios of phospho-Trk to total Trk (obtained by stripping and re-probing; not shown), with the “Trk alone (no PTP)” lanes arbitrarily set to 10. Data are from a single experiment done in duplicate.

To determine if PTPRO can effectively dephosphorylate TrkC, we co-expressed TrkC with several PTPs, including the non-receptor PTP, SHP-2, as well as 3 RPTPs—PTPRO, PTPRD, and PTP-μ/PTPRM. Expression of PTPRO substantially reduced tyrosine phosphorylation of TrkC, while both PTPRM and PTPRD were less effective (Fig. 7B). SHP-2, a cytoplasmic PTP that is known to modulate Trk responses (Okada et al. 1996, Araki et al. 2000, Chen et al. 2002), also efficiently dephosphorylated TrkC (Fig. 7B). Thus PTPRO is relatively effective at dephosphorylating TrkC.

The previous experiments were done with TrkC that was autophosphorylated through overexpression, which may not involve biologically relevant tyrosine residues. We therefore produced cell lines stably expressing TrkC, transiently transfected these cells with PTPRO, and lysed them after incubation in the presence and absence of the TrkC ligand, NT-3. Western blot analysis showed that expression of PTPRO, but not PTPRD, greatly diminished NT-3 induced TrkC phosphorylation (Fig. 7C). These results indicate that PTPRO can reduce activation of TrkC induced by its native ligand. Together with the results described above, they suggest that TrkC may be a relevant substrate for PTPRO.

NGF-induced dimerization of Trk-RO decreases its ability to dephosphorylate TrkC.

The finding that PTPRO can efficiently dephosphorylate TrkC provides an opportunity to test whether dimerization of Trk-RO influences its ability to dephosphorylate relevant substrates. Fluorescent microscopy of cells cotransfected with TrkC tagged with mVenus (TrkCv) and Trk-RO tagged with mCherry (Trk-ROc) demonstrated partially overlapping expression in intracellular puncta and on cell surfaces (Fig. 8A and data not shown). To ensure that the chimeric phosphatase could effectively dephosphorylate TrkC, we probed Western blots of lysates from cotransfected cells for phosphotyrosine and for TrkCv expression. As expected, expression of increasing amounts of Trk-ROc led to increasing dephosphorylation of autophosphorylated TrkCv (Fig. 8B and data not shown).

Fig. 8. The Trk-RO chimera dephosphorylates TrkC in intact cells.

Fig. 8.

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Cells were co-transfected with mVenus-tagged (TrkCv) and the Trk-RO chimera tagged with mCherry (TrkROc) (A) Fluorescent microscopy showed that the two proteins were partially co-localized in membrane compartments; non-overlapping expression was also readily observed. (B) Lysates from untransfected cells (lane a) or cells transfected with the TrkCv plasmid alone (b) or together with 0.375 μg (c), or 6 μg (d) of Trk-ROc plasmid were analyzed on Western blots probed with anti-GFP (GFP), to show expression levels of TrkCv, and with anti-phosphotyrosine (pY). Phosphorylation of both TrkCv (arrows) and an unidentified band at 115kD (arrowhead) decreased with increasing expression of Trk-ROc. Numbers in parentheses below the figure are ratios of the phospho-TrkC band to total TrkC, with TrkC alone set to 10. Similar results were seen in 2 independent replicates of this experiment, and intermediate phosphorylation levels were seen with 2 μg of Trk-ROc plasmid (not shown).

To determine if activated TrkC could be dephosphorylated (and therefore regulated) by the chimeric phosphatase, we co-expressed TrkCv and either Trk-ROc or a catalytically dead version (Trk-ROc-CS). We treated these cells with NT-3 (50ng/ml) to activate TrkC, and examined TrkC phosphorylation in the presence or absence of NGF (100ng/mL). As predicted, co-expression of Trk-ROc led to a decrease in NT-3 induced TrkC phosphorylation compared to that seen with expression of the inactive mutant (Fig. 9A). Treatment with NGF increased the relative phosphorylation of TrkC 2-fold, while NGF did not increase TrkC phosphorylation in the presence of the inactive mutant (Fig. 9B). Thus dimerization of Trk-ROc with NGF decreases its activity toward a putative substrate in intact cells.

Fig. 9. NGF binding to the Trk-ROc chimera decreases its ability to dephosphorylate TrkC.

Fig. 9.

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Cells were co-transfected with TrkCv and either Trk-ROc or a catalytically dead mutant (TrkROc-CS), and incubated in the presence of NT-3 alone or both NT-3 and NGF. Cell lysates were examined on Western blots probed with anti-pY (top panel) and anti-GFP (bottom panel). A. NGF treatment led to an increase in phosphotyrosine levels when wt Trk-ROc was expressed, while NGF did not affect levels with co-expression of the inactive Trk-ROc-CS mutant. B. Quantification of relative intensities of phospho-TrkC normalized to TrkC expression. *significantly different from the C-S mutant (p<0.01); and from TrkC alone (p<0.05); N=5 independent experiments. C. Cartoon illustrates working model. (Left) NT-3 activates TrkCv, and monomeric Trk-ROc dephosphorylates activated TrkCv. (Right) NGF dimerizes Trk-Roc, inactivating it, allowing TrkCv to remain phosphorylated. (Not shown) Dimerization of TrkROc-CS with NGF does not affect phosphorylation of TrkCv, since this protein is already inactive.

