Redox-regulated Rotational Coupling of Receptor Protein-tyrosine Phosphatase α Dimers
2003; Elsevier BV; Volume: 278; Issue: 16 Linguagem: Inglês
10.1074/jbc.m300632200
ISSN1083-351X
AutoresThea van der Wijk, Christophe Blanchetot, John Overvoorde, Jeroen den Hertog,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoReceptor protein-tyrosine phosphatase α (RPTPα) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTPα revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTPα and of constitutively dimeric, active RPTPα-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTPα-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 RPTPα that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTPα-D2). Here we report that H2O2 treatment of cells induced a change in rotational coupling in RPTPα dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys723 in RPTPα-D2, which was required for the conformational change of RPTPα-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 RPTPα led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling. Receptor protein-tyrosine phosphatase α (RPTPα) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTPα revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTPα and of constitutively dimeric, active RPTPα-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTPα-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 RPTPα that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTPα-D2). Here we report that H2O2 treatment of cells induced a change in rotational coupling in RPTPα dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys723 in RPTPα-D2, which was required for the conformational change of RPTPα-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 RPTPα led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling. Two antagonistically acting families of enzymes regulate phosphorylation of protein tyrosine residues, an important determinant for many cellular functions: protein-tyrosine kinases (PTKs) 1The abbreviations used are: PTKprotein-tyrosine kinaseHAhemagglutininPTPprotein-tyrosine phosphataseEGFRepidermal growth factor receptorHEKhuman embryonic kidneyLARleukocyte common antigen-relatedRPTKreceptor-like PTKRPTPreceptor-like PTPROSreactive oxygen species1The abbreviations used are: PTKprotein-tyrosine kinaseHAhemagglutininPTPprotein-tyrosine phosphataseEGFRepidermal growth factor receptorHEKhuman embryonic kidneyLARleukocyte common antigen-relatedRPTKreceptor-like PTKRPTPreceptor-like PTPROSreactive oxygen species 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. 1Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (593) Google Scholar). 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 (2Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (462) Google Scholar). Clear evidence for ligands that regulate RPTP activity remains elusive. In some cases, as for the interaction between RPTPβ, leukocyte common antigen-related (LAR), and PTPς 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 (3Aricescu A.R. McKinnell I.W. Halfter W. Stoker A.W. Mol. Cell. Biol. 2002; 22: 1881-1892Crossref PubMed Scopus (172) Google Scholar, 4O'Grady P. Thai T.C. Saito H. J. Cell Biol. 1998; 141: 1675-1684Crossref PubMed Scopus (118) Google Scholar, 5Peles E. Nativ M. Campbell P.L. Sakurai T. Martinez R. Lev S. Clary D.O. Schilling J. Barnea G. Plowman G.D. Cell. 1995; 82: 251-260Abstract Full Text PDF PubMed Scopus (368) Google Scholar). Sorby et al. (6Sorby M. Sandstrom J. Ostman A. Oncogene. 2001; 20: 5219-5224Crossref PubMed Scopus (34) Google Scholar) 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 RPTPβ/ζ activity, in that β-catenin phosphorylation was enhanced in cells expressing RPTPβ/ζ in response to pleiotrophin (7Meng K. Rodriguez-Pena A. Dimitrov T. Chen W. Yamin M. Noda M. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2603-2608Crossref PubMed Scopus (364) Google Scholar).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. 8Jiang G. Hunter T. Curr. Biol. 1999; 9: R568-R571Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar and 9Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). 