Interactive Fly, Drosophila

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

Two classes of protein tyrosine kinases (PTK) are utilized by the T cell antigen receptor (TcR)/CD3 complex for initiation of the signaling cascade, the Src-family PTK p56lck and p59fyn, and the Syk-family PTK p70zap and p72syk. In addition, the CD45 phosphotyrosine phosphatase (PTPase) is required for the induction of tyrosine phosphorylation by the TcR/CD3, presumably by positively regulating Src-family PTK. CD45 also regulates the Syk-family PTK p70zap (or ZAP-70). In CD45-negative T cells, p70zap is constitutively phosphorylated on tyrosine and co-immunoprecipitates with the TcR-zeta chain. In resting wild-type CD45-positive cells, p70zap is mainly unphosphorylated, but it is rapidly phosphorylated on tyrosine upon treatment of the cells with anti-CD3 or PTPase inhibitors. Finally, p70zap co-distributes with CD45 in intact T cells, and tyrosine phosphorylated p70zap is dephosphorylated by CD45 in vitro. These findings suggest that CD45 plays an important role, direct or indirect, in the regulation of p70zap and its function in TcR/CD3 signaling (Mustelin, 1995).

The CD45 protein tyrosine phosphatase (PTPase) has been shown to regulate the activity of Lck and Fyn protein tyrosine kinases in T cells. However, it is not clear that these constitute the only CD45 substrates. Moreover, the manner by which PTPase activity and substrate recruitment are regulated, is poorly understood. Previous in vitro studies have suggested that the first cytoplasmic PTPase domain (D1) of CD45 is the active PTPase, which may be regulated by an enzymatically inactive second PTPase domain (D2). However, the function of CD45 D2 in vivo is unknown. In this study, reconstitution of CD45(-) T cells with specific CD45 PTPase mutants allowed demonstration of a critical role for D2 in TCR-mediated interleukin (IL)-2 production. Specifically, replacement of CD45 D2 with that of the LAR PTPase to form a CD45/LAR:D2 chimera, abrogates CD45-dependent IL-2 production. This effect cannot be accounted for by loss of PTPase activity per se. The expression of D1 substrate-trapping mutants reveals an in vivo interaction between CD45 and TCR-zeta that is dependent on CD45 D2. Thus, cells expressing CD45 lacking D2 exhibit abnormal TCR-mediated signaling characterized by hyperphosphorylation of zeta and deficient ZAP-70 phosphorylation. These data suggest an essential role for CD45 D2 in TCR-regulated IL-2 production through substrate recruitment of the zeta chain (Kashio, 1998).

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. The phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules (Desai, 1994).

To further understand the functional interactions between CD45 and p56(lck) in T-cells, their expression was stably reconstituted in a nonlymphoid system. The results of this analyses demonstrates that CD45 can dephosphorylate tyrosine 505 of p56(lck) in NIH 3T3 fibroblasts. As is the case for T-cells, removal of the unique domain of p56(lck) interfers with dephosphorylation of tyrosine 505 in fibroblasts, further stressing the importance of this region in the interactions between CD45 and p56(lck). The ability of CD45 to dephosphorylate tyrosine 505 in NIH 3T3 cells is also greatly influenced by the catalytic activity of p56(lck). Indeed, whereas CD45 provokes dephosphorylation of kinase-defective Lck molecules in this system, it fails to stably dephosphorylate kinase-active p56(lck) polypeptides. These studies show that CD45 is also able to inhibit the oncogenic potential of a constitutively activated version of p56(lck) in NIH 3T3 cells. This effect does not require the Lck unique domain and apparently results from selective dephosphorylation of substrates of activated p56(lck) in fibroblasts. In addition to providing insights into the nature and regulation of the interactions between CD45 and p56(lck) in T-cells, these results indicated that CD45 clearly has the capacity to both positively and negatively regulate p56(lck)-mediated functions in vivo (Gervais, 1997).

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine that is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. CD45 is phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue is not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 is phosphorylated in intact T cells, and phosphorylation increases upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells causes tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 binds p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 causes a severalfold increase in its PTPase activity. These results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation can be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes (Autero, 1994).

