Expression domains of receptor tyrosine phosphatases

Migrating embryonic growth cones require multiple, membrane-associated signaling molecules to monitor and respond to guidance cues. Evidence is presented that vertebrate cell adhesion molecule-like protein tyrosine phosphatases are likely to be components of this signaling system. CRYP alpha, the gene for an avian cell adhesion molecule-like phosphatase, is strongly expressed in the embryonic nervous system. The protein has been immunolocalized in the early chick embryo and its predominant localization in axons of the central and peripheral nervous systems is demonstrated. This location suggests that the major, early role of the enzyme is in axonal development. In a study of sensory neurites in culture, this phosphatase is shown to localize in migrating growth cones, within both the lamellipodia and filopodia. The dependence of growth cone migration on both cell adhesion and signaling through phosphotyrosine turnover, places the cell adhesion molecule-like CRYP alpha phosphatase in a position to be a regulator of these processes (Stoker, 1995a).

The avian CRYP alpha gene is expressed in the embryonic nervous system and encodes a receptor-like protein tyrosine phosphatase with structural similarity to neural cell adhesion molecules. To gain further insight into the role of the CRYP alpha phosphatase in neural development, this study addresses the protein's cellular distribution in the well characterised embryonic visual system. High levels of CRYP alpha protein localize in retinal axons extending from the eye to the tectum throughout the major growth periods of these nerve processes. In addition, primitive inner plexiform layer processes in the retina, tectobulbar axons, and non-retinal fibers of the tectal stratum opticum, contain large amounts of CRYP alpha. Its presence in non-fasciculated processes suggests that CRYP alpha has a role other than in fasciculation in short range fibres. In contrast to CRYP alpha, NgCAM is confined largely to axon fascicles in the retina and tectum, consistent with its demonstrated role in fasciculation of cultured neurites. In cultured retinal neurons CRYP alpha proteins reside both in neurite processes and in growth cone membranes, implicating both of these as potential functional locations for the protein. Although CRYP alpha continues to be expressed in the later embryo, the strong, early expression suggests a significant developmental role in the initial growth or guidance of nerve processes. This applies both over the longer range in the retinotectal and tectobulbar projections and over the shorter range within plexiform layers (Stoker, 1995b).

Axonal growth and guidance, like other aspects of neuronal differentiation, can be regulated by changes in tyrosine phosphorylation. Although much is known concerning the role of tyrosine kinases in these processes, relatively little is known about the nature and function of the protein tyrosine phosphatases (PTPs) that may be involved. To identify the PTPs expressed in the embryonic chicken CNS at the time of axon growth, a polymerase chain reaction based 'screen' was performed using degenerate primers directed against conserved regions of the PTP catalytic domain. Five distinct PTP-related cDNAs, two of which code for novel PTPs were obtained. One, designated CRYP-2, is selectively expressed in the CNS. Full-length cloning of CRYP-2 reveals that it is a receptor-type PTP with an adhesion molecule-like extracellular region comprising fibronectin (FN) type III repeats and a single catalytic domain in the intracellular region. It is alternatively spliced in the juxtamembrane region, similar to other PTPs recently cloned. CRYP-2 mRNA is strongly expressed in the brain during the time of axon growth; it is downregulated toward the end of embryogenesis. Western blot analysis has identified a 330-kDa glycoprotein as CRYP-2 and confirms that the protein is downregulated after hatching. Immunostaining of cerebellar neurons in vitro reveal that CRYP-2 is expressed on neuronal cell bodies and processes, but not on glia. The CAM-like structure, developmental pattern of expression, and neuron-specific localization of the CRYP-2 PTP suggest that it is involved in neuronal differentiation, particularly axon growth (Bodden, 1996).

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. This study focuses on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, it has been shown that five RPTP genes (CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha) have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. In contrast, CRYPalpha and CRYP-2 mRNAs are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes (CRYPalpha, CRYP-2, and PTPmu) are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection (Ledig, 1999).