DISCUSSION:

We have shown that the type III RPTP, PTPRO, can dimerize, both in native tissue and in transfected cells. Further, a maneuver that increases dimerization of the PTPRO catalytic domain decreases its activity, unlike what occurs for PTPRJ, a closely related RPTP. Finally, we have provided evidence that the high-affinity receptor for NT-3, TrkC, is a likely substrate for PTPRO. These results not only further our understanding of the regulation and function of PTPRO, but also have general implications for RPTP regulation.

Dimerization, and not some unknown effect of NGF binding, is likely to mediate the decrease in activity of the Trk-RO chimera. First, the ECD of TrkA is sufficient to form dimers in the presence of NGF, and crystallographic studies of the TrkA ECD suggest that NGF-induced conformational changes in the monomer are unlikely (Wehrman et al. 2007). Second, chimeric receptors containing the ECD of TrkA can transduce signals from distinct receptors, each of which depends on multimerization but has no other features in common with PTPRO (Stein et al. 2001, Stein & Tessier-Lavigne 2001, Basile et al. 2004). Finally, it is unlikely that conformational changes induced by NGF in the TrkA ECD could be transmitted to the catalytic domain of PTPRO through a single transmembrane helix (Lemmon & Schlessinger 1998).

PTPRO dimerization can be regulated by disulfide bonds. Because the chimeric form of PTPRO (Trk-ROv) can form disulfide-linked dimers, at least one of the cysteine residues involved is likely to be located in PTPRO’s ICD (there are no cysteines in the TMD). The catalytic cysteine (C1187) appears not to be involved, since a similar percentage of dimerization was seen for wt Trk-RO and for the mutant form lacking this cysteine (Trk-RO-CS; data not shown). In general, catalytic cysteines in the active (D1) domains of RPTPs are not involved in dimerization (den Hertog et al. 2005). Disulfide bonding is a common modifier of dimerization for RPTPs, and can be regulated by oxidation state (Walchli et al. 2005, Lee et al. 2007, van der Wijk et al. 2004).

It is difficult to develop a general scheme of RPTP dimerization from available data. The ECD of PTPRH is required for dimerization (Walchli et al. 2005), and the ECDs of several other RPTPs are likely involved in dimerization (Jiang et al. 2000, Xu & Weiss 2002, Cismasiu et al. 2004). However, the TMD has been implicated in the dimerization of PTPRS (Lee et al. 2007), and of transmembrane forms of PTPRR (Noordman et al., 2007), and may also play a role in the dimerization of PTPRA (Jiang et al. 2000, Tertoolen et al. 2001), and possibly of PTPRO (Chin et al. 2005). Recently, it was shown that homodimerization of the PTP-γ/PTPRG ICD can take place in the absence of the other domains, but this was not seen for PTPRO or for a large number of other RPTPs examined (Barr et al. 2009).

In two distinct assays we found that NGF-induced dimerization of PTPRO’s TMD and ICD led to decreased enzymatic activity. This result is consistent with a scheme of RPTP regulation first proposed for CD45/PTPRC and PTP-α/PTPRA (Desai et al. 1993, Bilwes et al. 1996, Jiang et al. 1999, Xu & Weiss 2002, Takeda et al. 2004), and later for PTP-ζ/PTPRZ (Fukada et al. 2006). However, the RPTPs cited above belong to distinct subclasses with tandemly repeated catalytic domains (D1 and D2), while type III RPTPs like PTPRO have only one catalytic domain. Antibody-mediated dimerization of a second type III RPTP, CD148/PTPRJ, has been reported to increase catalytic activity(Takahashi et al. 2006), and our own studies with PTPRJ are consistent with these results. Using a different antibody from that used by Takahashi et al., we found that PTPRJ’s ability to dephosphorylate a co-transfected substrate (c-Met) increased upon incubation with a monoclonal antibody (data not shown). Thus results for PTPRJ and PTPRO appear contradictory, but it may be that the exact mode and context of dimerization are important in determining the functional consequences (Jiang et al. 1999, van der Wijk et al. 2003, Jiang & Hunter 1999) . Oligomerization of PTPRJ, for example, may decrease activity under some circumstances (Tangye et al. 1998). Further data are required before a general scheme for regulation of RPTPs by dimerization can be developed.

