Intra- and Intermolecular Interactions between Intracellular Domains of Receptor Protein-tyrosine Phosphatases
2002; Elsevier BV; Volume: 277; Issue: 49 Linguagem: Inglês
10.1074/jbc.m205810200
ISSN1083-351X
AutoresChristophe Blanchetot, Leon G.J. Tertoolen, John Overvoorde, Jeroen den Hertog,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoThe presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The generally inactive membrane-distal PTP domains (RPTP-D2s) bind and are proposed to regulate the membrane-proximal PTP domains (RPTP-D1s). We set out to characterize the interactions between RPTP-D1s and RPTP-D2s in vivo by co-immunoprecipitation of hemagglutinin-tagged fusion proteins encoding the transmembrane domain and RPTP-D1 and myc-tagged RPTP-D2. Seven RPTPs from four different subfamilies were used: RPTPα, RPTPε, LAR, RPTPς, RPTPδ, CD45, and RPTPμ. We found that RPTP-D2s bound to RPTPs with different affinities. The presence of intrinsic RPTP-D2 altered the binding specificity toward other RPTP-D2s positively or negatively, depending on the identity of the RPTPs. Furthermore, the C terminus of RPTP-D2s and the “wedge” in RPTP-D1s played a central role in binding specificity. Finally, full-length RPTPα and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPα-D2, the LAR-D2 conformation was affected by oxidative stress, suggesting a common regulatory mechanism for RPTP complex formation. Taken together, interactions between RPTP-D1s and RPTP-D2s are a common but specific mechanism that is likely to be regulated. The RPTP-D2s and the wedge structures are crucial determinants of binding specificity, thus regulating cross-talk between RPTPs. The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The generally inactive membrane-distal PTP domains (RPTP-D2s) bind and are proposed to regulate the membrane-proximal PTP domains (RPTP-D1s). We set out to characterize the interactions between RPTP-D1s and RPTP-D2s in vivo by co-immunoprecipitation of hemagglutinin-tagged fusion proteins encoding the transmembrane domain and RPTP-D1 and myc-tagged RPTP-D2. Seven RPTPs from four different subfamilies were used: RPTPα, RPTPε, LAR, RPTPς, RPTPδ, CD45, and RPTPμ. We found that RPTP-D2s bound to RPTPs with different affinities. The presence of intrinsic RPTP-D2 altered the binding specificity toward other RPTP-D2s positively or negatively, depending on the identity of the RPTPs. Furthermore, the C terminus of RPTP-D2s and the “wedge” in RPTP-D1s played a central role in binding specificity. Finally, full-length RPTPα and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPα-D2, the LAR-D2 conformation was affected by oxidative stress, suggesting a common regulatory mechanism for RPTP complex formation. Taken together, interactions between RPTP-D1s and RPTP-D2s are a common but specific mechanism that is likely to be regulated. The RPTP-D2s and the wedge structures are crucial determinants of binding specificity, thus regulating cross-talk between RPTPs. Protein-tyrosine phosphorylation is of major importance for cell migration, proliferation, differentiation, and transformation within higher eukaryotic organisms. A common way to transmit extracellular signals into the cytoplasm is through receptor protein-tyrosine kinase (RPTK) 1The abbreviations used are: (R)PTK, (receptor) protein-tyrosine kinase; (R)PTP, (receptor) protein-tyrosine phosphatase; D1, membrane-proximal PTP domain; D2, membrane-distal PTP domain; HA: hemagglutinin, FRET, fluorescence resonance energy transfer; aa, amino acid(s); PVDF, polyvinylidene difluoride; LAR, leukocyte common antigen related; CFP, cyan fluorescent protein; HARPTPα, hemagglutinin-tagged RPTPα; YFP, yellow fluorescent protein 1The abbreviations used are: (R)PTK, (receptor) protein-tyrosine kinase; (R)PTP, (receptor) protein-tyrosine phosphatase; D1, membrane-proximal PTP domain; D2, membrane-distal PTP domain; HA: hemagglutinin, FRET, fluorescence resonance energy transfer; aa, amino acid(s); PVDF, polyvinylidene difluoride; LAR, leukocyte common antigen related; CFP, cyan fluorescent protein; HARPTPα, hemagglutinin-tagged RPTPα; YFP, yellow fluorescent protein activation that consequently activates cytosolic proteins by protein-tyrosine phosphorylation. Tyrosine phosphorylation levels are negatively regulated by the protein-tyrosine phosphatases (PTPs) (1Hunter T. Cell. 1995; 80: 225-236Google Scholar). The family of PTPs is divided into two large groups, the cytosolic PTPs and the transmembrane, receptor-like PTPs (RPTPs) (2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Google Scholar, 3den Hertog J. Mech. Dev. 1999; 85: 3-14Google Scholar). Most of the RPTPs include two PTP domains, of which the membrane proximal domain (RPTP-D1) contains all or most of the PTP activity. An understanding of the conservation of the membrane distal PTP domain (RPTP-D2) has remained elusive for a long time. Recent reports suggest a regulatory instead of a catalytic function. Indeed, most of the RPTP-D2s are inactive or very weakly active (4Wang Y. Pallen C.J. EMBO J. 1991; 10: 3231-3237Google Scholar). However, the structure of these RPTP-D2s is similar to that of RPTP-D1s (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar), and mutation of only two residues, which are otherwise highly conserved in active PTPs, restored catalytic activity in several RPTP-D2s (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar, 6Lim K.L. Ng C.H. Pallen C.J. Biochim. Biophys. Acta. 1999; 1434: 275-283Google Scholar, 7Lim K.L. Kolatkar P.R. Ng K.P. Ng C.H. Pallen C.J. J. Biol. Chem. 1998; 273: 28986-28993Google Scholar, 8Buist A. Zhang Y.L. Keng Y.F. Wu L. Zhang Z.Y. den Hertog J. Biochemistry. 