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 (10Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Abstract Full Text PDF PubMed Scopus (241) Google Scholar). In line with this, there is evidence for the regulation of RPTPα by dimerization. The crystal structure of RPTPα-D1 provided structural evidence that dimerization leads to inhibition of the catalytic activity of RPTPα because a wedge-like structure of one monomer inserts into the catalytic cleft of the other, thereby occluding the catalytic site (11Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (291) Google Scholar). 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 RPTPα leading to the formation of a stable intermolecular disulfide bridge (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar). A disulfide bridge at position 137 (RPTPα-P137C) resulted in reduced substrate dephosphorylation, whereas constructs with a disulfide bridge at position 135 (RPTPα-F135C) had activities similar to the wild type construct. These data indicate that dimerization of RPTPα 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 (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar). Point mutations in the wedge of EGFR-CD45 chimeras (13Majeti R. Bilwes A.M. Noel J.P. Hunter T. Weiss A. Science. 1998; 279: 88-91Crossref PubMed Scopus (218) Google Scholar) and constitutive dimer mutants of RPTPα (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar) 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 (14Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Crossref PubMed Scopus (106) Google Scholar) and fluorescence resonance energy transfer analysis (15Tertoolen L.G. Blanchetot C. Jiang G. Overvoorde J. Gadella T.W.J. Hunter T. den Hertog J. BMC Cell Biol. 2001; 2: 8Crossref PubMed Scopus (75) Google Scholar) indicate that RPTPα 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 (16Dornan S. Sebestyen Z. Gamble J. Nagy P. Bodnar A. Alldridge L. Doe S. Holmes N. Goff L.K. Beverley P. Szollosi J. Alexander D.R. J. Biol. Chem. 2002; 277: 1912-1918Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Takeda A. Wu J.J. Maizel A.L. J. Biol. Chem. 1992; 267: 16651-16659Abstract Full Text PDF PubMed Google Scholar, 18Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Crossref PubMed Scopus (222) Google Scholar). Interestingly, dimerization of distinct alternatively spliced isoforms of CD45 is different (18Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Crossref PubMed Scopus (222) Google Scholar), 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 RPTPα, RPTPα-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 (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar). Importantly, as shown by bis[sulfosuccinimidyl]suberate cross-linking experiments, H2O2 does not induce de novo dimerization (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar). The H2O2-induced conformational change is dependent on the catalytic site cysteine in RPTPα-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 RPTPα reflected the state of rotational coupling. Although the epitope tag was masked in wild type RPTPα and constitutively dimeric, active RPTPα-F135C with a single cysteine in the extracellular domain, the epitope tag in inactive dimeric RPTPα-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 RPTPα. Masking of the epitope tag was dependent on residues 20–42 in the extracellular domain and on dimerization of RPTPα. RPTPα-D2 and more specifically the catalytic site Cys in RPTPα-D2, Cys723, were required for H2O2-induced HA tag accessibility. Our results suggest that a conformational change in the cytoplasmic domain of RPTPα leads to a change in the conformation of the ectodomain of RPTPα and therefore provide evidence that RPTPs have the potential for inside-out signaling.DISCUSSIONHere we report that the conformation of the extracellular domain of RPTPα dimers changed in response to H2O2treatment, reflecting the catalytic state of RPTPα, active or inactive. We provide evidence that the catalytic site cysteine in the membrane-distal PTP domain, RPTPα-D2, is required for this effect. Our results suggest that redox signaling regulates rotational coupling of RPTPα dimers, thereby reversibly switching RPTPα from an active to an inactive dimeric state.We developed an assay to assess accessibility of the epitope tag in the ectodomain of HA-RPTPα on living cells. Surprisingly, this assay allowed us to discriminate between active and inactive constitutively dimeric conformations of RPTPα (Fig. 2). Moreover, H2O2 treatment of cells significantly increased the accessibility of the HA tag in HA-RPTPα (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 RPTPα-D2 (Fig. 