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.     

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.     

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.     

Functional significance of the LAR receptor protein tyrosine phosphatase family in development and diseases.

The protein tyrosine phosphatases (PTPs) have emerged as critical players in diverse cellular functions. The focus of this review is the leukocyte common antigen-related (LAR) subfamily of receptor PTPs (RPTPs). This subfamily is composed of three vertebrate homologs, LAR, RPTP-sigma, and RPTP-delta, as well as few invertebrates orthologs such as Dlar. LAR-RPTPs have a predominant function in nervous system development that is conserved throughout evolution. Proteolytic cleavage of LAR-RPTP proproteins results in the noncovalent association of an extracellular domain resembling cell adhesion molecules and intracellular tandem PTPs domains, which is likely regulated via dimerization. Their receptor-like structures allow them to sense the extracellular environment and transduce signals intracellularly via their cytosolic PTP domains. Although many interacting partners of the LAR-RPTPs have been identified and suggest a role for the LAR-RPTPs in actin remodeling, very little is known about the mechanisms of action of RPTPs. LAR-RPTPs recently raised a lot of interest when they were shown to regulate neurite growth and nerve regeneration in transgenic animal models. In addition, LAR-RPTPs have also been implicated in metabolic regulation and cancer. This RPTP subfamily is likely to become important as drug targets in these various human pathologies, but further understanding of their complex signal transduction cascades will be required.     

Expression of alpha 1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo.

In this study, we have examined the spatiotemporal distribution of the alpha 1 integrin subunit, a putative laminin and collagen receptor, in avian embryos, using immunofluorescence microscopy and immunoblotting techniques. We used an antibody raised against a gizzard 175 x 10(3) M(r) membrane protein which was described previously and which we found to be immunologically identical to the chicken alpha 1 integrin subunit. In adult avian tissues, alpha 1 integrin exhibited a very restricted pattern of expression; it was detected only in smooth muscle and in capillary endothelial cells. In the developing embryo, alpha 1 integrin subunit expression was discovered in addition to smooth muscle and capillary endothelial cells, transiently, in both central and peripheral nervous systems and in striated muscles, in association with laminin and collagen IV. alpha 1 integrin was practically absent from most epithelial tissues, including the liver, pancreas and kidney tubules, and was weakly expressed by tissues that were not associated with laminin and collagen IV. In the nervous system, alpha 1 integrin subunit expression occurred predominantly at the time of early neuronal differentiation. During skeletal muscle development, alpha 1 integrin was expressed on myogenic precursors, during myoblast migration, and in differentiating myotubes. alpha 1 integrin disappeared from skeletal muscle cells as they became contractile. In visceral and vascular smooth muscles, alpha 1 integrin appeared specifically during early smooth muscle cell differentiation and, later, was permanently expressed after cell maturation. These results indicate that (i) the expression pattern of alpha 1 integrin is consistent with a function as a laminin/collagen IV receptor; (ii) during avian development, expression of the alpha 1 integrin subunit is spatially and temporally regulated; (iii) during myogenesis and neurogenesis, expression of alpha 1 integrin is transient and correlates with cell migration and differentiation.     

Expression of the receptor protein-tyrosine phosphatase, PTPmu, restores E-cadherin-dependent adhesion in human prostate carcinoma cells.

Normal prostate expresses the receptor protein-tyrosine phosphatase, PTPmu, whereas LNCaP prostate carcinoma cells do not. PTPmu has been shown previously to interact with the E-cadherin complex. LNCaP cells express normal levels of E-cadherin and catenins but do not mediate either PTPmu- or E-cadherin-dependent adhesion. Re-expression of PTPmu restored cell adhesion to PTPmu and to E-cadherin. A mutant form of PTPmu that is catalytically inactive was re-expressed, and it also restored adhesion to PTPmu and to E-cadherin. Expression of PTPmu-extra (which lacks most of the cytoplasmic domain) induced adhesion to PTPmu but not to E-cadherin, demonstrating a requirement for the presence of the intracellular domains of PTPmu to restore E-cadherin-mediated adhesion. We previously observed a direct interaction between the intracellular domain of PTPmu and RACK1, a receptor for activated protein kinase C (PKC). We demonstrate that RACK1 binds to both the catalytically active and inactive mutant form of PTPmu. In addition, we determined that RACK1 binds to the PKCdelta isoform in LNCaP cells. We tested whether PKC could be playing a role in the ability of PTPmu to restore E-cadherin-dependent adhesion. Activation of PKC reversed the adhesion of PTPmuWT-expressing cells to E-cadherin, whereas treatment of parental LNCaP cells with a PKCdelta-specific inhibitor induced adhesion to E-cadherin. Together, these studies suggest that PTPmu regulates the PKC pathway to restore E-cadherin-dependent adhesion via its interaction with RACK1.     

Regulation of receptor protein-tyrosine phosphatase dimerization

Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.     