1999; 38: 914-922Google Scholar), suggesting that there is evolutionary pressure to keep RPTP-D2s inactive (8Buist A. Zhang Y.L. Keng Y.F. Wu L. Zhang Z.Y. den Hertog J. Biochemistry. 1999; 38: 914-922Google Scholar).Biochemical and structural studies show that RPTP-D2s bind to RPTP-D1s in an intra- and intermolecular fashion. RPTPδ-D2 was found to directly inhibit RPTPς-D1 activity through binding to the juxtamembrane region of RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar). In addition, RPTPα binds to various RPTP-D2s, suggesting that cross-talk between RPTPs may be a shared mechanism of regulation (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Inter- and intramolecular interactions between purified CD45-D1 and CD45-D2 suggest they are direct. CD45-D2 binding to CD45-D1 may disrupt CD45-D1/CD45-D1 homodimerization, perhaps leading to the CD45-D1 activation detected (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Recently, the juxtamembrane region of RPTPμ was shown to bind in an intramolecular fashion with both PTP domains of RPTPμ, thus regulating RPTPμ-D1 activity (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 13Aricescu A.R. Fulga T.A. Cismasiu V. Goody R.S. Szedlacsek S.E. Biochem. Biophys. Res. Commun. 2001; 280: 319-327Google Scholar). Finally, the crystal structure of the complete cytoplasmic domain of LAR, containing LAR-D1 and LAR-D2, provided some structural evidence for intramolecular D1/D2 binding (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar). LAR-D1 interacts extensively with LAR-D2 through the spacer region. Using intramolecular FRET, the conformation of RPTPα-D2 was found to change from a “closed” to an “open” conformation in an oxidative stress-dependent manner. The change in conformation has functional consequences, because only the open RPTPα-D2 binds intermolecularly with RPTP domains (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). However, whether RPTP-D1/RPTP-D2 binding is a general mechanism and, if so, with what specificity, is not known.The function of RPTP-D1/RPTP-D2 binding may be to regulate RPTP-D1 activity, either directly or through regulation of RPTP-D1/RPTP-D1 dimerization. Several reports suggest that RPTP-D1 activity of RPTPα and CD45 is negatively regulated by dimerization (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar, 16Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Google Scholar, 17Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Google Scholar, 18Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Google Scholar, 19Majeti R. Bilwes A.M. Noel J.P. Hunter T. Weiss A. Science. 1998; 279: 88-91Google Scholar). The crystal structure of RPTPα-D1 shows a direct reciprocal interaction of a helix-loop-helix “wedge” structure in the juxtamembrane region with the catalytic site of the opposing monomer (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar). The juxtamembrane domain of RPTPς and the wedge of RPTPα are involved in binding to RPTP-D2s (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Furthermore, CD45-D1 showed an increase in activity when fused to CD45-D2, presumably due to the increase in monomerization (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). The involvement of the wedge in RPTP-D1/RPTP-D2 binding suggested a possible role for RPTP-D2s in the regulation of RPTP-D1/RPTP-D1 dimerization (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar) or in direct regulation of the catalytic activity (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar).Like RPTKs, RPTPs may form heterodimers in vivo. Studies on the Erb family of RPTKs showed that heterodimerization induced specific downstream events different than the ones induced by homodimerization (20Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Google Scholar, 21Riese D.J. Stern D.F. Bioessays. 1998; 20: 41-48Google Scholar). To get basic insight into the relationship between RPTPs, we set out to identify binding between various RPTPs, including RPTPα, RPTPε, LAR, RPTPδ, RPTPς, RPTPμ, and CD45, and their RPTP-D2sin vivo. We found that specific RPTP-D1/RPTP-D2 interactions were favored. The presence of intrinsic RPTP-D2 affected the binding specificity. Furthermore, we found that the C-terminal sequence, at least in RPTPδ-D2, and the wedge in RPTPα were critical to direct RPTPδ-D2 binding specificity suggesting multiple sites of interaction. Finally, we also show heterodimerization between full-length RPTPα and LAR in an oxidative stress-dependent manner. Taken together, we show that binding of RPTPs to RPTP-D2s are common but specific and that RPTP-D2s contain all the features necessary to drive the specificity of RPTP dimerization. Furthermore, our results suggest a specific mechanism of cross-talk between RPTPs.DISCUSSIONLike RPTKs, RPTPs may be regulated by dimerization (27Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Google Scholar). Even though multiple regions may be involved in RPTP dimerization, an important component for dimerization is RPTP-D2. RPTPδ-D2 binds to RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), different RPTP-D2s bind to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and CD45-D2 and RPTPμ-D2 bind to their respective RPTP-D1s. Furthermore, the