Redox regulation of dimerization of the receptor protein-tyrosine phosphatases RPTPalpha, LAR, RPTPmu and CD45

Whether dimerization is a general regulatory mechanism of receptor protein-tyrosine phosphatases (RPTPs) is a subject of debate. Biochemical evidence demonstrates that RPTPalpha and cluster of differentiation (CD)45 dimerize. Their catalytic activity is regulated by dimerization and structural evidence from RPTPalpha supports dimerization-induced inhibition of catalytic activity. The crystal structures of CD45 and leukocyte common antigen related (LAR) indicate that dimerization would result in a steric clash. Here, we investigate dimerization of four RPTPs. We demonstrate that LAR and RPTPmu dimerized constitutively, which is likely to be due to their ectodomains. To investigate the role of the cytoplasmic domain in dimerization we generated RPTPalpha ectodomain (EDalpha)/RPTP chimeras and found that -- similarly to native RPTPalpha -- oxidation stabilized their dimerization. Limited tryptic proteolysis demonstrated that oxidation induced conformational changes in the cytoplasmic domains of these RPTPs, indicating that the cytoplasmic domains are not rigid structures, but rather that there is flexibility. Moreover, oxidation induced changes in the rotational coupling of dimers of full length EDalpha/RPTP chimeras in living cells, which were largely dependent on the catalytic cysteine in the membrane-distal protein-tyrosine phosphatase domain of RPTPalpha and LAR. Our results provide new evidence for redox regulation of dimerized RPTPs.     

H2O2-induced intermolecular disulfide bond formation between receptor protein-tyrosine phosphatases

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.     

Receptor-like protein tyrosine phosphatase alpha homodimerizes on the cell surface

We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase alpha (RPTPalpha) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTPsigma D1 catalytic domain. Most recently, we have also shown that enforced dimerization inhibits the biological activity of full-length RPTPalpha in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTPalpha dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTPalpha exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTPalpha biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTPalpha homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization. Finally, we also provide evidence that both the RPTPalpha extracellular domain and the transmembrane domain were independently able to homodimerize. These results lead us to propose a zipper model in which inactive RPTPalpha dimers are stabilized by multiple, relatively weak dimerization interfaces. Dimerization in this manner would provide a potential mechanism for negative regulation of RPTPalpha. Such RPTPalpha dimers could be activated by extracellular ligands or intracellular binding proteins that induce monomerization or by intracellular signaling events that induce an open conformation of the dimer.     

Reversible oxidation of the membrane distal domain of receptor PTPalpha is mediated by a cyclic sulfenamide

Protein tyrosine phosphatases (PTPs) are fundamental to the regulation of cellular signalling cascades triggered by protein tyrosine kinases. Most receptor-like PTPs (RPTPs) comprise two tandem PTP domains, with only the membrane proximal domains (D1) having significant phosphatase activity; the membrane distal domains (D2) display little to no catalytic activity. Intriguingly, however, many RPTP D2s share the catalytically essential Cys and Arg residues of D1s. D2 of RPTPalpha may function as a redox sensor that mediates regulation of D1 via reactive oxygen species. Oxidation of Cys723 of RPTPalpha D2 (equivalent to PTP catalytic Cys residues) stabilizes RPTPalpha dimers, induces rotational coupling, and is required for inactivation of D1 phosphatase activity. Here, we investigated the structural consequences of RPTPalpha D2 oxidation. Exposure of RPTPalpha D2 to oxidants promotes formation of a cyclic sulfenamide species, a reversibly oxidized state of Cys723, accompanied by conformational changes of the D2 catalytic site. The cyclic sulfenamide is highly resistant to terminal oxidation to sulfinic and sulfonic acids. Conformational changes associated with RPTPalpha D2 oxidation have implications for RPTPalpha quaternary structure and allosteric regulation of D1 phosphatase activity.     

Receptor protein-tyrosine phosphatase signalling in development

Receptor Protein-Tyrosine Phosphatases (RPTPs) belong to the superfamily of protein-tyrosine phosphatases and have the intrinsic ability to transduce signals across the cell membrane. We are beginning to understand the role of RPTPs in development of invertebrates, due to elegant genetic studies. In contrast, relatively little is known about the role of RPTPs in vertebrate development. Signalling by RPTPs has predominantly been studied in mammalian cell systems, which has led to important insights into potential ligands, into regulation of RPTP activity and into potential RPTP substrates. Here, we will introduce the RPTPs, and discuss the function of the LAR-subfamily of RPTPs. In addition, we focus on the function and signalling of the haematopoietic RPTP, CD45. Finally, we will discuss the structure and function of RPTPalpha, the RPTP that is the subject of our studies.     