CD100 is a 150-kDa human surface glycoprotein implicated in T cell activation. CD100 is associated with CD45 and this association has functional significance. The association was demonstrated using coimmunoprecipitation and detection of CD45 enzymatic PTPase activity. Furthermore, the association is increased during T cell activation: triggering CD45 molecules through discrete epitopes induces the down-modulation of CD100 molecules at the cell surface. This modulation can be attributed to the shedding of a soluble form of CD100 in the culture supernatant. One of the functional consequences of this T cell activation-induced CD100-CD45 association is revealed by the finding that CD100 mAbs have an effect on CD45-induced T cell aggregation (Herold, 1996).

CD45-phosphotyrosine phosphatase (PTPase) constitutes the major portion of the PTPase activity within plasma membranes of neutrophilic leukocytes, where it regulates signals leading to functional activation. The catalytic component of neutrophil plasma membrane CD45-PTPASE is regulated by a cytosolic inactivator which itself is attenuated upon cellular stimulation, allowing enzyme translocated from granule stores to express full activity. The present study investigated mechanisms of cytosolic inactivator attenuation. Preincubation of plasma membranes of stimulated neutrophils with cytosol from resting cells results in a rapid loss of membrane-associated PTPase activity. Phosphatidic acid has no direct effect on plasma membrane PTPase activity but blunts in a dose dependent manner the effects of the PTPase inactivator. Inactivator attenuation is not observed with equivalent concentrations of either diacylglycerol or lysophosphatidic acid. Optimal attenuation of inactivator activity is obtained with long chain, soluble ligands, such as dicapryl phosphatidic acid. Inhibitors of neutrophil plasma membrane ecto-phosphatidic acid phosphohydrolase do not block inactivator attenuation, suggesting that phosphatidic acid and not one of its metabolites is the entity responsible. In conclusion, neutrophil plasma membrane PTPase is dynamically regulated by a cytosolic inactivator, the inhibition of which may potentiate the effects of PTPase translocated during cellular stimulation. Phosphatidic acid generated as a consequence of cellular stimulation may mediate this inhibition and thereby regulate the effects of tyrosine kinases activated during the initial phases of cell stimulation (Cui, 1997).

Phenotypic effects of receptor tyrosine phosphatase mutation

Receptor-like protein-tyrosine phosphatases (RPTPs) form a diverse family of cell surface molecules whose functions remain poorly understood. The LAR subfamily of RPTPs has been implicated in axon guidance and neural development. This study reports the molecular and genetic analysis of the C. elegans LAR subfamily member PTP-3. PTP-3 isoforms are expressed in many tissues in early embryogenesis, and later become localized to neuronal processes and to epithelial adherens junctions. Loss of function in ptp-3 causes low-penetrance defects in gastrulation and epidermal development similar to those of VAB-1 Eph receptor tyrosine kinase mutants. Loss of function in ptp-3 synergistically enhances phenotypes of mutations in the C. elegans Eph receptor VAB-1 and a subset of its ephrin ligands, but does not show specific interactions with several other RTKs or morphogenetic mutants. The genetic interaction of vab-1 and ptp-3 suggests that LAR-like RPTPs and Eph receptors have related and partly redundant functions in C. elegans morphogenesis (Harrington, 2002).

The LAR-like RPTPs have been highly conserved in animal evolution. LAR-like RPTPs have been identified in vertebrates, Drosophila (DLar), leeches (HmLAR1, HmLAR2), protochordates, and now in nematodes. In all vertebrate species examined, three LAR subfamily RPTPs are expressed: LAR itself, and the closely related proteins PTPsigma and PTPdelta. The Drosophila and C. elegans genomes each contain a single LAR-like gene, whereas the leech Hirudo medicinalis expresses two LAR-family genes: HmLAR1 and HmLAR2. Ancestral metazoans may thus have expressed a single LAR-like gene that became duplicated in the annelid and vertebrate lineages (Harrington, 2002).