fact, that the so-called wedge structure, which interacts with the catalytic site of RPTP-D1 in the RPTPα dimer (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar), also binds to RPTP-D2s (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), suggests that RPTP-D2s may be involved in the regulation of dimerization (in trans) (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Our results form the basis of a matrix of in vivo binding between RPTPs and RPTP-D2s. Such a matrix shows that all RPTP-D2s tested bound to membrane-localized RPTP-D1s but with different affinities. Apparently, four different patterns exist for RPTP/RPTP-D2 binding (Table I): 1) RPTPε-D2 and RPTPς-D2 always bound relatively well to all RPTP-D1s tested; 2) RPTPδ-D2 bound either strongly (to RPTPα, RPTPε, or LAR-D1D2) or not at all (to the other RPTPs); 3) RPTPα-D2 and RPTPμ-D2 bound with relatively similar but weak affinity to all RPTP-D1s tested; and 4) LAR-D2 and CD45-D2 also have a similar binding pattern and bound weakly to some but not to other RPTPs. Very closely related RPTP-D2s have significantly different binding properties. RPTPε-D2 bound strongly to all RPTP-D1s, whereas RPTPα-D2, the closest homologue of RPTPε-D2, bound with much less affinity. Furthermore, LAR-D2, RPTPς-D2, and RPTPδ-D2 have very different binding affinities, while they all share very high sequence homology. LAR-D2 bound weakly but specifically to RPTPs. RPTPς-D2 bound strongly to all RPTPs tested, whereas RPTPδ-D2 bound strongly to some RPTPs, but not at all to others. Taken together, our results show that RPTP-D2s have all the characteristics for being at the base of the specificity of RPTP dimerization. Furthermore, our results show that accurate predictions of RPTP-D1/RPTP-D2 interactions based on sequence comparison is still not possible, putting emphasis on our experimental approach to unravel RPTP-D1/RPTP-D2 interactions and function.The presence or absence of intrinsic RPTP-D2 affected the binding efficiency between the remaining RPTP-D1 and other RPTP-D2s. For instance, LAR-D1D2 preferentially bound RPTPδ-D2 while LAR-D1 preferentially bound RPTPς-D2 (Fig. 3 B). These results are consistent with the model that intramolecular D1/D2 binding regulates intermolecular D1/D2 interactions. Indeed, it was shown for CD45 and RPTPμ that the juxtamembrane region and the spacer region, when fused to RPTP-D1, alter the binding specificity of D1 toward RPTP-D2 (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar,30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar). Furthermore, we previously showed that the spacer region and the C-terminal region of RPTPα-D2 bind intramolecularly and regulate the interaction between RPTPα-D1 and RPTPα-D2 (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Because the binding specificity of RPTP-D1s is different than RPTP-D1D2s, our results suggest that stimuli may induce a reshuffling of RPTP dimers.We have previously shown that the C terminus of RPTPα-D2 was involved in binding to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Using RPTPδ-D2, we further show the importance of the C terminus in RPTP-D2s and of the wedge in RPTP-D1s. Replacement of the C-terminal sequence of RPTPδ-D2 changed the binding specificity and affinity. The corresponding region in LAR forms a helix that is localized at the interface between LAR-D1 and LAR-D2 in the crystal structure (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar), providing structural support that this region is somehow involved in intramolecular RPTPD1/RPTPD2 binding. The fact that mutation of this region positively and negatively altered the binding efficiency of different RPTPs may suggest that the C terminus forms (part of) the binding site for RPTP-D1s or plays an indirect role (by regulating RPTP-D2 opening). Other sites are involved in RPTP-D1/RPTP-D2 binding as well, because replacement of the C terminus increased binding in some cases. These results suggest that multiple binding sites between RPTP-D1 and RPTP-D2s exist and may compete and/or cooperate. The juxtamembrane region (Fig. 4 C and Refs. 9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar and10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), the C-terminal region (Fig. 4 and Ref. 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and the spacer region (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar, 12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar, 30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar) are all involved in RPTP-D1/RPTP-D2 binding. The differences in binding between constructs may be due to differences in binding efficiency to one or more binding sites. A matrix such as the one described here may help to pinpoint specific binding sites or help to define these sites.Homodimerization of RPTPα and CD45 is now well established. However, we found that oxidative stress stimulation was necessary to allow co-immunoprecipitation of full-length RPTPα dimers (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Oxidative stress induced a change in the conformation of RPTPα-D2 that released RPTPα from a closed to an open conformation allowing stabilization of dimers. Here we show that oxidative stress induced formation of heterodimers between full-length RPTPα and LAR as well. Using FRET, we demonstrated that oxidative stress induced a conformational change in LAR-D2 as well, albeit to a lesser extent than in RPTPα-D2. Although this was the first time that full-length RPTP heterodimers were detected, cross-talk between RPTPs has been proposed before. Importantly, several elaborate studies on Drosophila axon pathfinding clearly indicate functional cooperation and competition between RPTPs (28Desai C.J. Krueger N.X. Saito H. Zinn K. Development. 