8), suggesting that the conformational change in RPTPα-D2 that we reported previously (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar) 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 RPTPε, masked the epitope tag in RPTPα-Δ43–142, whereas the ectodomain of RPTPε 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 RPTPα 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 RPTPα (14Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Crossref PubMed Scopus (106) Google Scholar) specifically abolished masking of the epitope tag (Fig. 5). Moreover, the short extracellular domain of RPTPε did not mask the RPTPα ectodomain in RPTPα-RPTPε heterodimers. Therefore, we conclude that intermolecular interactions, likely mediated by direct interactions of the ectodomains in RPTPα dimers, mask the epitope tag in the extracellular domain of RPTPα.Taken together, based on our results, we propose a model for the effects of H2O2 on the conformation of RPTPα dimers (Fig. 9). In the prestimulation state RPTPα exists as a preformed, active dimer. Using fluorescence resonance energy transfer and chemical cross-linkers, we have demonstrated that RPTPα dimerization is extensive in the prestimulation state (15Tertoolen L.G. Blanchetot C. Jiang G. Overvoorde J. Gadella T.W.J. Hunter T. den Hertog J. BMC Cell Biol. 2001; 2: 8Crossref PubMed Scopus (75) Google Scholar). The fact that the epitope tag is not accessible in the prestimulation state again suggests that most RPTPα is dimeric. In addition to a conformational change in RPTPα-D2 leading to stabilization of RPTPα dimers (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar), we demonstrate here that H2O2 treatment alters the conformation of the extracellular domain from a state resembling active RPTPα-F135C to a state resembling inactive RPTPα-P137C. Our results suggest that oxidative stress induces a rapid change in rotational coupling, which slowly reverts to the prestimulation state upon reduction of RPTPα.The function of the extracellular domain of RPTPα remains to be determined. Both RPTPα and RPTPε 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 RPTPα and RPTPε and their corresponding cytoplasmic forms (24Andersen J.N. Elson A. Lammers R. Romer J. Clausen J.T. Moller K.B. Moller N.P. Biochem. J. 2001. 2001; 354: 581-590Google Scholar) and in studies addressing the effect of calpain-induced cleavage of RPTPα and RPTPε that occurs in the intracellular juxtamembrane domain of the RPTPs (25Gil-Henn H. Volohonsky G. Elson A. J. Biol. Chem. 2001; 276: 31772-31779Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, previous studies with EGFR-RPTPα chimeras suggest that the RPTPα ectodomain does not solely serve as a membrane localizer but may also confer ligand-dependent regulatory mechanisms to RPTPα (26Blanchetot C. den Hertog J. FEBS Lett. 2000; 484: 235-240Crossref PubMed Scopus (7) Google Scholar).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 RPTPα to its putative ligand. Conversely, ligands may bind to the extracellular domain of RPTPα, 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 RPTPα activity. However, bona fide ligands have not been reported yet for RPTPα. Only the GPI-linked protein contactin was reported to bind to the ectodomain of RPTPα in cis (27Zeng L. D'Alessandri L. Kalousek M.B. Vaughan L. Pallen C.J. J. Cell Biol. 1999; 147: 707-714Crossref PubMed Scopus (98) Google Scholar). However, co-transfection of contactin did not affect accessibility of RPTPα in our hands (data not shown). Our results suggest that RPTPα 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. 28Miranti C.K. Brugge J.S. Nat. Cell Biol. 2002; 4: E83-E90Crossref PubMed Scopus (685) Google Scholar and 29Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). 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 (30Xu D. Rovira I.I. Finkel T. Dev. Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Many PTPs have been demonstrated to be inactivated by oxidation of their catalytic site cysteines (31Caselli A. Marzocchini R. Camici G. Manao G. Moneti G. Pieraccini G. Ramponi G. J. Biol. Chem. 1998; 273: 32554-32560Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 32Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (817) Google Scholar, 33Fauman E.B. Cogswell J.P. Lovejoy B. Rocque W.J. Holmes W. Montana V.G. Piwnica-Worms H. Rink M.J. Saper M.A. Cell. 1998; 93: 617-625Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 34Knebel A. Rahmsdorf H.J. Ullrich A. Herrlich P. EMBO J. 1996; 15: 5314-5325Crossref PubMed Scopus (465) Google Scholar). 