The decrease in activity caused by dimerization of PTPRA appears to depend on interactions between a region near the D1 catalytic domain (the “inhibitory wedge”) and the catalytic core of its neighbor (Jiang et al. 1999, Bilwes et al. 1996). This model has also been proposed to hold for CD45/PTPRC (Majeti et al. 1998, Xu & Weiss 2002), and for the type III RPTP, PTPRH (Matozo et al. 2007). However, the wedge model appears incompatible with structural information developed for PTPRK, PTPRM, or even for PTPRC (Hoffmann et al. 1997, Nam et al. 2005, Eswaran et al. 2006). Indeed, recent structural data on a large number of PTPs from many different subfamilies, including PTPRO, suggest that the inhibitory wedge model is inconsistent with the structures of RPTPs other than PTPRA (Barr et al. 2009). Thus, it is unclear which mechanisms account for dimerization-induced decreases in activity for different RPTPs, and there is no structure-based proposal for the increase in activity observed with PTPRJ dimerization. In this respect it is interesting that despite their overall similarity, the crystal structures of the PTPRO and PTPRJ catalytic domains show the latter in a “closed” (active) position, while the former is in an “atypically open” position (Barr et al. 2009).

Our data on the relationship between PTPRO dimerization and activity stem from studies of the Trk-RO chimera, but we would argue that our chimeric protein is relevant to native RPTP. First, a great deal has been learned from the study of similar RTK chimeras (Weidner et al. 1993, Zhu et al. 1994, Marron et al. 2000, Stein & Tessier-Lavigne 2001, Kelly-Spratt et al. 2002, Hong et al. 1999, Riedel et al. 1986), as well as a chimera produced from CD45/PTPRC (Desai et al. 1993). Second, our chimera contains not only the ICD, but also the TMD of PTPRO; this seems even more likely than the chimera produced by Desai et al. to model the behavior of the full-length protein. Third, the Trk-RO chimera and the full-length protein dimerized to a similar extent when expressed in cell lines (data not shown). Finally, the fact that we obtained a functional effect with NGF treatment suggests that “rotational coupling” between induced dimerization and productive protein-protein interactions (Blanchetot et al. 2002, Jiang et al. 1999) was occurring with our chimeric PTPRO. Although much can be learned from studies using chimeric approaches to dimerization, a full appreciation of regulatory events will require the study of natural ligands of RPTPs.

Elucidation of the signaling mechanisms employed by PTPRO requires identification of its physiological substrates. Previous work from our lab identified NPCD (neuronal pentraxin with chromo domain) as a PTPRO substrate (Chen & Bixby 2005a, Chen & Bixby 2005b), and more recent work implicates Eph receptors as substrates of PTPRO (Shintani et al. 2006). In this study we provide evidence that TrkC is a substrate for PTPRO. TrkC and PTPRO can be shown to interact, and coexpression of TrkC and PTPRO leads to efficient dephosphorylation of TrkC. Further, TrkC phosphorylation levels can be regulated by dimerization of PTPRO. Since TrkC and PTPRO are co-expressed in both central and peripheral neurons (Beltran et al. 2003), TrkC is a plausible PTPRO substrate. Interestingly, axon guidance from TrkC-expressing sensory neurons is perturbed in mice lacking full-length PTPRO (M. Gonzalez-Brito and JLB, unpublished).

In summary, our results show that the TMD and ICD of PTPRO are involved in a functional dimerization that leads to a decrease in enzymatic activity toward both an artificial peptide and a putative natural substrate. These results contrast with those for PTPRJ, in which dimerization leads to an increase in enzymatic activity. It will be important to identify the structural basis for the inhibition of PTPRO activity with dimerization, since the “inhibitory wedge” structure described for PTPRA does not appear generalizable. Understanding how these receptors function will pave the way for strategies to regulate their activity, both experimentally and therapeutically.

ACKNOWLEDGEMENTS:

This work was supported by grants to JLB from the NIH/NINDS (NS38920) and Amgen, Inc. AEH was a Lois Pope LIFE Fellow. We thank Drs. Martijn Gebbink, Atsushi Miyawaki, Arthur Weiss, Morag Park, Louis Reichardt, Roger Tsien, and Pantelis Tsoulfas for their generous gifts of antibodies and cDNAs. We thank Dr. Roger Wiggins for the gift of PTPRO−/− mice. We also thank Dr. Tsoulfas for technical advice, Xuan Le for excellent technical assistance, and Drs. Vance Lemmon and Ellen Barrett for helpful comments on the manuscript.

Abbreviations used:

ECD

extracellular domain

flROflag

flag-tagged full-length PTPRO

flROQADA

full-length PTPRO, Q to A and D to A mutation

ICD

intracellular domain

mVenus

monomeric Venus

NGF

nerve growth factor

PTP

protein tyrosine phosphatase

PTPase

protein tyrosine phosphatase

RPTP

receptor-type tyrosine phosphatase

RTK

receptor tyrosine kinase

TMD

transmembrane domain

Trk A

tropomyosin-related kinase A

Trk-RO-CS

catalytically inactive Trk-RO

Trk-RO

TrkA-PTPRO chimera

Trk-ROf (c,v)

flag- (Cherry-, Venus-) tagged Trk-RO

Footnotes

Names of Phosphatases: PTPRA, RPTP-α; PTPRB, RPTP-β; PTPRC, CD45; PTPRG, RPTP-γ; PTPRH, Sap-1; PTPRJ, DEP-1/CD148; PTPRK, RPTP-κ; PTPRM, RPTP-μ; PTPRO, GLEPP-1/CRYP-2; PTPRR, PTP-SL/PTPBR7; PTPRS, RPTP-σ, PTPRZ, RPTP-ζ

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