1997; 124: 1941-1952Google Scholar, 29Sun Q. Schindelholz B. Knirr M. Schmid A. Zinn K. Mol. Cell. Neurosci. 2001; 17: 274-291Google Scholar). Although these studies only show genetic interactions, it is now tempting to speculate that cross-talk betweenDrosophila RPTPs may be mediated by direct, stimulation-dependent heterodimerization.A burning question that remains is: what is the effect of RPTP-D2 on RPTP-D1 activity? RPTPδ-D2 binding inhibits RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), and RPTPμ-D2 binding decreases RPTPμ-D1 catalytic activity (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 13Aricescu A.R. Fulga T.A. Cismasiu V. Goody R.S. Szedlacsek S.E. Biochem. Biophys. Res. Commun. 2001; 280: 319-327Google Scholar). In contrast, the presence of CD45-D2 fused to CD45-D1 led to an increase in total PTP catalytic activity, presumably due to an increase in monomerization (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Furthermore, in vitro no changes in RPTPα-D1 activity were detected after addition of RPTPα-D2 (data not shown). Taken together, these results suggest that the effect of RPTP-D2s on RPTP-D1s may depend on the RPTP, or on the RPTP-D1/RPTP-D2 combination. Indeed, the strength and site(s) of the interaction may be important. Strong binding of RPTP-D2 to the wedge may lead to inactivation of RPTP-D1, whereas weak and dynamic RPTP-D2 binding to the wedge may lead to monomerization and activation of RPTP-D1. For these reasons, all RPTP-D1/RPTP-D2 combinations will need to be studied for changes in activity. However, these activity assays are technically difficult, because the stoichiometry of binding of RPTP-D1 to RPTP-D2 will influence the effect of RPTP-D2 on RPTP-D1 activity in assaysin vitro. A much-preferred configuration would require analysis of dephosphorylation of physiological substrates in living cells, which is not feasible yet for all RPTPs.In conclusion, our results suggest that specific and regulated heterodimerization between RPTPs occurs in vivo. RPTP-D2s have specific affinity for RPTP-D1s and consequently may be at the base of functional and regulatory cross-talk between RPTPs. Protein-tyrosine phosphorylation is of major importance for cell migration, proliferation, differentiation, and transformation within higher eukaryotic organisms. A common way to transmit extracellular signals into the cytoplasm is through receptor protein-tyrosine kinase (RPTK) 1The abbreviations used are: (R)PTK, (receptor) protein-tyrosine kinase; (R)PTP, (receptor) protein-tyrosine phosphatase; D1, membrane-proximal PTP domain; D2, membrane-distal PTP domain; HA: hemagglutinin, FRET, fluorescence resonance energy transfer; aa, amino acid(s); PVDF, polyvinylidene difluoride; LAR, leukocyte common antigen related; CFP, cyan fluorescent protein; HARPTPα, hemagglutinin-tagged RPTPα; YFP, yellow fluorescent protein 1The abbreviations used are: (R)PTK, (receptor) protein-tyrosine kinase; (R)PTP, (receptor) protein-tyrosine phosphatase; D1, membrane-proximal PTP domain; D2, membrane-distal PTP domain; HA: hemagglutinin, FRET, fluorescence resonance energy transfer; aa, amino acid(s); PVDF, polyvinylidene difluoride; LAR, leukocyte common antigen related; CFP, cyan fluorescent protein; HARPTPα, hemagglutinin-tagged RPTPα; YFP, yellow fluorescent protein activation that consequently activates cytosolic proteins by protein-tyrosine phosphorylation. Tyrosine phosphorylation levels are negatively regulated by the protein-tyrosine phosphatases (PTPs) (1Hunter T. Cell. 1995; 80: 225-236Google Scholar). The family of PTPs is divided into two large groups, the cytosolic PTPs and the transmembrane, receptor-like PTPs (RPTPs) (2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Google Scholar, 3den Hertog J. Mech. Dev. 1999; 85: 3-14Google Scholar). Most of the RPTPs include two PTP domains, of which the membrane proximal domain (RPTP-D1) contains all or most of the PTP activity. An understanding of the conservation of the membrane distal PTP domain (RPTP-D2) has remained elusive for a long time. Recent reports suggest a regulatory instead of a catalytic function. Indeed, most of the RPTP-D2s are inactive or very weakly active (4Wang Y. Pallen C.J. EMBO J. 1991; 10: 3231-3237Google Scholar). However, the structure of these RPTP-D2s is similar to that of RPTP-D1s (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar), and mutation of only two residues, which are otherwise highly conserved in active PTPs, restored catalytic activity in several RPTP-D2s (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar, 6Lim K.L. Ng C.H. Pallen C.J. Biochim. Biophys. Acta. 1999; 1434: 275-283Google Scholar, 7Lim K.L. Kolatkar P.R. Ng K.P. Ng C.H. Pallen C.J. J. Biol. Chem. 1998; 273: 28986-28993Google Scholar, 8Buist A. Zhang Y.L. Keng Y.F. Wu L. Zhang Z.Y. den Hertog J. Biochemistry. 1999; 38: 914-922Google Scholar), suggesting that there is evolutionary pressure to keep RPTP-D2s inactive (8Buist A. Zhang Y.L. Keng Y.F. Wu L. Zhang Z.Y. den Hertog J. Biochemistry. 