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 (35Chiarugi P. Fiaschi T. Taddei M.L. Talini D. Giannoni E. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 33478-33487Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 36Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar, 37Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 38Meng T.C. Fukada T. Tonks N.K. Mol. Cell. 2002; 9: 387-399Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar). Regulation of PTPs by ROS is rapid and reversible. It appears that regulation of RPTPα and perhaps other RPTPs by ROS is more complex, involving not only direct oxidation of the catalytic site cysteine. Previously, we demonstrated that H2O2induced stabilization of RPTPα dimerization, which is responsible for complete inactivation of RPTPα. Here, we demonstrate a change in the ectodomain of RPTPα 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 RPTPα in an inactive conformation.Not only RPTPα 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 (39Blanchetot C. Tertoolen L.G. Overvoorde J. den Hertog J. J. Biol. Chem. 2002; 277: 47263-47269Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 RPTPα implicating that RPTPα has the capacity for inside-out signaling. It will be interesting to see whether redox signaling regulates rotational coupling of other RPTPs as well. Two antagonistically acting families of enzymes regulate phosphorylation of protein tyrosine residues, an important determinant for many cellular functions: protein-tyrosine kinases (PTKs) 1The abbreviations used are: PTKprotein-tyrosine kinaseHAhemagglutininPTPprotein-tyrosine phosphataseEGFRepidermal growth factor receptorHEKhuman embryonic kidneyLARleukocyte common antigen-relatedRPTKreceptor-like PTKRPTPreceptor-like PTPROSreactive oxygen species1The abbreviations used are: PTKprotein-tyrosine kinaseHAhemagglutininPTPprotein-tyrosine phosphataseEGFRepidermal growth factor receptorHEKhuman embryonic kidneyLARleukocyte common antigen-relatedRPTKreceptor-like PTKRPTPreceptor-like PTPROSreactive oxygen species 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. 1Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (593) Google Scholar). 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 (2Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (462) Google Scholar). Clear evidence for ligands that regulate RPTP activity remains elusive. In some cases, as for the interaction between RPTPβ, leukocyte common antigen-related (LAR), and PTPς 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 (3Aricescu A.R. McKinnell I.W. Halfter W. Stoker A.W. Mol. Cell. Biol. 2002; 22: 1881-1892Crossref PubMed Scopus (172) Google Scholar, 4O'Grady P. Thai T.C. Saito H. J. Cell Biol. 1998; 141: 1675-1684Crossref PubMed Scopus (118) Google Scholar, 5Peles E. Nativ M. Campbell P.L. Sakurai T. Martinez R. Lev S. Clary D.O. Schilling J. Barnea G. Plowman G.D. Cell. 1995; 82: 251-260Abstract Full Text PDF PubMed Scopus (368) Google Scholar). Sorby et al. (6Sorby M. Sandstrom J. Ostman A. Oncogene. 2001; 20: 5219-5224Crossref PubMed Scopus (34) Google Scholar) 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 RPTPβ/ζ activity, in that β-catenin phosphorylation was enhanced in cells expressing RPTPβ/ζ in response to pleiotrophin (7Meng K. Rodriguez-Pena A. Dimitrov T. Chen W. Yamin M. Noda M. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2603-2608Crossref PubMed Scopus (364) Google Scholar). protein-tyrosine kinase hemagglutinin protein-tyrosine phosphatase epidermal growth factor receptor human embryonic kidney leukocyte common antigen-related receptor-like PTK receptor-like PTP reactive oxygen species protein-tyrosine kinase hemagglutinin protein-tyrosine phosphatase epidermal growth factor receptor human embryonic kidney leukocyte common antigen-related receptor-like PTK receptor-like PTP reactive oxygen species 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. 8Jiang G. Hunter T. Curr. Biol. 1999; 9: R568-R571Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar and 9Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). 