Vertebrate LAR subfamily genes are expressed in distinct but partly overlapping patterns, both in the developing and adult nervous systems and in a variety of non-neuronal tissues. Like its vertebrate orthologs, PTP-3 displays widespread, almost ubiquitous expression in early C. elegans embryos. PTP-3 later becomes localized to neuronal processes, as found for other vertebrate and invertebrate LAR family members. Outside the nervous system LAR-like proteins are often found in proliferating epithelia, such as those of the lung and gut. In some mature C. elegans epidermal cells PTP3 appears to localize to adherens junctions; vertebrate LAR and PTPsigma proteins are also found in adherens junctions, where they interact with ß-catenin. The potential role of PTP-3 in epidermal adherens junctions is unclear, since ptp-3 mutant phenotypes do not resemble those resulting from loss of function in the catenin/cadherin complex; furthermore, loss of function in ptp-3 does not enhance or suppress the phenotypes of loss-of-function mutations in other adherens junction proteins such as HMP-1 (Harrington, 2002).

The C. elegans ptp-3 gene encodes at least two isoforms, PTP-3A and PTP-3B, by use of alternative promoters; this genomic organization has so far not been found in other LAR genes. Anti-PTP-3 antibodies, which should recognize both isoforms, show staining weaker than that of an isoform-specific PTP-3B::GFP transgene but otherwise indistinguishable, implying that both isoforms are expressed in similar patterns. The ptp-3(op147) insertion allele affects a phosphatase domain common to both isoforms and should decrease the function of both isoforms, although it may not affect phosphatase-independent functions of PTP-3, as proposed for Drosophila Lar. Transgenes encoding only PTP-3B can rescue most or all of the defects observed in ptp-3 mutants, suggesting that PTP-3B function is necessary for morphogenesis. Consistent with this hypothesis, a deletion mutation that specifically disrupts the PTP-3A isoform does not cause defects in embryonic morphogenesis and does not synergize with vab-1 mutations. PTP-3A might have no function in embryonic morphogenesis, or its functions might be redundant with PTP-3B, such that only mutations disrupting both isoforms cause morphogenetic defects (Harrington, 2002).

In C. elegans LAR plays a subtle role in early neural and epidermal development, as reflected by the mild defects of ptp-3 mutants. The mild phenotypes of LAR mutants in Drosophila, mice and C. elegans suggest the possibility that these proteins function in highly redundant signaling processes (Harrington, 2002).

Analysis of ptp-3 mutant phenotypes has revealed that PTP-3 and Eph signaling are required in similar processes of embryogenesis. Loss of function in PTP-3, in the Eph receptor VAB-1, or in the ephrin ligand EFN-1, causes incompletely penetrant defects in neuroblast movements during closure of the gastrulation cleft, and in later epidermal morphogenesis. In vab-1 ptp-3 double mutants the penetrance and severity of these defects are dramatically enhanced, although no new defects are seen in the double mutants. The simplest interpretation of this synergistic genetic interaction is that PTP-3 and VAB-1 function in closely related pathways, and that these pathways have partly redundant functions in controlling neuroblast movements. In contrast to the extremely variable defects of vab-1(0) or ptp-3 single mutants, vab-1(0) ptp-3 double mutants display a consistent arrest at early epidermal enclosure. This suggests that the variability of the single mutant phenotypes reflects compensation by the other pathway; that is, the variability of the Vab-1 null phenotype reflects the ability of PTP-3 signaling to partly compensate for lack of VAB-1, and vice versa. The synergistic interaction of ptp-3 with efn-1 mutations is consistent with data showing that EFN-1 functions in the VAB-1 pathway in embryonic morphogenesis. ptp-3 does not show similar synergistic lethal interactions with ephrins efn-2 and efn-3, consistent with their relatively minor roles in embryogenesis (Harrington, 2002).

Because vab-1 and ptp-3 mutants display defects in the movements of ventral neuroblasts during gastrulation cleft closure, the hypothesis that VAB-1 and PTP-3 function redundantly within the same sets of neuronal precursors is preferred. This hypothesis is supported by tissue-specific expression data. Significant rescue of vab-1 ptp-3 mutant phenotypes was only observed when PTP-3B was expressed using a pan-neural promoter and not when using epithelial or epidermal-specific promoters. While these experiments do not address whether VAB-1 and PTP-3 function in the same individual neurons, they are consistent with VAB-1 and PTP-3 functioning in the same tissue (Harrington, 2002).