Antibody-induced dimerization of HARPTPalpha-EGFR chimera suggests a ligand dependent mechanism of regulation for RPTPalpha

We developed a system to study the function of the ectodomain of RPTPalpha, a transmembrane protein-tyrosine phosphatase, by fusing the HA-epitope tagged ectodomain of RPTPalpha to the transmembrane and intracellular domain of the epidermal growth factor receptor, EGFR, a receptor protein-tyrosine kinase that is activated by dimerization. Although the use of chemical crosslinkers shows that preformed HARPTPalpha-EGFR dimers exist, bivalent anti-HA-tag antibody activated HARPTPalpha-EGFR chimeras, suggesting this system may be used to study regulation of dimerization. We used this system to show that newborn calf serum may contain (a) potential ligand(s) for RPTPalpha. Our results suggest that RPTPalpha dimerization and thus activity may be affected by ligand binding.     

Multimerization of the protein-tyrosine phosphatase (PTP)-like insulin-dependent diabetes mellitus autoantigens IA-2 and IA-2beta with receptor PTPs (RPTPs). Inhibition of RPTPalpha enzymatic activity

Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains, and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2beta, major autoantigens in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2beta are involved in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that IA-2 and IA-2beta are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPalpha and RPTPepsilon, and down-regulate RPTPalpha enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2beta.     

Regulation of receptor protein-tyrosine phosphatase alpha by oxidative stress

The presence of two protein-tyrosine phosphatase (PTP) domains is a striking feature in most transmembrane receptor PTPs (RPTPs). The function of the generally inactive membrane-distal PTP domain (RPTP-D2) is unknown. Here we report that an intramolecular interaction between the spacer region (Sp) and the C-terminus in RPTPalpha prohibited intermolecular interactions. Interestingly, stress factors such as H(2)O(2), UV and heat shock induced reversible, free radical-dependent, intermolecular interactions between RPTPalpha and RPTPalpha-SpD2, suggesting an inducible switch in conformation and binding. The catalytic site cysteine of RPTPalpha-SpD2, Cys723, was required for the H(2)O(2) effect on RPTPalpha. H(2)O(2) induced a rapid, reversible, Cys723-dependent conformational change in vivo, as detected by fluorescence resonance energy transfer, with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) flanking RPTPalpha-SpD2 in a single chimeric protein. Importantly, H(2)O(2) treatment stabilized RPTPalpha dimers, resulting in inactivation. We propose a model in which oxidative stress induces a conformational change in RPTPalpha-D2, leading to stabilization of RPTPalpha dimers, and thus to inhibition of RPTPalpha activity.     

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

Background  

Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined.     

Novel mechanism for suppression of hyperpolarization-activated cyclic nucleotide-gated pacemaker channels by receptor-like tyrosine phosphatase-alpha

We have previously reported an important role of increased tyrosine phosphorylation activity by Src in the modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we provide evidence showing a novel mechanism of decreased tyrosine phosphorylation on HCN channel properties. We found that the receptor-like protein-tyrosine phosphatase-alpha (RPTPalpha) significantly inhibited or eliminated HCN2 channel expression in HEK293 cells. Biochemical evidence showed that the surface expression of HCN2 was remarkably reduced by RPTPalpha, which was in parallel to the decreased tyrosine phosphorylation of the channel protein. Confocal imaging confirmed that the membrane surface distribution of the HCN2 channel was inhibited by RPTPalpha. Moreover, we detected the presence of RPTPalpha proteins in cardiac ventricles with expression levels changed during development. Inhibition of tyrosine phosphatase activity by phenylarsine oxide or sodium orthovanadate shifted ventricular hyperpolarization-activated current (I(f), generated by HCN channels) activation from nonphysiological voltages into physiological voltages associated with accelerated activation kinetics. In conclusion, we showed a critical role RPTPalpha plays in HCN channel function via tyrosine dephosphorylation. These findings are also important to neurons where HCN and RPTPalpha are richly expressed.     