1999; 38: 914-922Google Scholar). Biochemical and structural studies show that RPTP-D2s bind to RPTP-D1s in an intra- and intermolecular fashion. RPTPδ-D2 was found to directly inhibit RPTPς-D1 activity through binding to the juxtamembrane region of RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar). In addition, RPTPα binds to various RPTP-D2s, suggesting that cross-talk between RPTPs may be a shared mechanism of regulation (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Inter- and intramolecular interactions between purified CD45-D1 and CD45-D2 suggest they are direct. CD45-D2 binding to CD45-D1 may disrupt CD45-D1/CD45-D1 homodimerization, perhaps leading to the CD45-D1 activation detected (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Recently, the juxtamembrane region of RPTPμ was shown to bind in an intramolecular fashion with both PTP domains of RPTPμ, thus regulating RPTPμ-D1 activity (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 13Aricescu A.R. Fulga T.A. Cismasiu V. Goody R.S. Szedlacsek S.E. Biochem. Biophys. Res. Commun. 2001; 280: 319-327Google Scholar). Finally, the crystal structure of the complete cytoplasmic domain of LAR, containing LAR-D1 and LAR-D2, provided some structural evidence for intramolecular D1/D2 binding (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar). LAR-D1 interacts extensively with LAR-D2 through the spacer region. Using intramolecular FRET, the conformation of RPTPα-D2 was found to change from a “closed” to an “open” conformation in an oxidative stress-dependent manner. The change in conformation has functional consequences, because only the open RPTPα-D2 binds intermolecularly with RPTP domains (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). However, whether RPTP-D1/RPTP-D2 binding is a general mechanism and, if so, with what specificity, is not known. The function of RPTP-D1/RPTP-D2 binding may be to regulate RPTP-D1 activity, either directly or through regulation of RPTP-D1/RPTP-D1 dimerization. Several reports suggest that RPTP-D1 activity of RPTPα and CD45 is negatively regulated by dimerization (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar, 16Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Google Scholar, 17Jiang G. den Hertog J. Su J. Noel J. Sap J. Hunter T. Nature. 1999; 401: 606-610Google Scholar, 18Jiang G. den Hertog J. Hunter T. Mol. Cell. Biol. 2000; 20: 5917-5929Google Scholar, 19Majeti R. Bilwes A.M. Noel J.P. Hunter T. Weiss A. Science. 1998; 279: 88-91Google Scholar). The crystal structure of RPTPα-D1 shows a direct reciprocal interaction of a helix-loop-helix “wedge” structure in the juxtamembrane region with the catalytic site of the opposing monomer (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar). The juxtamembrane domain of RPTPς and the wedge of RPTPα are involved in binding to RPTP-D2s (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Furthermore, CD45-D1 showed an increase in activity when fused to CD45-D2, presumably due to the increase in monomerization (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). The involvement of the wedge in RPTP-D1/RPTP-D2 binding suggested a possible role for RPTP-D2s in the regulation of RPTP-D1/RPTP-D1 dimerization (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar) or in direct regulation of the catalytic activity (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Like RPTKs, RPTPs may form heterodimers in vivo. Studies on the Erb family of RPTKs showed that heterodimerization induced specific downstream events different than the ones induced by homodimerization (20Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Google Scholar, 21Riese D.J. Stern D.F. Bioessays. 1998; 20: 41-48Google Scholar). To get basic insight into the relationship between RPTPs, we set out to identify binding between various RPTPs, including RPTPα, RPTPε, LAR, RPTPδ, RPTPς, RPTPμ, and CD45, and their RPTP-D2sin vivo. We found that specific RPTP-D1/RPTP-D2 interactions were favored. The presence of intrinsic RPTP-D2 affected the binding specificity. Furthermore, we found that the C-terminal sequence, at least in RPTPδ-D2, and the wedge in RPTPα were critical to direct RPTPδ-D2 binding specificity suggesting multiple sites of interaction. Finally, we also show heterodimerization between full-length RPTPα and LAR in an oxidative stress-dependent manner. Taken together, we show that binding of RPTPs to RPTP-D2s are common but specific and that RPTP-D2s contain all the features necessary to drive the specificity of RPTP dimerization. Furthermore, our results suggest a specific mechanism of cross-talk between RPTPs. DISCUSSIONLike RPTKs, RPTPs may be regulated by dimerization (27Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Google Scholar). Even though multiple regions may be involved in RPTP dimerization, an important component for dimerization is RPTP-D2. RPTPδ-D2 binds to RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), different RPTP-D2s bind to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and CD45-D2 and RPTPμ-D2 bind to their respective RPTP-D1s. Furthermore, the fact, that the so-called wedge structure, which interacts with the catalytic site of RPTP-D1 in the RPTPα dimer (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar), also binds to RPTP-D2s (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), suggests that RPTP-D2s may be involved in the regulation of dimerization (in trans) (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Our results form the basis of a matrix of in vivo binding between RPTPs and RPTP-D2s. Such