Alternative splicing of receptor tyrosine phosphatases

Transgenic mice and Drosophila mutant studies demonstrate that the leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTPase) receptor is required for formation of neural networks. The hypothesis that alternative splicing of the LAR extracellular region contributes to this function was assessed by establishing temporospatial expression patterns of LAR isoforms containing an alternatively spliced extracellular nine amino acid segment (LAR alternatively spliced element-c; LASE-c). LASE-c is present in multiple alternatively spliced and truncated LAR transcripts. In contrast to LAR isoforms without LASE-c, levels of LAR transcripts and protein isoforms containing LASE-c are primarily present during development, suggesting a mechanism for developmental regulation of LAR function. In situ analysis demonstrates increasingly region- and cell-specific expression of LASE-c during maturation. Immunostaining reveals LASE-c-containing LAR protein along neurites and in growth cones. The discovery of highly regulated, temporospatial extracellular domain alternative splicing of LAR-type PTPase receptors points to a novel mechanism by which these receptors might influence network formation (Zhang, 1998).

Examination of null-mutant Drosophila and leukocyte common antigen-related (LAR)-deficient transgenic mice has demonstrated that the LAR protein tyrosine phosphatase (PTP) receptor promotes neurite outgrowth. In the absence of known ligands, the mechanisms of regulation of LAR-type PTP receptors are unknown. It is hypothesized that an alternatively spliced eleven amino acid proximal membrane segment of LAR (LAR alternatively spliced element-a; LASE-a) contributes to regulation of LAR function. Human, rat and mouse LAR cDNA sequences demonstrate that the predicted eleven amino acid inserts in rat and mouse are identical and share nine of eleven residues with the human insert. LASE-a splicing leads to the introduction of a Ser residue into LAR at a position analogous to Ser residues undergoing regulated phosphorylation in other PTPs. In-situ studies reveal increasingly region-specific expression of LASE-a containing LAR transcripts during postnatal development. RT-PCR analysis of cortical and hippocampal tissue confirms that the proportion of LAR transcripts containing LASE-a decreases during development. Immunostaining of cultured PC12 cells, cerebellar granule neurons, dorsal root ganglia and sciatic nerve sections with antibody directed against the LASE-a insert demonstrate signal in cell bodies but little if any along neurites. In contrast, staining with antibody directed to a separate domain of LAR shows accumulation of LAR along neurites. The findings that LASE-a splicing is conserved across human, rat and mouse, that the LASE-a insert introduces a Ser at a site likely to be targeted for regulated phosphorylation and that developmentally regulated splicing is coordinated with specific regional and intraneuronal localization point to important novel potential mechanisms regulating LAR-type tyrosine phosphatase receptor function in the nervous system (Honkaniemi, 1998).

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, thymic T cell activation responses were compared by elevating expression of two CD45 isoforms normally found on quiescent T cells. CD45RABC significantly increases CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhances Ca2+ mobilization and phosphotyrosine accumulation, and suppresses the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 does not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and requires a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified (Chui, 1994).

The CD45 family of transmembrane protein-tyrosine phosphatases plays a critical role in T cell activation signaling by regulating the tyrosine phosphorylation of protein-tyrosine kinases and their substrates. Multiple alternatively spliced CD45 isoforms, differing only in their extracellular domains, are differentially expressed by subsets of T cells with distinct functional repertoires. However, the physiological function of the various isoforms remains elusive. Using a novel panel of Jurkat T cell clones that uniquely express either the smallest [CD45(0)] or the largest [CD45(ABC)] isoform, CD45 isoform-specific differences have been demonstrated in interleukin-2 secretion and tyrosine phosphorylation of Vav. Differential activation-induced tyrosine phosphorylation of a 76-kDa Vav-associated protein (pp76) by cells expressing distinct CD45 isoforms has also been demonstrated. The tyrosine phosphorylation of Vav and associated pp76 follow parallel kinetics. pp76 interacts with the SH2 and SH3 domains of Vav. The pp76 protein has been identified as SLP-76, a recently cloned Grb2-binding protein. After activation with anti-CD3, CD45(ABC) transfectants demonstrate increased tyrosine phosphorylation and physical association of SLP-76 with Vav, when compared to transfectants expressing CD45(0). These results establish a novel physical link between Vav and SLP-76 that is differentially regulated by CD45 isoform expression (Onodera, 1996).