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 (10Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Abstract Full Text PDF PubMed Scopus (241) Google Scholar). In line with this, there is evidence for the regulation of RPTPα by dimerization. The crystal structure of RPTPα-D1 provided structural evidence that dimerization leads to inhibition of the catalytic activity of RPTPα because a wedge-like structure of one monomer inserts into the catalytic cleft of the other, thereby occluding the catalytic site (11Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (291) Google Scholar). 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 RPTPα leading to the formation of a stable intermolecular disulfide bridge (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar). A disulfide bridge at position 137 (RPTPα-P137C) resulted in reduced substrate dephosphorylation, whereas constructs with a disulfide bridge at position 135 (RPTPα-F135C) had activities similar to the wild type construct. These data indicate that dimerization of RPTPα 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 (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar). Point mutations in the wedge of EGFR-CD45 chimeras (13Majeti R. Bilwes A.M. Noel J.P. Hunter T. Weiss A. Science. 1998; 279: 88-91Crossref PubMed Scopus (218) Google Scholar) and constitutive dimer mutants of RPTPα (12Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Crossref PubMed Scopus (156) Google Scholar) 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 (14Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Crossref PubMed Scopus (106) Google Scholar) and fluorescence resonance energy transfer analysis (15Tertoolen L.G. Blanchetot C. Jiang G. Overvoorde J. Gadella T.W.J. Hunter T. den Hertog J. BMC Cell Biol. 2001; 2: 8Crossref PubMed Scopus (75) Google Scholar) indicate that RPTPα 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 (16Dornan S. Sebestyen Z. Gamble J. Nagy P. Bodnar A. Alldridge L. Doe S. Holmes N. Goff L.K. Beverley P. Szollosi J. Alexander D.R. J. Biol. Chem. 2002; 277: 1912-1918Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 17Takeda A. Wu J.J. Maizel A.L. J. Biol. Chem. 1992; 267: 16651-16659Abstract Full Text PDF PubMed Google Scholar, 18Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Crossref PubMed Scopus (222) Google Scholar). Interestingly, dimerization of distinct alternatively spliced isoforms of CD45 is different (18Xu Z. Weiss A. Nat. Immunol. 2002; 3: 764-771Crossref PubMed Scopus (222) Google Scholar), 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 RPTPα, RPTPα-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 (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar). Importantly, as shown by bis[sulfosuccinimidyl]suberate cross-linking experiments, H2O2 does not induce de novo dimerization (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar). The H2O2-induced conformational change is dependent on the catalytic site cysteine in RPTPα-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 RPTPα reflected the state of rotational coupling. Although the epitope tag was masked in wild type RPTPα and constitutively dimeric, active RPTPα-F135C with a single cysteine in the extracellular domain, the epitope tag in inactive dimeric RPTPα-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 RPTPα. Masking of the epitope tag was dependent on residues 20–42 in the extracellular domain and on dimerization of RPTPα. RPTPα-D2 and more specifically the catalytic site Cys in RPTPα-D2, Cys723, were required for H2O2-induced HA tag accessibility. Our results suggest that a conformational change in the cytoplasmic domain of RPTPα leads to a change in the conformation of the ectodomain of RPTPα and therefore provide evidence that RPTPs have the potential for inside-out signaling. DISCUSSIONHere we report that the conformation of the extracellular domain of RPTPα dimers changed in response to H2O2treatment, reflecting the catalytic state of RPTPα, active or inactive. We provide evidence that the catalytic site cysteine in the membrane-distal PTP domain, RPTPα-D2, is required for this effect. Our results suggest that redox signaling regulates rotational coupling of RPTPα dimers, thereby reversibly switching RPTPα from an active to an inactive dimeric state.We developed an assay to assess accessibility of the epitope tag in the ectodomain of HA-RPTPα on living cells. Surprisingly, this assay allowed us to discriminate between active and inactive constitutively dimeric