a matrix shows that all RPTP-D2s tested bound to membrane-localized RPTP-D1s but with different affinities. Apparently, four different patterns exist for RPTP/RPTP-D2 binding (Table I): 1) RPTPε-D2 and RPTPς-D2 always bound relatively well to all RPTP-D1s tested; 2) RPTPδ-D2 bound either strongly (to RPTPα, RPTPε, or LAR-D1D2) or not at all (to the other RPTPs); 3) RPTPα-D2 and RPTPμ-D2 bound with relatively similar but weak affinity to all RPTP-D1s tested; and 4) LAR-D2 and CD45-D2 also have a similar binding pattern and bound weakly to some but not to other RPTPs. Very closely related RPTP-D2s have significantly different binding properties. RPTPε-D2 bound strongly to all RPTP-D1s, whereas RPTPα-D2, the closest homologue of RPTPε-D2, bound with much less affinity. Furthermore, LAR-D2, RPTPς-D2, and RPTPδ-D2 have very different binding affinities, while they all share very high sequence homology. LAR-D2 bound weakly but specifically to RPTPs. RPTPς-D2 bound strongly to all RPTPs tested, whereas RPTPδ-D2 bound strongly to some RPTPs, but not at all to others. Taken together, our results show that RPTP-D2s have all the characteristics for being at the base of the specificity of RPTP dimerization. Furthermore, our results show that accurate predictions of RPTP-D1/RPTP-D2 interactions based on sequence comparison is still not possible, putting emphasis on our experimental approach to unravel RPTP-D1/RPTP-D2 interactions and function.The presence or absence of intrinsic RPTP-D2 affected the binding efficiency between the remaining RPTP-D1 and other RPTP-D2s. For instance, LAR-D1D2 preferentially bound RPTPδ-D2 while LAR-D1 preferentially bound RPTPς-D2 (Fig. 3 B). These results are consistent with the model that intramolecular D1/D2 binding regulates intermolecular D1/D2 interactions. Indeed, it was shown for CD45 and RPTPμ that the juxtamembrane region and the spacer region, when fused to RPTP-D1, alter the binding specificity of D1 toward RPTP-D2 (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar,30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar). Furthermore, we previously showed that the spacer region and the C-terminal region of RPTPα-D2 bind intramolecularly and regulate the interaction between RPTPα-D1 and RPTPα-D2 (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Because the binding specificity of RPTP-D1s is different than RPTP-D1D2s, our results suggest that stimuli may induce a reshuffling of RPTP dimers.We have previously shown that the C terminus of RPTPα-D2 was involved in binding to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Using RPTPδ-D2, we further show the importance of the C terminus in RPTP-D2s and of the wedge in RPTP-D1s. Replacement of the C-terminal sequence of RPTPδ-D2 changed the binding specificity and affinity. The corresponding region in LAR forms a helix that is localized at the interface between LAR-D1 and LAR-D2 in the crystal structure (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar), providing structural support that this region is somehow involved in intramolecular RPTPD1/RPTPD2 binding. The fact that mutation of this region positively and negatively altered the binding efficiency of different RPTPs may suggest that the C terminus forms (part of) the binding site for RPTP-D1s or plays an indirect role (by regulating RPTP-D2 opening). Other sites are involved in RPTP-D1/RPTP-D2 binding as well, because replacement of the C terminus increased binding in some cases. These results suggest that multiple binding sites between RPTP-D1 and RPTP-D2s exist and may compete and/or cooperate. The juxtamembrane region (Fig. 4 C and Refs. 9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar and10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), the C-terminal region (Fig. 4 and Ref. 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and the spacer region (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar, 12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar, 30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar) are all involved in RPTP-D1/RPTP-D2 binding. The differences in binding between constructs may be due to differences in binding efficiency to one or more binding sites. A matrix such as the one described here may help to pinpoint specific binding sites or help to define these sites.Homodimerization of RPTPα and CD45 is now well established. However, we found that oxidative stress stimulation was necessary to allow co-immunoprecipitation of full-length RPTPα dimers (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Oxidative stress induced a change in the conformation of RPTPα-D2 that released RPTPα from a closed to an open conformation allowing stabilization of dimers. Here we show that oxidative stress induced formation of heterodimers between full-length RPTPα and LAR as well. Using FRET, we demonstrated that oxidative stress induced a conformational change in LAR-D2 as well, albeit to a lesser extent than in RPTPα-D2. Although this was the first time that full-length RPTP heterodimers were detected, cross-talk between RPTPs has been proposed before. Importantly, several elaborate studies on Drosophila axon pathfinding clearly indicate functional cooperation and competition between RPTPs (28Desai C.J. Krueger N.X. Saito H. Zinn K. Development. 1997; 124: 1941-1952Google Scholar, 29Sun Q. Schindelholz B. Knirr M. Schmid A. Zinn K. Mol. Cell. Neurosci. 