Structure of Lar extracellular domain

Elucidation of mechanisms by which receptor protein tyrosine phosphatases (PTPs) regulate neurite outgrowth will require characterization of ligand-receptor interactions and identification of ligand-induced signalling components mediating neurite outgrowth. The first identified ligand of the leucocyte common antigen-related (LAR) receptor PTP consists of a 99-residue ectodomain isoform, termed LARFN5C, which undergoes homophilic binding to LAR and promotes neurite outgrowth. Peptide mapping of LARFN5C was used to identify an active neurite-promoting domain of LAR. A peptide mimetic consisting of 37 residues (L59) and corresponding to the fifth LAR fibronectin type III (FNIII) domain prevents LARFN5C homophilic binding, demonstrates homophilic binding to itself and promotes neurite outgrowth of mouse E16-17 hippocampal neurons and of dorsal root ganglia explants. Response to L59 is partially lost when using neurons derived from LAR-deficient mice or neurons treated with LAR siRNA, consistent with homophilic interaction of L59 with LAR. L59 neurite-promoting activity is decreased in the presence of inhibitors of Src, Trk, PLCgamma, PKC, PI3K and MAPK. L59 activates Src (a known substrate of LAR), FAK and TrkB and also activates downstream signalling intermediates including PKC, ERK, AKT and CREB. BDNF augments the maximal neurite-promoting activity of L59, a finding consistent with the presence of shared and distinct signalling pathways activated by L59 with BDNF and L59 with TrkB. These studies are the first to identify an ectodomain of LAR (located within the fifth FNIII domain) capable of promoting neurite outgrowth and point to novel approaches for promotion of neurite outgrowth (Yang, 2005).

Different functional roles are played by the two PTP domains of receptor tyrosine phosphatases

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR reveals that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) confers a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution (Nam, 1999).

CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear. In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains. A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) is catalytically active and found to exist primarily as a dimer. In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) exists as a monomer and has no catalytic activity against any of the substrates tested. Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicates that rD1/D2 is 2-3-fold more catalytically active, is more thermostable, and exists primarily as a monomer. Limited trypsin digestion of rD1/D2 provides evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction. Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay. Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45. In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation (Felberg, 1998).

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTP delta, and PTP sigma, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. A yeast two-hybrid screen was performed with the first catalytic domain of PTP sigma (PTP sigma-D1) as bait to identify interacting regulatory proteins. Using this screen, the second catalytic domain of PTP delta (PTP delta-D2) was identified as an interactor of PTP sigma-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells reveal strong binding between PTP sigma-D1 and PTP delta-D2, an association which requires the presence of the wedge sequence in PTP sigma-D1, a sequence that mediates D1-D1 homodimerization in the phosphatase RPTP alpha. This interaction is not reciprocal, because PTP delta-D1 does not bind PTP sigma-D2. Addition of a glutathione S-transferase (GST)-PTP delta-D2 fusion protein (but not GST alone) to GST-PTP sigma-D1 leads to approximately 50% inhibition of the catalytic activity of PTP sigma-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTP sigma-D1 activity is obtained with coimmunoprecipitated PTP delta-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTP sigma (PTP sigma-D2), very similar in sequence to PTP delta-D2, bind poorly to PTP sigma-D1. PTP delta-D1 and LAR-D1 are also able to bind PTP delta-D2, but more weakly than PTP sigma-D1, with a binding hierarchy of PTP sigma-D1 >> PTP delta-D1 > LAR-D1. These results suggest that association between PTP sigma-D1 and PTP delta-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and probably other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains (Wallace, 1998).

Protein interactions of receptor tyrosine phosphatases

Leukocyte antigen-related protein (LAR) is a prototype for members of a family of transmembrane protein tyrosine phosphatases whose extracellular domains are is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4-8 has been observed. The extracellular matrix laminin-nidogen complex has been identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR-laminin-nidogen binding is regulated by the alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence results in disruption of the laminin-nidogen-binding activity. Long cellular processes are observed when HeLa cells are plated on laminin-nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining reveals high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin-nidogen. Antibody-induced cross-linking of LAR inhibits formation of these cellular processes, and inhibition is correlated with changes in cellular actin cytoskeletal structure. Thus, LAR-laminin-nidogen binding may play a role in regulating cell signaling induced by laminin-nidogen, resulting in cell morphological changes (O'Grady, 1998).