Intra- and intermolecular interactions between intracellular domains of receptor protein-tyrosine phosphatases

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: RPTPalpha, RPTPepsilon, LAR, RPTPvarsigma, RPTPdelta, CD45, and RPTP(mu). 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 RPTPalpha and LAR heterodimerized in an oxidative stress-dependent manner. Like RPTPalpha-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.     

Multiple interactions between receptor protein-tyrosine phosphatase (RPTP) alpha and membrane-distal protein-tyrosine phosphatase domains of various RPTPs

Receptor protein-tyrosine phosphatase (RPTP) alpha belongs to the large family of receptor protein-tyrosine phosphatases containing two tandem phosphatase domains. Most of the catalytic activity is retained in the first, membrane-proximal domain (RPTPalpha-D1), and little is known about the function of the second, membrane-distal domain (RPTPalpha-D2). We investigated whether proteins bound to RPTPalpha using the two-hybrid system and found that the second domain of RPTPsigma interacted with the juxtamembrane domain of RPTPalpha. We confirmed this interaction by co-immunoprecipitation experiments. Furthermore, RPTPalpha not only interacted with RPTPsigma-D2 but also with RPTPalpha-D2, LAR-D2, RPTPdelta-D2, and RPTPmu-D2, members of various RPTP subfamilies, although with different affinities. In the yeast two-hybrid system and in glutathione S-transferase pull-down assays, we show that the RPTP-D2s interacted directly with the wedge structure of RPTPalpha-D1 that has been demonstrated to be involved in inactivation of the RPTPalpha-D1/RPTPalpha-D1 homodimer. The interaction was specific because the equivalent wedge structure in LAR was unable to interact with RPTPalpha-D2 or LAR-D2. In vivo, we show that other interaction sites exist as well, including the C terminus of RPTPalpha-D2. The observation that RPTPalpha, but not LAR, bound to multiple RPTP-D2s with varying affinities suggests a specific mechanism of cross-talk between RPTPs that may regulate their biological function.     

Expression of receptor protein-tyrosine phosphatase alpha, sigma and LAR during development of the zebrafish embryo.

Receptor protein-tyrosine phosphatases (RPTPs) are key players in Drosophila development. To study the role of RPTPs in vertebrate development, we have cloned zebrafish (zf) RPTPs, including RPTP alpha (RPTPalpha), RPTP sigma (RPTPsigma) and LAR. These three RPTPs are broadly transcribed in early development. At 24h post fertilisation (hpf), all three genes are expressed in the nervous system in partially overlapping patterns. At 3 days post fertilisation zf-RPTPalpha and zf-LAR show similar expression patterns in the central nervous system (CNS), the pharyngeal arches, the pectoral fins and the spinal cord. Interestingly, zf-LAR is uniquely expressed in the neuromast cells, whereas zf-RPTPsigma expression is confined to the central nervous system.     

Dimerization of protein tyrosine phosphatase sigma governs both ligand binding and isoform specificity

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase sigma (PTPsigma) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTPsigma forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTPsigma ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTPsigma can alter the ligand binding specificities of PTPsigma ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.     

Transmembrane homodimerization of receptor-like protein tyrosine phosphatases

Receptor-like protein tyrosine phosphatases (RPTPs) are type I integral membrane proteins. Together with protein tyrosine kinases, RPTPs regulate the phosphotyrosine levels in the cell. Studies of two RPTPs, CD45 and PTPalpha, have provided strong evidence that dimerization leads to inactivation of the receptors, and that the dimerization of PTPalpha involves interactions in the transmembrane domain (TMD). Using the TOXCAT assay, a genetic approach for analyzing TM interactions in Escherichia coli membranes, we show that the TMD of RPTPs interact in the membrane, albeit to different extents. Using fusion proteins of TMDs, we also observe an equilibrium between monomer and dimer in sodium dodecyl sulfate (SDS) micelles. Through a mutational study of the DEP1 TMD, we demonstrate that these interactions are specific. Taken together, our results define a subset of the RPTP family in which TM homodimerization may act as a mediator of protein function.     