2001; 17: 274-291Google Scholar). Although these studies only show genetic interactions, it is now tempting to speculate that cross-talk betweenDrosophila RPTPs may be mediated by direct, stimulation-dependent heterodimerization.A burning question that remains is: what is the effect of RPTP-D2 on RPTP-D1 activity? RPTPδ-D2 binding inhibits RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), and RPTPμ-D2 binding decreases RPTPμ-D1 catalytic activity (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 13Aricescu A.R. Fulga T.A. Cismasiu V. Goody R.S. Szedlacsek S.E. Biochem. Biophys. Res. Commun. 2001; 280: 319-327Google Scholar). In contrast, the presence of CD45-D2 fused to CD45-D1 led to an increase in total PTP catalytic activity, presumably due to an increase in monomerization (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Furthermore, in vitro no changes in RPTPα-D1 activity were detected after addition of RPTPα-D2 (data not shown). Taken together, these results suggest that the effect of RPTP-D2s on RPTP-D1s may depend on the RPTP, or on the RPTP-D1/RPTP-D2 combination. Indeed, the strength and site(s) of the interaction may be important. Strong binding of RPTP-D2 to the wedge may lead to inactivation of RPTP-D1, whereas weak and dynamic RPTP-D2 binding to the wedge may lead to monomerization and activation of RPTP-D1. For these reasons, all RPTP-D1/RPTP-D2 combinations will need to be studied for changes in activity. However, these activity assays are technically difficult, because the stoichiometry of binding of RPTP-D1 to RPTP-D2 will influence the effect of RPTP-D2 on RPTP-D1 activity in assaysin vitro. A much-preferred configuration would require analysis of dephosphorylation of physiological substrates in living cells, which is not feasible yet for all RPTPs.In conclusion, our results suggest that specific and regulated heterodimerization between RPTPs occurs in vivo. RPTP-D2s have specific affinity for RPTP-D1s and consequently may be at the base of functional and regulatory cross-talk between RPTPs. Like RPTKs, RPTPs may be regulated by dimerization (27Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Google Scholar). Even though multiple regions may be involved in RPTP dimerization, an important component for dimerization is RPTP-D2. RPTPδ-D2 binds to RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), different RPTP-D2s bind to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and CD45-D2 and RPTPμ-D2 bind to their respective RPTP-D1s. Furthermore, the fact, that the so-called wedge structure, which interacts with the catalytic site of RPTP-D1 in the RPTPα dimer (15Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Google Scholar), also binds to RPTP-D2s (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar, 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), suggests that RPTP-D2s may be involved in the regulation of dimerization (in trans) (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Our results form the basis of a matrix of in vivo binding between RPTPs and RPTP-D2s. Such a matrix shows that all RPTP-D2s tested bound to membrane-localized RPTP-D1s but with different affinities. Apparently, four different patterns exist for RPTP/RPTP-D2 binding (Table I): 1) RPTPε-D2 and RPTPς-D2 always bound relatively well to all RPTP-D1s tested; 2) RPTPδ-D2 bound either strongly (to RPTPα, RPTPε, or LAR-D1D2) or not at all (to the other RPTPs); 3) RPTPα-D2 and RPTPμ-D2 bound with relatively similar but weak affinity to all RPTP-D1s tested; and 4) LAR-D2 and CD45-D2 also have a similar binding pattern and bound weakly to some but not to other RPTPs. Very closely related RPTP-D2s have significantly different binding properties. RPTPε-D2 bound strongly to all RPTP-D1s, whereas RPTPα-D2, the closest homologue of RPTPε-D2, bound with much less affinity. Furthermore, LAR-D2, RPTPς-D2, and RPTPδ-D2 have very different binding affinities, while they all share very high sequence homology. LAR-D2 bound weakly but specifically to RPTPs. RPTPς-D2 bound strongly to all RPTPs tested, whereas RPTPδ-D2 bound strongly to some RPTPs, but not at all to others. Taken together, our results show that RPTP-D2s have all the characteristics for being at the base of the specificity of RPTP dimerization. Furthermore, our results show that accurate predictions of RPTP-D1/RPTP-D2 interactions based on sequence comparison is still not possible, putting emphasis on our experimental approach to unravel RPTP-D1/RPTP-D2 interactions and function. The presence or absence of intrinsic RPTP-D2 affected the binding efficiency between the remaining RPTP-D1 and other RPTP-D2s. For instance, LAR-D1D2 preferentially bound RPTPδ-D2 while LAR-D1 preferentially bound RPTPς-D2 (Fig. 3 B). These results are consistent with the model that intramolecular D1/D2 binding regulates intermolecular D1/D2 interactions. Indeed, it was shown for CD45 and RPTPμ that the juxtamembrane region and the spacer region, when fused to RPTP-D1, alter the binding specificity of D1 toward RPTP-D2 (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar,30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar). Furthermore, we previously showed that the spacer region and the C-terminal region of RPTPα-D2 bind intramolecularly and regulate the interaction between RPTPα-D1 and RPTPα-D2 (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Because the binding specificity of RPTP-D1s is different than RPTP-D1D2s, our results suggest that stimuli may induce a reshuffling of RPTP