conformations of RPTPα (Fig. 2). Moreover, H2O2 treatment of cells significantly increased the accessibility of the HA tag in HA-RPTPα (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 RPTPα-D2 (Fig. 8), suggesting that the conformational change in RPTPα-D2 that we reported previously (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar) 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 RPTPε, masked the epitope tag in RPTPα-Δ43–142, whereas the ectodomain of RPTPε 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 RPTPα 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 RPTPα (14Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Crossref PubMed Scopus (106) Google Scholar) specifically abolished masking of the epitope tag (Fig. 5). Moreover, the short extracellular domain of RPTPε did not mask the RPTPα ectodomain in RPTPα-RPTPε heterodimers. Therefore, we conclude that intermolecular interactions, likely mediated by direct interactions of the ectodomains in RPTPα dimers, mask the epitope tag in the extracellular domain of RPTPα.Taken together, based on our results, we propose a model for the effects of H2O2 on the conformation of RPTPα dimers (Fig. 9). In the prestimulation state RPTPα exists as a preformed, active dimer. Using fluorescence resonance energy transfer and chemical cross-linkers, we have demonstrated that RPTPα dimerization is extensive in the prestimulation state (15Tertoolen L.G. Blanchetot C. Jiang G. Overvoorde J. Gadella T.W.J. Hunter T. den Hertog J. BMC Cell Biol. 2001; 2: 8Crossref PubMed Scopus (75) Google Scholar). The fact that the epitope tag is not accessible in the prestimulation state again suggests that most RPTPα is dimeric. In addition to a conformational change in RPTPα-D2 leading to stabilization of RPTPα dimers (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar), we demonstrate here that H2O2 treatment alters the conformation of the extracellular domain from a state resembling active RPTPα-F135C to a state resembling inactive RPTPα-P137C. Our results suggest that oxidative stress induces a rapid change in rotational coupling, which slowly reverts to the prestimulation state upon reduction of RPTPα.The function of the extracellular domain of RPTPα remains to be determined. Both RPTPα and RPTPε 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 RPTPα and RPTPε and their corresponding cytoplasmic forms (24Andersen J.N. Elson A. Lammers R. Romer J. Clausen J.T. Moller K.B. Moller N.P. Biochem. J. 2001. 2001; 354: 581-590Google Scholar) and in studies addressing the effect of calpain-induced cleavage of RPTPα and RPTPε that occurs in the intracellular juxtamembrane domain of the RPTPs (25Gil-Henn H. Volohonsky G. Elson A. J. Biol. Chem. 2001; 276: 31772-31779Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, previous studies with EGFR-RPTPα chimeras suggest that the RPTPα ectodomain does not solely serve as a membrane localizer but may also confer ligand-dependent regulatory mechanisms to RPTPα (26Blanchetot C. den Hertog J. FEBS Lett. 2000; 484: 235-240Crossref PubMed Scopus (7) Google Scholar).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 RPTPα to its putative ligand. Conversely, ligands may bind to the extracellular domain of RPTPα, 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 RPTPα activity. However, bona fide ligands have not been reported yet for RPTPα. Only the GPI-linked protein contactin was reported to bind to the ectodomain of RPTPα in cis (27Zeng L. D'Alessandri L. Kalousek M.B. Vaughan L. Pallen C.J. J. Cell Biol. 1999; 147: 707-714Crossref PubMed Scopus (98) Google Scholar). However, co-transfection of contactin did not affect accessibility of RPTPα in our hands (data not shown). Our results suggest that RPTPα 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. 28Miranti C.K. Brugge J.S. Nat. Cell Biol. 2002; 4: E83-E90Crossref PubMed Scopus (685) Google Scholar and 29Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). 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 (30Xu D. Rovira I.I. Finkel T. Dev. Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Many PTPs have been demonstrated to be inactivated by oxidation of their catalytic site cysteines (31Caselli A. Marzocchini R. Camici G. Manao G. Moneti G. Pieraccini G. Ramponi G. J. Biol. Chem. 1998; 273: 32554-32560Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 32Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (817) Google Scholar, 33Fauman E.B. Cogswell J.P. Lovejoy B. Rocque W.J. Holmes W. Montana V.G. Piwnica-Worms H. Rink M.J. Saper M.A. Cell. 1998; 93: 617-625Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 34Knebel A. Rahmsdorf H.J. Ullrich A. Herrlich P. EMBO J. 1996; 15: 5314-5325Crossref PubMed Scopus (465) Google Scholar). 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 (35Chiarugi P. Fiaschi T. Taddei M.L. Talini D. Giannoni E. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 33478-33487Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 36Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar, 37Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 38Meng T.C. Fukada T. Tonks N.K. Mol. Cell. 2002; 9: 387-399Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar). Regulation of PTPs by ROS is rapid and reversible. It appears that regulation of RPTPα and perhaps other RPTPs by ROS is more complex, involving not only direct oxidation of the catalytic site cysteine. Previously, we demonstrated that H2O2induced stabilization of RPTPα dimerization, which is responsible for complete inactivation of RPTPα. Here, we demonstrate a change in the ectodomain of RPTPα 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 RPTPα in an inactive conformation.Not only RPTPα 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 (39Blanchetot C. Tertoolen L.G. Overvoorde J. den Hertog J. J. Biol. Chem. 2002; 277: 47263-47269Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 RPTPα implicating that RPTPα has the capacity for inside-out signaling. It will be interesting to see whether redox signaling regulates rotational coupling of other RPTPs as well. Here we report that the conformation of the extracellular domain of RPTPα dimers changed in response to H2O2treatment, reflecting the catalytic state of RPTPα, active or inactive. We provide evidence that the catalytic site cysteine in the membrane-distal PTP domain, RPTPα-D2, is required for this effect. Our results suggest that redox signaling regulates rotational coupling of RPTPα dimers, thereby reversibly switching RPTPα from an active to an inactive dimeric state. We developed an assay to assess accessibility of the epitope tag in the ectodomain of HA-RPTPα on living cells. Surprisingly, this assay allowed us to discriminate between active and inactive constitutively dimeric conformations of RPTPα (Fig. 2). Moreover, H2O2 treatment of cells significantly increased the accessibility of the HA tag in HA-RPTPα (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 RPTPα-D2 (Fig. 8), suggesting that the conformational change in RPTPα-D2 that we reported previously (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar) 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 RPTPε, masked the epitope tag in RPTPα-Δ43–142, whereas the ectodomain of RPTPε 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 RPTPα 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 RPTPα (14Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Crossref PubMed Scopus (106) Google Scholar) specifically abolished masking of the epitope tag (Fig. 5). Moreover, the short extracellular domain of RPTPε did not mask the RPTPα ectodomain in RPTPα-RPTPε heterodimers. Therefore, we conclude that intermolecular interactions, likely mediated by direct interactions of the ectodomains in RPTPα dimers, mask the epitope tag in the extracellular domain of RPTPα. Taken together, based on our results, we propose a model for the effects of H2O2 on the conformation of RPTPα dimers (Fig. 9). In the prestimulation state RPTPα exists as a preformed, active dimer. Using fluorescence resonance energy transfer and chemical cross-linkers, we have demonstrated that RPTPα dimerization is extensive in the prestimulation state (15Tertoolen L.G. Blanchetot C. Jiang G. Overvoorde J. Gadella T.W.J. Hunter T. den Hertog J. BMC Cell Biol. 2001; 2: 8Crossref PubMed Scopus (75) Google Scholar). The fact that the epitope tag is not accessible in the prestimulation state again suggests that most RPTPα is dimeric. In addition to a conformational change in RPTPα-D2 leading to stabilization of RPTPα dimers (19Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Crossref PubMed Scopus (130) Google Scholar), we demonstrate here that H2O2 treatment alters the conformation of the extracellular domain from a state resembling active RPTPα-F135C to a state resembling inactive RPTPα-P137C. Our results suggest that oxidative stress induces a rapid change in rotational coupling, which slowly reverts to the prestimulation state upon reduction of RPTPα. The function of the extracellular domain of RPTPα remains to be determined. Both RPTPα and RPTPε 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 RPTPα and RPTPε and their corresponding cytoplasmic forms (24Andersen J.N. Elson A. Lammers R. Romer J. Clausen J.T. Moller K.B. Moller N.P. Biochem. J. 2001. 