Human LAR associates with the cadherin-catenin complex. This association requires the amino-terminal domain of ß-catenin but does not require the armadillo repeats, which mediate association with cadherins. The association is not mediated by alpha-catenin or by cadherins. LAR-protein tyrosine phosphatases are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. It is proposed that changes in tyrosine phosphorylation of ß-catenin, mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth (Kypta, 1996).

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Epithelial cell migration is accompanied by tyrosine phosphorylation of beta-catenin and an increase of its free cytoplasmic pool. The protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with beta-catenin and plakoglobin. Interestingly, ectopic expression of LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of beta-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of beta-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis (Muller, 1999).

Rho-like GTP binding proteins (see Drosophila Rho1) play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of Rho bound GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), a cDNA was isolated encoding a 2861-amino acid protein termed Trio, containing three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, it is proposed that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration (Debant, 1996).

LAR is a transmembrane receptor-like protein tyrosine phosphatase (PTP). Genetic studies of Drosophila LAR suggest that LAR may function to regulate cell adhesions or adhesion-mediated signal transduction. The over-expression of LAR in mammalian tissue culture cells does not affect cell adhesion but induces caspase-dependent apoptosis. This study investigates molecular mechanisms of LAR-induced apoptosis by searching for in vivo substrates of LAR that are responsible for LAR-induced apoptosis. The over-expression of LAR in tissue culture cells specifically decreases the steady state protein level of p130Cas, a multifunctional signal assembly protein in signal transduction, by reducing the tyrosine phosphorylation and protein stability of p130Cas. The reduction of p130Cas protein level can be inhibited by tyrosine phosphatase inhibitors. Phosphatase domain-deleted mutant LARs have no effect on p130Cas. LAR also preferentially dephosphorylates p130Cas in vitro. Subcellularly, LAR and p130Cas are co-localized along stress fibers and at focal adhesions. LAR over-expression eliminates p130Cas from focal adhesions without affecting focal adhesion assembly. Restoring the level of p130Cas alleviates LAR-induced apoptosis. It is concluded that p130Cas is an in vivo substrate of LAR. LAR specifically dephosphorylates and destabilizes p130Cas and may play a role in regulating cell adhesion-mediated cell survival. The function of p130Cas in focal adhesions may not be to regulate focal adhesion assembly and cell adhesion but rather to transduce the cell adhesion-generated signals which are essential for cell survival (Weng, 1999).

LAR is a widely expressed receptor-like protein tyrosine phosphatase that is implicated in regulation of intracellular signaling triggered by both cell adhesion and peptide growth factors. Genetic studies have revealed that LAR regulates neuron axon path finding in Drosophila and mammary gland epithelial cell differentiation in mice. The molecular mechanism underlying the tissue specific function of LAR has not been clearly understood. The role and mechanism of LAR has been studied in peptide growth factors EGF and FGF signaling in human tissue culture cells in which the expression of LAR is under the control of an inducible promoter. Although both EGF and FGF induce activation of mitogen-activated protein kinase (MAPK), LAR only inhibits FGF-induced MAPK activation. LAR does not interact directly with the peptide growth factor receptors, since the ligand-induced autophosphorylation of growth factor receptors was not affected by induction of LAR. The specific effect of LAR on FGF-induced MAPK activation appears to be mediated by specific inhibition of the phosphorylation of two signal transducers that act downstream of the FGF receptor, FRS2 and a 180 kDa protein, and by prevention of their interaction with the adaptor protein GRB2. In contrast, LAR selectively inhibits the epidermal growth factor (EGF)-induced phosphorylation of p130CAS and the formation of the complex between p130CAS (see CAS/CSE1 segregation protein) and GRB2 but this effect does not influence the activation of MAPK by EGF. These data suggest that LAR and similar receptor-like protein tyrosine phosphatases may contribute to the regulation of transmembrane signaling by selectively inhibiting the tyrosine phosphorylation of specific signal transducers that act downstream of the plasma membrane-associated tyrosine kinases. The consequent inhibition of the formation of signaling complexes by these proteins may contrbute to the specificity of the signals generated by specific peptide growth factors as well as extracellular matrix proteins (Wang, 2000).