Identification of p130cas as an in vivo substrate of receptor protein-tyrosine phosphatase alpha

We have employed a substrate trapping strategy to identify physiological substrates of the receptor protein-tyrosine phosphatase alpha (RPTPalpha). Here we report that a substrate-trapping mutant of the RPTPalpha membrane proximal catalytic domain (D1), RPTPalpha-D1-C433S, specifically bound to tyrosine-phosphorylated proteins from pervanadate-treated cells. The membrane distal catalytic domain of RPTPalpha (D2) and mutants thereof did not bind to tyrosine-phosphorylated proteins. The pattern of tyrosine-phosphorylated proteins that bound to RPTPalpha-D1-C433S varied between cell lines, but a protein of approximately 130 kDa was pulled down from every cell line. This protein was identified as p130(cas). Tyrosine-phosphorylated p130(cas) from fibronectin-stimulated NIH3T3 cells bound to RPTPalpha-D1-C433S as well, suggesting that p130(cas) is a physiological substrate of RPTPalpha. RPTPalpha dephosphorylated p130(cas) in vitro, and RPTPalpha co-localized with a subpopulation of p130(cas) to the plasma membrane. Co-transfection experiments with activated SrcY529F, p130(cas), and RPTPalpha or inactive, mutant RPTPalpha indicated that RPTPalpha dephosphorylated p130(cas) in vivo. Tyrosine-phosphorylated epidermal growth factor receptor was not dephosphorylated by RPTPalpha under these conditions, suggesting that p130(cas) is a specific substrate of RPTPalpha in living cells. In conclusion, our results provide evidence that p130(cas) is a physiological substrate of RPTPalpha in vivo.     

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

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

Involvement of the membrane distal catalytic domain in pervanadate-induced tyrosine phosphorylation of receptor protein-tyrosine phosphatase alpha

Receptor protein-tyrosine phosphatase alpha, RPTPalpha, is a typical transmembrane protein-tyrosine phosphatase (PTP) with two cytoplasmic catalytic domains. RPTPalpha became strongly phosphorylated on tyrosine upon treatment of cells with the PTP inhibitor pervanadate. Surprisingly, mutation of the catalytic site Cys in the membrane distal PTP domain (D2), but not of the membrane proximal PTP domain (D1) that harbors the majority of the PTP activity, almost completely abolished pervanadate-induced tyrosine phosphorylation. Pervanadate-induced RPTPalpha tyrosine phosphorylation was not restricted to Tyr789, a known phosphorylation site. Cotransfection of wild-type RPTPalpha did not potentiate tyrosine phosphorylation of inactive RPTPalpha-C433SC723S, suggesting that RPTPalpha-mediated activation of kinase(s) does not underlie the observed effects. Mapping experiments indicated that pervanadate-induced tyrosine phosphorylation sites localized predominantly, but not exclusively, to the C-terminus. Our results demonstrate that RPTPalpha-D2 played a role in pervanadate-induced tyrosine phosphorylation of RPTPalpha, which may suggest that RPTPalpha-D2 is involved in protein-protein interactions.     

Redox-regulated rotational coupling of receptor protein-tyrosine phosphatase alpha dimers

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) constitutively forms dimers in the membrane, and activity studies with forced dimer mutants of RPTP alpha revealed that rotational coupling of the dimer defines its activity. The hemagglutinin (HA) tag of wild type RPTP alpha and of constitutively dimeric, active RPTP alpha-F135C with a disulfide bond in the extracellular domain was not accessible for antibody, whereas the HA tag of constitutively dimeric, inactive RPTP alpha-P137C was. All three proteins were expressed on the plasma membrane to a similar extent, and the accessibility of their extracellular domains did not differ as determined by biotinylation studies. Dimerization was required for masking the HA tag, and we identified a region in the N terminus of RPTP alpha that was essential for the effect. Oxidative stress has been shown to induce a conformational change of the membrane distal PTP domain (RPTP alpha-D2). Here we report that H(2)O(2) treatment of cells induced a change in rotational coupling in RPTP alpha dimers as detected using accessibility of an HA tag in the extracellular domain as a read-out. The catalytic site Cys(723) in RPTP alpha-D2, which was required for the conformational change of RPTP alpha-D2 upon H(2)O(2) treatment, was essential for the H(2)O(2)-induced increase in accessibility. These results show for the first time that a conformational change in the intracellular domain of RPTP alpha led to a change in conformation of the extracellular domains, indicating that RPTPs have the capacity for inside-out signaling.     

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.