Several PTPs, including LMW-PTP, FAP-1, and SHP-2, appear to function downstream of Eph receptors. Although PTP-3 is unlikely to function directly in the VAB-1/Eph receptor signaling pathway, this is the first indication of a specific genetic interaction between a receptor-like PTP and Eph pathways. The proposal that a receptor PTP and receptor PTK function redundantly in promoting signaling through a common pathway raises the question of the mechanism by which this may be achieved by two apparently antagonistic enzymes. While there are abundant examples of PTPs antagonizing PTK-dependent signaling pathways, there are also many examples of PTPs that function positively to promote signaling. For example, CD45, the prototypic receptor PTP, plays an essential positive role in signaling through T and B cell receptors and PTP-alpha promotes signaling events associated with cell growth. In both cases, the PTPs appear to dephosphorylate an inhibitory site of tyrosine phosphorylation at the C terminus of Src family PTKs, activating the kinase. Thus, a PTP can function in concert with a PTK to promote tyrosine phosphorylation. The SH2-domain-containing phosphatase SHP-2, and its Drosophila homolog Csw, function positively in several RTK pathways. In the case of the RTK Torso, Csw can promote signaling by dephosphorylation of inhibitory phosphotyrosines in the Torso cytoplasmic domain. It is thought unlikely that PTP-3 acts via dephosphorylation of VAB-1, since PTP-3 mutations have dramatic effects in a VAB-1 null mutant background. PTP-3 might promote signaling in the VAB-1 pathway by dephosphorylation of a downstream substrate; elucidation of the substrates of PTP-3 will be required to test this possibility (Harrington, 2002).

LAR-like RPTPs and Eph RTKs have been implicated in related aspects of cellular behavior in other organisms. Both Eph RTKs and LAR can cause axonal growth cone collapse, and thus can promote repulsive interactions between growth cones and their substrates. However, in some situations LAR-like RPTPs may promote cell adhesion or growth-cone attraction, rather than repulsion, suggesting that Eph signaling and LAR signaling could play antagonistic or synergistic roles depending on the specific cellular context. The finding that Eph signaling and LAR play related and partly redundant roles in C. elegans morphogenesis suggests that these two pathways may also be intimately connected in other organisms (Harrington, 2002).

Leukocyte-common antigen related (LAR)-like phosphatase receptors are conserved cell adhesion molecules that function in multiple developmental processes. The Caenorhabditis elegans ptp-3 gene encodes two LAR family isoforms that differ in the extracellular domain. The long isoform, PTP-3A, localizes specifically at synapses and the short isoform, PTP-3B, is extrasynaptic. Mutations in ptp-3 cause defects in axon guidance that can be rescued by PTP-3B but not by PTP-3A. Mutations that specifically affect ptp-3A do not affect axon guidance but instead cause alterations in synapse morphology. Genetic double-mutant analysis is consistent with ptp-3A acting with the extracellular matrix component nidogen, nid-1, and the intracellular adaptor alpha-liprin (See Drosophila Liprin-alpha), syd-2. nid-1 and syd-2 are required for the recruitment and stability of PTP-3A at synapses, and mutations in ptp-3 or nid-1 result in aberrant localization of SYD-2. Overexpression of PTP-3A is able to bypass the requirement for nid-1 for the localization of SYD-2 and RIM. It is proposed that PTP-3A acts as a molecular link between the extracellular matrix and alpha-liprin during synaptogenesis (Ackley, 2005).