2001; 354: 581-590Google Scholar) and in studies addressing the effect of calpain-induced cleavage of RPTPα and RPTPε that occurs in the intracellular juxtamembrane domain of the RPTPs (25Gil-Henn H. Volohonsky G. Elson A. J. Biol. Chem. 2001; 276: 31772-31779Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, previous studies with EGFR-RPTPα chimeras suggest that the RPTPα ectodomain does not solely serve as a membrane localizer but may also confer ligand-dependent regulatory mechanisms to RPTPα (26Blanchetot C. den Hertog J. FEBS Lett. 2000; 484: 235-240Crossref PubMed Scopus (7) Google Scholar). 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 RPTPα to its putative ligand. Conversely, ligands may bind to the extracellular domain of RPTPα, 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 RPTPα activity. However, bona fide ligands have not been reported yet for RPTPα. Only the GPI-linked protein contactin was reported to bind to the ectodomain of RPTPα in cis (27Zeng L. D'Alessandri L. Kalousek M.B. Vaughan L. Pallen C.J. J. Cell Biol. 1999; 147: 707-714Crossref PubMed Scopus (98) Google Scholar). However, co-transfection of contactin did not affect accessibility of RPTPα in our hands (data not shown). Our results suggest that RPTPα 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. 28Miranti C.K. Brugge J.S. Nat. Cell Biol. 2002; 4: E83-E90Crossref PubMed Scopus (685) Google Scholar and 29Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). 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 (30Xu D. Rovira I.I. Finkel T. Dev. Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Many PTPs have been demonstrated to be inactivated by oxidation of their catalytic site cysteines (31Caselli A. Marzocchini R. Camici G. Manao G. Moneti G. Pieraccini G. Ramponi G. J. Biol. Chem. 1998; 273: 32554-32560Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 32Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (817) Google Scholar, 33Fauman E.B. Cogswell J.P. Lovejoy B. Rocque W.J. Holmes W. Montana V.G. Piwnica-Worms H. Rink M.J. Saper M.A. Cell. 1998; 93: 617-625Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 34Knebel A. Rahmsdorf H.J. Ullrich A. Herrlich P. EMBO J. 1996; 15: 5314-5325Crossref PubMed Scopus (465) Google Scholar). 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 (35Chiarugi P. Fiaschi T. Taddei M.L. Talini D. Giannoni E. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 33478-33487Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 36Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2300) Google Scholar, 37Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 38Meng T.C. Fukada T. Tonks N.K. Mol. Cell. 2002; 9: 387-399Abstract Full Text Full Text PDF PubMed Scopus (881) Google Scholar). Regulation of PTPs by ROS is rapid and reversible. It appears that regulation of RPTPα and perhaps other RPTPs by ROS is more complex, involving not only direct oxidation of the catalytic site cysteine. Previously, we demonstrated that H2O2induced stabilization of RPTPα dimerization, which is responsible for complete inactivation of RPTPα. Here, we demonstrate a change in the ectodomain of RPTPα 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 RPTPα in an inactive conformation. Not only RPTPα 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 (39Blanchetot C. Tertoolen L.G. Overvoorde J. den Hertog J. J. Biol. Chem. 2002; 277: 47263-47269Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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 RPTPα implicating that RPTPα has the capacity for inside-out signaling. It will be interesting to see whether redox signaling regulates rotational coupling of other RPTPs as well. We thank Jan Sap for the RPTPα−/− mouse embryo fibroblasts and Ari Elson for the RPTPε cDNA.
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