Contact inhibition of hepatocyte growth regulated by functional association of the c-Met/hepatocyte growth factor receptor and LAR protein-tyrosine phosphatase

Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, we investigated the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF), focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at a sparse cell density, HGF stimulation induced prolonged c-Met tyrosine phosphorylation for over 5 h and a marked mitogenic response. In contrast, HGF stimulation induced transient c-Met tyrosine phosphorylation in <3 h and failed to induce mitogenic response in hepatocytes cultured at a confluent cell density. Treatment of the confluent cells with HGF plus orthovanadate, a broad spectrum protein-tyrosine phosphatase inhibitor, however, prolonged c-Met tyrosine phosphorylation for over 5 h and permitted the subsequent mitogenic response. The mitogenic response to HGF was associated with the duration of c-Met tyrosine phosphorylation even in the sparse cells. We found that the activity and expression of the protein-tyrosine phosphatase LAR increased following HGF stimulation specifically in confluent hepatocytes and not in sparse hepatocytes. LAR and c-Met were associated, and purified LAR dephosphorylated tyrosine-phosphorylated c-Met in in vitro phosphatase reactions. Furthermore, antisense oligonucleotides specific for LAR mRNA suppressed the expression of LAR, allowed prolonged c-Met tyrosine phosphorylation, and led to acquisition of a mitogenic response in hepatocytes even under the confluent condition. Thus functional association of LAR and c-Met underlies the inhibition of c-Met-mediated mitogenic signaling through the dephosphorylation of c-Met, which specifically occurs under the confluent condition.     

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.     

Splice variant 3, but not 2 of receptor protein-tyrosine phosphatase sigma can mediate stimulation of insulin-secretion by alpha-latrotoxin

Alpha-latrotoxin (alpha-LTX) binds to several cell surface receptors including receptor protein-tyrosine phosphatase sigma (RPTPsigma). Here we demonstrate that transient overexpression of the short splice variant 3 conferred alpha-LTX induced secretion to hamster insulinoma (HIT-15) cells. In contrast, the long splice variant 2 containing four additional extracellular fibronectin-III domains was inactive in secretion or in a single cell assay. Toxin-sensitive (MIN6) and toxin-insensitive (HIT-T15) insulinoma cell lines as well as PC12 cells expressed similar amounts of endogenous short RPTPsigma splice variant suggesting that this receptor does not play a role for toxin-sensitivity.     

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.     

Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha

cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase alpha (PTPalpha) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPalpha-D2 in a Ca(2+)-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPalpha, to the two tandem catalytic domains (D1D2) of PTPalpha, nor to the closely related D2 domain of PTPepsilon. Calmodulin bound to PTPalpha-D2 with high affinity, exhibiting a K(D) approximately 3 nm. The calmodulin-binding site was localized to amino acids 520-538 in the N-terminal region of D2. Site-directed mutagenesis showed that Lys-521 and Asn-534 were required for optimum calmodulin binding and that restoration of these amino acids to the counterpart PTPepsilon sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPalpha-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) approximately 340 nm), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPalpha-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPalpha and PTPalpha lacking only D1, but not full-length PTPepsilon, bound to calmodulin beads in the presence of Ca(2+). Also, PTPalpha was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPalpha in vivo but not with PTPalpha-D1D2 in vitro, highlighting a potential conformational difference between these forms of the tandem catalytic domains. The above findings suggest that calmodulin is a possible specific modulator of PTPalpha-D2 and, via D2, of PTPalpha.     

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.     

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.     

Expression of a truncated protein-tyrosine phosphatase mRNA in human lung.

Protein-tyrosine phosphatases (PTPases) are becoming an important family of enzymes that might regulate key events in cell growth and transformation. While isolating a new member of this family via amplification of human lung cDNA by the polymerase chain reaction, we found a clone identical to but truncated at the 3'-end of the coding region of human PTPase beta (HPTP beta) mRNA. This difference in sequence is situated in the most conserved part of the catalytic domain of the enzyme. The expression level of the truncated form of HPTP beta mRNA in human lung was lower than its normal form.     

Expression of the chaperonin 10 gene during zebrafish development

We have isolated a cDNA encoding chaperonin 10 (cpn10) from the zebrafish. Using northern, western, and in situ hybridization analysis, we observed that the cpn10 gene is expressed uniformly and ubiquitously throughout embryonic development of the zebrafish. Upregulation of cpn10 expression was observed following exposure of zebrafish embryos to a heat shock of 1 hour at 37 degrees C compared to control embryos raised at 27 degrees C. The extracellular form of Cpn10 called early pregnancy factor (EPF), found in the serum of pregnant mammals, was not detected in the serum of either male or female zebrafish. These expression studies suggest that Cpn10 plays a general role in zebrafish development as well as being consistent with the hypothesis that EPF is involved in the embryo implantation process in mammals.     

Expression analysis of the aquaporins during zebrafish embryonic development

The aquaporins are integral membrane proteins from a larger family of major intrinsic protein (MIP) that form pores in the membrane of cells. These proteins selectively transport water and other small uncharged solutes across cell plasma membranes. The organization of water within cells and tissues is fundamental to life, and the aquaporins play an important role in serving as the plumbing system for cells. As many as thirteen mammalian AQPs have been characterized, which have been shown to be vital for the regulation of water homeostasis in most tissues, such as renal water balance and brain-fluid homeostasis. However, complete expression patterns of most of the aquaporins in lower vertebrate at embryo stages has not been elucidated. Currently, we systematically described the temporal-spatial expression pattern of nine zebrafish aquaporins, using whole amount in situ hybridization. The results of whole mount in situ hybridization revealed that members of aquaporins family displayed diverse expression pattern, each of aquaporins has its unique distribution in different cell types and tissues, suggesting that they might play distinct roles in the embryonic development. Overall, current study will provide new insight into the expression of vertebrate quaporins and an important basis for the functional analysis of aquaporins in zebrafish development.     

Metalloproteinase- and gamma-secretase-mediated cleavage of protein-tyrosine phosphatase receptor type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/gamma-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system.     

Expression of five frizzleds during zebrafish craniofacial development

Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post-fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.