The protein tyrosine phosphatase family comprises transmembrane receptor-like and cytosolic forms. Although the exact biological functions of these enzymes are largely unknown, they are believed to counter-balance the effects of protein tyrosine kinases. Expression of a mammalian transmembrane protein tyrosine phosphatase called LAR (leukocyte common antigen related gene) is often associated with proliferating epithelial cells or epithelial progenitor cells. This study investigates the potential role of LAR in the regulation of cell growth and death in mammals. In cultured mammalian cells, either the full-length wild-type LAR or a truncation mutant containing only the extracellular domain of the molecule are overexpressed. Although the truncated LAR can be readily overexpressed in various cell lines, cells overexpressing the wild-type LAR are negatively selected. Using an inducible expression system, it has been demonstrated that overexpression of the wild-type LAR, but not the truncated LAR, activates the caspase pathway directly and induces p53-independent apoptosis. This suggests that LAR might regulate cellular signals essential for cell survival. Overproduction of LAR may tilt the balance between the tyrosine phosphorylation and dephosphorylation of proteins whose activities are critical for cell survival, and therefore lead to cell death. In addition, the observation that overexpression of LAR induces cell death without affecting cell adhesion suggests that LAR may activate the caspase pathway and induce cell death directly. This work is the first example of the involvement of a receptor-like protein tyrosine phosphatase in cell-death control and provides the basis for searching for molecules and mechanisms linking signal transduction by protein tyrosine phosphorylation to the caspase-mediated cell-death pathway (Weng, 1998).

The LAR receptor-like protein tyrosine phosphatase is composed of two intracellular tyrosine phosphatase domains and a cell adhesion molecule-like extracellular region containing three immunoglobulin-like domains in combination with eight fibronectin type-III-like repeats. This architecture suggests that LAR may function in cellular signaling by the regulation of tyrosine phosphorylation through cell-cell or cell-matrix interactions. Gene targeting was used in mouse embryonic stem cells to generate mice lacking sequences encoding both LAR phosphatase domains. Northern blot analysis of various tissues reveals the presence of a truncated LAR mRNA lacking the cytoplasmic tyrosine phosphatase domains and indicates that this LAR mutation is not accompanied by obvious changes in the expression levels of either one of the LAR-like receptor tyrosine phosphatases (PTPdelta or PTPsigma). LAR-/- mice develop and grow normally and display no appreciable histological tissue abnormalities. However, upon breeding, an abnormal neonatal death rate was observed for pups from LAR-/- females. Mammary glands of LAR-/- females are incapable of delivering milk due to an impaired terminal differentiation of alveoli at late pregnancy. As a result, the glands fail to switch to a lactational state and show a rapid involution postpartum. In wild-type mice, LAR expression is regulated during pregnancy reaching maximum levels around Day 16 of gestation. Taken together, these findings suggest an important role for LAR-mediated signaling in mammary gland development and function (Schaapveld, 1997).

Protein tyrosine phosphatase sigma (PTP-sigma, encoded by the Ptprs gene) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases that is highly expressed during mammalian embryonic development in the germinal cell layer lining the lateral ventricles of the developing brain, dorsal root ganglia, Rathke's pouch, olfactory epithelium, retina and developing lung and heart. On the basis of its expression and homology with the Drosophila melanogaster orthologs DPTP99 and DPTP100A, which have roles in the targeting of axonal growth cones, it is hypothesized that PTP-sigma may also have a modulating function in cell-cell interactions, as well as in axon guidance during mammalian embryogenesis. To investigate its function in vivo, Ptprs-deficient mice were generated. The resulting Ptprs-/-animals display retarded growth, increased neonatal mortality, hyposmia and hypofecundity. Anatomical and histological analyses have shown a decrease in overall brain size with a severe depletion of luteinizing hormone-releasing hormone-immunoreactive cells in Ptprs-/- hypothalamus. Ptprs-/- mice have an enlarged intermediate pituitary lobe, but smaller anterior and posterior lobes. These results suggest that tyrosine phosphorylation-dependent signaling pathways regulated by PTP-sigma influence the proliferation and/or adhesiveness of various cell types in the developing hypothalamo-pituitary axis (Elchebly, 1999).

The leukocyte common antigen-related (LAR) receptor is known to be present in rodent dorsal root ganglion (DRG) neurons; the well established model of postcrush sciatic nerve regeneration was used to test the hypothesis that LAR is required for neurite outgrowth in the adult mammalian nervous system. In uninjured sciatic nerves, no differences in nerve morphology and sensory function were detected between wild-type and LAR-deficient littermate transgenic mice. Sciatic nerve crush results in increased LAR protein expression in DRG neurons. In addition, nerve injury leads to an increase in the proportion of LAR protein isoforms known to have increased binding affinity to neurite-promoting laminin-nidogen complexes. Two weeks after nerve crush, morphological analysis of distal nerve segments in LAR-deficient transgenic mice demonstrates significantly decreased densities of myelinated fibers, decreased axonal areas, and increased myelin/axon area ratios compared with littermate controls. Electron microscopy analysis reveals a significant twofold reduction in the density of regenerating unmyelinated fibers in LAR-/- nerves distal to the crush site. Sensory testing at the 2 week time point reveals a corresponding 3 mm lag in the proximal-to-distal progression of functioning sensory fibers along the distal nerve segment. These studies introduce PTP receptors as a major new gene family regulating regenerative neurite outgrowth in vivo in the adult mammalian system (Xie, 2001).