The leucocyte common antigen-related phosphatase (LAR) has been implicated in receptor tyrosine kinase signalling pathways while also displaying cell-density-dependency and localization to adherens junctions. Whereas physiological substrates for LAR have not been identified unequivocally, beta-catenin associates with LAR and is a substrate in vitro. With the implication that LAR may play a role in regulating E-cadherin-dependent cell-cell communication and contact inhibition, the relationship of LAR with E-cadherin was investigated. LAR expression increases with cell density in the human breast cancer cell line MCF-7 and in Ln 3 cells derived from the 13762NF rat mammary adenocarcinoma. LAR protein levels decrease rapidly when cells are replated at a low density after attaining high expression of LAR at high cell density. COS-7 cells display comparable density-dependent regulation of LAR expression when transiently expressing exogenous LAR under the control of a constitutively active promoter, indicating that the regulation of expression is not at the level of gene regulation. Disrupting homophilic E-cadherin complexes by chelating extracellular calcium causes a marked decrease in LAR protein levels. Similarly, blocking E-cadherin interactions with saturating amounts of E-cadherin antibody (HECD-1) also leads to a rapid and pronounced loss of cellular LAR. In contrast, mimicking cell-surface E-cadherin engagement by plating cells at low density on to dishes coated with HECD-1 results in a 2-fold increase in LAR expression compared with controls. These results suggest that density-dependent regulation of LAR expression is mediated by functional E-cadherin and may play a role in density-dependent contact inhibition by regulating tyrosine phosphorylation in E-cadherin complexes (Symons, 2002).

During axon guidance, the ventral guidance of the Caenorhabditis elegans anterior ventral microtubule axon is controlled by two cues, the UNC-6/netrin attractant recognized by the UNC-40/DCC receptor and the SLT-1/slit repellent recognized by the SAX-3/robo receptor. Loss-of-function mutations in clr-1 enhance netrin-dependent attraction, suppressing ventral guidance defects in slt-1 mutants. clr-1 encodes a transmembrane receptor protein tyrosine phosphatase (RPTP) that functions in AVM to inhibit signaling through the DCC family receptor UNC-40 and its effector, UNC-34/enabled. The known effects of other RPTPs in axon guidance could result from modulation of guidance receptors like UNC-40/DCC (Chang, 2004).

Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF) was investigated, focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at a sparse cell density, HGF stimulation induces prolonged c-Met tyrosine phosphorylation for over 5 h and a marked mitogenic response. In contrast, HGF stimulation induces transient c-Met tyrosine phosphorylation in less than 3 h and fails 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, prolongs c-Met tyrosine phosphorylation for over 5 h and permits the subsequent mitogenic response. The mitogenic response to HGF is associated with duration of c-Met tyrosine phosphorylation even in the sparse cells. The activity and expression of the protein tyrosine phosphatase, LAR increases following HGF-stimulation specifically in confluent hepatocytes and not in sparse hepatocytes. LAR and c-Met are associated, and purified LAR dephosphorylates tyrosine-phosphorylated c-Met in in vitro phosphatase reactions. Furthermore, antisense oligonucleotides specific for LAR mRNA suppress the expression of LAR, allow prolonged c-Met tyrosine phosphorylation, and lead 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 (Machide, 2006).

PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. This study describes an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS (see Drosophila Lar), a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK (see Drosophila Rolled). PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling (Davis, 2018).

LAR interaction with liprins

LAR family transmembrane protein-tyrosine phosphatases function in axon guidance and mammary gland development. In cultured cells, LAR binds to the intracellular, coiled coil LAR-interacting protein at discrete ends of focal adhesions, implicating these proteins in the regulation of cell-matrix interactions. Seven LAR-interacting protein-like genes are described in humans and Caenorhabditis elegans that form the liprin gene family (see Drosophila Liprin-alpha). Based on sequence similarities and binding characteristics, liprins may be subdivided into either alpha- or beta-type. The C-terminal, non-coiled coil regions of alpha-liprins bind to the membrane-distal phosphatase domains of LAR family members, as well as to the C-terminal, non-coiled coil region of beta-liprins. Both alpha- and beta-liprins homodimerize via their N-terminal, coiled coil regions. Liprins are thus multivalent proteins that potentially form complex structures. Some liprins have broad mRNA tissue distributions, whereas others are predominately expressed in the brain. Co-expression studies indicate that liprin-alpha2 alters LAR cellular localization and induces LAR clustering. It is proposed that liprins function to localize LAR family tyrosine phosphatases at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates (Serra-Pages, 1998).