dimers. We have previously shown that the C terminus of RPTPα-D2 was involved in binding to RPTPα (10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar). Using RPTPδ-D2, we further show the importance of the C terminus in RPTP-D2s and of the wedge in RPTP-D1s. Replacement of the C-terminal sequence of RPTPδ-D2 changed the binding specificity and affinity. The corresponding region in LAR forms a helix that is localized at the interface between LAR-D1 and LAR-D2 in the crystal structure (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar), providing structural support that this region is somehow involved in intramolecular RPTPD1/RPTPD2 binding. The fact that mutation of this region positively and negatively altered the binding efficiency of different RPTPs may suggest that the C terminus forms (part of) the binding site for RPTP-D1s or plays an indirect role (by regulating RPTP-D2 opening). Other sites are involved in RPTP-D1/RPTP-D2 binding as well, because replacement of the C terminus increased binding in some cases. These results suggest that multiple binding sites between RPTP-D1 and RPTP-D2s exist and may compete and/or cooperate. The juxtamembrane region (Fig. 4 C and Refs. 9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar and10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), the C-terminal region (Fig. 4 and Ref. 10Blanchetot C. den Hertog J. J. Biol. Chem. 2000; 275: 12446-12452Google Scholar), and the spacer region (5Nam H.J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Google Scholar, 12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar, 30Hayami-Noumi K. Tsuchiya T. Moriyama Y. Noumi T. FEBS Lett. 2000; 468: 68-72Google Scholar) are all involved in RPTP-D1/RPTP-D2 binding. The differences in binding between constructs may be due to differences in binding efficiency to one or more binding sites. A matrix such as the one described here may help to pinpoint specific binding sites or help to define these sites. Homodimerization of RPTPα and CD45 is now well established. However, we found that oxidative stress stimulation was necessary to allow co-immunoprecipitation of full-length RPTPα dimers (14Blanchetot C. Tertoolen L.G. den Hertog J. EMBO J. 2002; 21: 493-503Google Scholar). Oxidative stress induced a change in the conformation of RPTPα-D2 that released RPTPα from a closed to an open conformation allowing stabilization of dimers. Here we show that oxidative stress induced formation of heterodimers between full-length RPTPα and LAR as well. Using FRET, we demonstrated that oxidative stress induced a conformational change in LAR-D2 as well, albeit to a lesser extent than in RPTPα-D2. Although this was the first time that full-length RPTP heterodimers were detected, cross-talk between RPTPs has been proposed before. Importantly, several elaborate studies on Drosophila axon pathfinding clearly indicate functional cooperation and competition between RPTPs (28Desai C.J. Krueger N.X. Saito H. Zinn K. Development. 1997; 124: 1941-1952Google Scholar, 29Sun Q. Schindelholz B. Knirr M. Schmid A. Zinn K. Mol. Cell. Neurosci. 2001; 17: 274-291Google Scholar). Although these studies only show genetic interactions, it is now tempting to speculate that cross-talk betweenDrosophila RPTPs may be mediated by direct, stimulation-dependent heterodimerization. A burning question that remains is: what is the effect of RPTP-D2 on RPTP-D1 activity? RPTPδ-D2 binding inhibits RPTPς-D1 (9Wallace M.J. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Google Scholar), and RPTPμ-D2 binding decreases RPTPμ-D1 catalytic activity (12Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Google Scholar, 13Aricescu A.R. Fulga T.A. Cismasiu V. Goody R.S. Szedlacsek S.E. Biochem. Biophys. Res. Commun. 2001; 280: 319-327Google Scholar). In contrast, the presence of CD45-D2 fused to CD45-D1 led to an increase in total PTP catalytic activity, presumably due to an increase in monomerization (11Felberg J. Johnson P. J. Biol. Chem. 1998; 273: 17839-17845Google Scholar). Furthermore, in vitro no changes in RPTPα-D1 activity were detected after addition of RPTPα-D2 (data not shown). Taken together, these results suggest that the effect of RPTP-D2s on RPTP-D1s may depend on the RPTP, or on the RPTP-D1/RPTP-D2 combination. Indeed, the strength and site(s) of the interaction may be important. Strong binding of RPTP-D2 to the wedge may lead to inactivation of RPTP-D1, whereas weak and dynamic RPTP-D2 binding to the wedge may lead to monomerization and activation of RPTP-D1. For these reasons, all RPTP-D1/RPTP-D2 combinations will need to be studied for changes in activity. However, these activity assays are technically difficult, because the stoichiometry of binding of RPTP-D1 to RPTP-D2 will influence the effect of RPTP-D2 on RPTP-D1 activity in assaysin vitro. A much-preferred configuration would require analysis of dephosphorylation of physiological substrates in living cells, which is not feasible yet for all RPTPs. In conclusion, our results suggest that specific and regulated heterodimerization between RPTPs occurs in vivo. RPTP-D2s have specific affinity for RPTP-D1s and consequently may be at the base of functional and regulatory cross-talk between RPTPs. We thank Wiljan Hendriks for providing the hLAR cDNA, Ari Elson for the mRPTPε cDNA, Wouter Moolenaar for the RPTPμ cDNA, and Thea van der Wijk and Jaap van Hellemond for helpful discussions.
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