Receptor-type protein tyrosine phosphatases (RPTPs) are required for appropriate growth of axons during nervous system development in Drosophila. In the vertebrate, type IIa RPTPs [protein tyrosine phosphatase (PTP)-dekta, PTP-sigma, and LAR (leukocyte common-antigen-related)] and the type III RPTP, PTP receptor type O (PTPRO), have been implicated in the regulation of axon growth, but their roles in developmental axon guidance are unclear. PTPRO, PTP-delta, and PTP-sugma are each expressed in chick motor neurons during the period of axonogenesis. To examine potential roles of RPTPs in axon growth and guidance in vivo, double-stranded RNA (dsRNA) interference combined with in ovo electroporation were used to knock down RPTP expression levels in the embryonic chick lumbar spinal cord. Although most branches of the developing limb nerves appeared grossly normal, a dorsal nerve identified as the anterior iliotibialis was clearly affected by dsRNA knock-down of RPTPs. In experimental embryos treated with dsRNA targeting PTP-delta, PTP-sigma, or PTPRO, this nerve showed abnormal fasciculation, was reduced in size, or was missing entirely; interference with PTPRO produced the most severe phenotypes. Control embryos electroporated with vehicle, or with dsRNA targeting choline acetyltransferase or axonin-1, did not exhibit this phenotype. Surprisingly, embryos electroporated with dsRNA targeting PTP-delta together with PTPRO, or all three RPTPs combined, had less severe phenotypes than embryos treated with PTPRO alone. This result suggests that competition between type IIa and type III RPTPs can regulate motor axon outgrowth, consistent with findings in Drosophila. These results indicate that RPTPs, and especially PTPRO, are required for axon growth and guidance in the developing vertebrate limb (Stepanek, 2005).

Growth factors stimulating neurogenesis act through protein tyrosine kinases that are counterbalanced by protein tyrosine phosphatases (PTPs); thus, downregulation of progenitor PTP function might provide a novel strategy for promoting neurogenesis. The hypotheses were tested that the leukocyte common antigen-related (LAR) PTP is present in adult dentate gyrus progenitors, and that its downregulation would promote neurogenesis. In adult mice, LAR immunostaining is present in Ki-67- and PCNA-positive subgranular zone cells. At 1 h post-BrdU administration, LAR-/- mice demonstrate an approximately 3-fold increase in BrdU- and PCNA-positive cells, indicating increased progenitor proliferation. At 1 day and 4 weeks following 6 days of BrdU administration, LAR-/- mice exhibit a significant increase in BrdU and NeuN colabeled cells consistent with increased neurogenesis. In association with increased neurogenesis in LAR-/- mice, stereological analysis revealed a significant 37% increase in the number of neurons present in the granule cell layer. In cultured progenitor clones derived from LAR+/+ mice, LAR immunostaining was present in PCNA- and BrdU-positive cells. Progenitor clones derived from adult LAR-/- hippocampus or LAR+/+ clones, made LAR-deficient with LAR siRNA, demonstrate increased proliferation and, under differentiation conditions, increased proportions of Tuj1- and MAP2-positive cells. These studies introduce LAR as the first PTP found to be expressed in dentate progenitors and point to inhibition of LAR as a potential strategy for promoting neurogenesis. These findings also provide a rare in vivo demonstration of an association between increased dentate neurogenesis and an expanded population of granule cell layer neurons (Bernabeu, 2006).

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. It is proposed that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP (Dunah, 2005).