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors. GRIP binds to the liprin-alpha/SYD2 family of proteins that interacts with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation (Wyszynski, 2002).

The LAR transmembrane tyrosine phosphatase associates with liprin-alpha proteins and colocalizes with liprin-alpha1 at focal adhesions. LAR has been implicated in axon guidance, and liprins are involved in synapse formation and synapse protein trafficking. Several liprin mutants have weaker binding to LAR as assessed by yeast interaction trap assays, and the extent of in vitro and in vivo phosphorylation of these mutants was reduced relative to that of wild-type liprin-alpha1. Treatment of liprin-alpha1 with calf intestinal phosphatase weakens its interaction with the recombinant GST-LAR protein. A liprin LH region mutant that inhibits liprin phosphorylation does not bind to LAR as assessed by coprecipitation studies. Endogenous LAR binds phosphorylated liprin-alpha1 from MDA-486 cells labeled in vivo with [32P]orthophosphate. In further characterizing the phosphorylation of liprin, immunoprecipitates of liprin-alpha1 expressed in COS-7 cells were found to incorporate phosphate after washes of up to 4 M NaCl. Additionally, purified liprin-alpha1 derived from Sf-9 insect cells retains the ability to incorporate phosphate in in vitro phosphorylation assays, and a liprin-alpha1 truncation mutant incorporates phosphate after denaturation and/or renaturation in SDS gels. Finally, binding assays show that liprin binds to ATP-agarose and that the interaction is challenged by free ATP, but not by free GTP. Moreover, liprin LH region mutations that inhibit liprin phosphorylation stabilize the association of liprin with ATP-agarose. Taken together, these results suggest that liprin autophosphorylation regulates its association with LAR (Serra-Pages, 2005).

Subcellular localization of receptor tyrosine phosphatases

Focal adhesions are sites of cell-extracellular matrix interactions that function in anchoring stress fibers to the plasma membrane and in adhesion-mediated signal transduction. Both focal adhesion structure and signaling ability involve protein tyrosine phosphorylation. LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion-like ectodomain and two intracellular protein tyrosine phosphatase domains. A novel cytoplasmic 160 kDa phosphoserine protein termed LAR-interacting protein 1 (LIP.1) has been identified, which binds to the LAR membrane-distal D2 protein tyrosine phosphatase domain and appears to localize LAR to focal adhesions. Both LAR and LIP.1 decorate the ends of focal adhesions most proximal to the cell nucleus and are excluded from the distal ends of focal adhesions, thus localizing to regions of focal adhesions presumably undergoing disassembly. It is proposed that LAR and LIP.1 may regulate the disassembly of focal adhesions and thus help orchestrate cell-matrix interactions (Serra-Pages, 1995).

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. The functional significance of processing was studied for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resemble that of the related PTPase LAR and is expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma undergo further proteolytic processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells requires overexpression of PKCalpha. Induced proteolysis results in shedding of the extracellular domains of both PTPases. This is in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies have identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, direct association of plakoglobin and beta-catenin is found with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases are internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which leads to cell contact disruption, also results in internalization but is not associated with prior proteolytic cleavage and shedding of the extracellular domain. It is concluded that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of both of their extracellular domains and one of which involves the presence of intact cell-cell contacts (Aicher, 1997).

Proteolysis of LAR

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. The epidermal growth factor receptor (EGFR) has been identified as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines have been shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage does not require PKC-activity. For both stimuli, however, processing of the P-subunit turns out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and is completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways (Ruhe, 2006).

Protein interactions of CD45, a receptor tyrosine phosphatase involved in the immune response

Continued: see Dlar: Evolutionary homologs part 2/2

Leukocyte-antigen-related-like/Dlar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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