Protein tyrosine phosphatase delta (PTPdelta) is a receptor-type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. PTPdelta-deficiency generated by gene targeting in mice is semi-lethal due to insufficient food intake. Mutant mice also exhibited learning impairment in the Morris water maze, reinforced T-maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long-term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPdelta-deficient mice, with augmented paired-pulse facilitation in the CA1 region. Thus, PTPdelta plays important roles in regulating hippocampal LTP and learning processes, and hippocampal LTP does not necessarily positively correlate with spatial learning ability. This is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory (Uetani, 2000).

The leukocyte common antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs), LAR, RPTP-sigma, and RPTP-Δ, regulate neuroendocrine development, axonal regeneration, and hippocampal long-term potentiation in mammals. In Drosophila, RPTPs are required for appropriate axon targeting during embryonic development. In contrast, deletion of any one of the three LAR-RPTP family members in mammals does not result in gross axon targeting defects. Both RPTP-sigma and RPTP-Δ are highly expressed in the developing mammalian nervous system, suggesting they might be functionally redundant. To test this hypothesis, RPTP-sigma and RPTP-Δ (RPTP-sigma/Δ) double-mutant mice were generated. Although embryonic day 18.5 RPTP-sigma and RPTP-Δ single-mutant embryos are viable, RPTP-sigma/Δ double mutants are paralyzed, are never observed to draw a breath, and die shortly after cesarean section. RPTP-sigma/Δ double mutants exhibit severe muscle dysgenesis and severe loss of motoneurons in the spinal cord. Detailed analysis of the projections of phrenic nerves in RPTP-sigma/Δ double mutants indicated that these motoneuron axons emerge normally from the cervical spinal cord, but stall on reaching the diaphragm. The results demonstrate that RPTP-sigma and RPTP-Δ complement each other functionally during mammalian development, and reveal an essential contribution of RPTP-sigma and RPTP-Δ to appropriate motoneuron axon targeting during mammalian axonogenesis (Uetani, 2006: full text of article).

Receptor tyrosine phosphatases and neurite outgrowth

In Drosophila, several receptor tyrosine phosphatases (rPTPs), including DLAR, have been shown to participate in directing neurite outgrowth. As yet, however, it is not known how rPTPs act to affect navigation. To gain insight into the mechanisms of rPTP-mediated outgrowth guidance, the role of HmLAR2, a Hirudo medicinalis homolog of DLAR, has been investigated in process outgrowth. HmLAR2 is expressed by, among other cells, a transient neuron-like template cell, the Comb cell. HmLAR2 protein becomes concentrated within the growth cones at a stage when C cell processes undergo rapid outgrowth. When antibodies raised against the extracellular domain of HmLAR2 are injected into intact embryos, they bind specifically to the C cell surface at growth cones and along processes and cause the partial internalization of HmLAR2 receptors. Moreover, the C cell processes were found to project aberrantly, to deviate from their normally highly regular trajectories and to extend shorter distances in the presence of the antibodies. It is proposed that HmLAR2 is required by the C cell for guidance and extension and suggest that it functions via its ectodomain to transduce extracellular guidance cues (Gershon, 1998a).

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. Two Hirudo medicinalis (leech) rPTPs have been cloned and sequenced that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. HmLAR2 is expressed on the cell bodies, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, it is hypothesized that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. It is hypothesized that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. It is also proposed that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2 (Gershon, 1998b).

The receptor-like protein tyrosine phosphatase (RPTP) PTPsigma controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPsigma have been thought to be cell-autonomous, the possibility that RPTPs related to PTPsigma also have non-cell-autonomous signaling functions during axon development has also been supported genetically. This study reports that a cell culture substrate made from purified PTPsigma ectodomains supports retinal neurite outgrowth in cell culture. A receptor for PTPsigma must exist on retinal axons and binding of PTPsigma to this receptor does not require the known, heparin binding properties of PTPsigma. The neurite-promoting potential of PTPsigma ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPsigma. Furthermore, heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPsigma substrate, by competing for binding to this same domain. This is the first direct evidence of a non-cell-autonomous, neurite-promoting function of PTPsigma and of a potential role for heparin-related oligosaccharides in modulating neurite promotion by an RPTP (Sajnani, 2005).

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

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