Transmembrane Protein Tyrosine Phosphatase Research Articles

Differential Regulation of CD45 Expression on Granulocytes, Lymphocytes, and Monocytes in COVID-19.

CD45 is a transmembrane glycoprotein and protein tyrosine phosphatase expressed on the surface of all nucleated hematopoietic cells. While there is increasing evidence demonstrating the involvement of CD45 in immune system regulation, no information on CD45 expression in inflammation and sepsis is currently available. Therefore, we determined the CD45 surface expression on granulocytes, lymphocytes, and monocytes in patients with COVID-19 and healthy volunteers in both absence and presence of lipopolysaccharide (LPS). Following approval by the local ethics committee, whole blood samples were obtained from patients with COVID-19 infection on day 1 of hospital admission and healthy volunteers. Samples were incubated in absence and presence of LPS and CD45 was measured in granulocytes, lymphocytes, and monocytes using flow cytometry. In comparison with healthy individuals, COVID-19 patients showed an increased CD45 expression on the surface of granulocytes (+35%, p < 0.02) and lymphocytes (+39%, p < 0.0001), but a reduced CD45 expression on monocytes (−35%, p < 0.0001). LPS incubation of whole blood from healthy individuals increased the CD45 expression on granulocytes (+430%, p < 0.0001), lymphocytes (+32%, p = 0.0012), and monocytes (+36%, p = 0.0005), respectively. LPS incubation of whole blood samples from COVID-19 patients increased the CD45 expression on granulocytes and monocytes, and decreased the CD45 expression on lymphocytes. In conclusion, CD45 expression on leucocytes is altered: (1) in COVID-19 patients, and (2) in in vitro endotoxemia in a complex cell-specific way, thus representing a new immunoregulatory mechanism.

The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

BackgroundCD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied.AimWe aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling.MethodsCD148 forms, wild‐type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin‐1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme‐linked immunosorbent assay, and Western blotting.ResultsBoth CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF‐induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)‐expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells.ConclusionOur data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1‐CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.

Agonistic anti-CD148 monoclonal antibody attenuates diabetic nephropathy in mice.

CD148 is a transmembrane protein tyrosine phosphatase (PTP) that is expressed in the renal vasculature, including the glomerulus. Previous studies have shown that CD148 plays a role in the negative regulation of growth factor signals (including epidermal growth factor and vascular endothelial growth factor), suppressing cell proliferation and transformation. However, the role of CD148 in kidney disease remains unknown. Here, we generated an agonistic anti-CD148 antibody and evaluated its effects in murine diabetic nephropathy (DN). Monoclonal antibodies (mAbs) against the mouse CD148 ectodomain sequence were generated by immunizing CD148 knockout (CD148KO) mice. The mAbs that increased CD148 activity were selected by biological (proliferation) and biochemical (PTP activity) assays. The mAb (18E1) that showed strong agonistic activity was injected (10 mg/kg ip) in streptozotocin-induced wild-type and CD148KO diabetic mice for 6 wk, and the renal phenotype was then assessed. The effects of 18E1 mAb in podocyte growth factor signals were also assessed in culture. Compared with control IgG, 18E1 mAb significantly decreased albuminuria and mesangial expansion without altering hyperglycemia and blood pressure in wild-type diabetic mice. Immunohistochemical evaluation showed that 18E1 mAb significantly prevented the reduction of podocyte number and nephrin expression and decreased glomerular fibronectin expression and renal macrophage infiltration. The 18E1 mAb showed no effects in CD148KO diabetic mice. Furthermore, we demonstrated that 18E1 mAb reduces podocyte epidermal growth factor receptor signals in culture and in diabetic mice. These findings suggest that agonistic anti-CD148 mAb attenuates DN in mice, in part by reducing epidermal growth factor receptor signals in podocytes. This antibody may be used for the treatment of early DN.

A non-canonical Notch complex regulates adherens junctions and vascular barrier function.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and hemostasis1. Hemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here, using an engineered organotypic model of perfused microvessels and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain – the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling.

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology.

Determination of the CD148-Interacting Region in Thrombospondin-1.

CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types, including vascular endothelial cells and duct epithelial cells. Previous studies have shown a prominent role of CD148 to reduce growth factor signals and suppress cell proliferation and transformation. Further, we have recently shown that thrombospondin-1 (TSP1) serves as a functionally important ligand for CD148. TSP1 has multiple structural elements and interacts with various cell surface receptors that exhibit differing effects. In order to create the CD148-specific TSP1 fragment, here we investigated the CD148-interacting region in TSP1 using a series of TSP1 fragments and biochemical and biological assays. Our results demonstrate that: 1) CD148 binds to the 1st type 1 repeat in TSP1; 2) Trimeric TSP1 fragments that contain the 1st type repeat inhibit cell proliferation in A431D cells that stably express wild-type CD148 (A431D/CD148wt cells), while they show no effects in A431D cells that lack CD148 or express a catalytically inactive form of CD148. The anti-proliferative effect of the TSP1 fragment in A431D/CD148wt cells was largely abolished by CD148 knockdown and antagonized by the 1st, but not the 2nd and 3rd, type 1 repeat fragment. Furthermore, the trimeric TSP1 fragments containing the 1st type repeat increased the catalytic activity of CD148 and reduced phospho-tyrosine contents of EGFR and ERK1/2, defined CD148 substrates. These effects were not observed in the TSP1 fragments that lack the 1st type 1 repeat. Last, we demonstrate that the trimeric TSP1 fragment containing the 1st type 1 repeat inhibits endothelial cell proliferation in culture and angiogenesis in vivo. These effects were largely abolished by CD148 knockdown or deficiency. Collectively, these findings indicate that the 1st type 1 repeat interacts with CD148, reducing growth factor signals and inhibiting epithelial or endothelial cell proliferation and angiogenesis.

Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain

BackgroundICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in β-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512–RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18). MethodsPure recombinant ICA512–RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512–GFP to the membrane of insulinoma cells was monitored by immunofluorescence. ResultsICA512–RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512–RESP18HD binds with high-affinity to insulin and proinsulin. ConclusionsRESP18HD is required for efficient sorting of ICA512 to secretory granules. General significanceRESP18HD is a key determinant for ICA512 granule targeting.

In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions.

Spatiotemporal aspects of protein-tyrosine phosphatase (PTP) activity and interaction partners for many PTPs are elusive. We describe here an elegant and relatively simple method, in situ proximity ligation assay (in situ PLA), which can be used to address these issues. The possibility to detect endogenous unmodified proteins in situ and to visualize individual interactions with spatial resolution is the major advantage of this technique. We provide protocols suitable to monitor association of the transmembrane PTPs PTPRJ/DEP-1/CD148 and PTPRB/VE-PTP with their substrates, the receptor tyrosine kinases FMS-like tyrosine kinase 3 (FLT3/CD135), and Tie2 and vascular endothelial growth factor receptor 2 (VEGFR2), respectively. Detailed description of method development and reagents as well as highlighting of critical factors will enable the reader to apply the method successfully to other PTP-protein interactions.

Role of SHP2 in invasion and metastasis of breast cancer

Src homology 2 domain-containing phosphatase (SHP2), which is encoded by proto-oncogene PTPN11, is one of the transmembrane protein-tyrosine phosphatases. SHP2 has an important function in signal transduction pathways and activities of cells through the regulation of tyrosine phosphorylation level of intracellular proteins. The status of SHP2 activation is closely connected with the regulation of hormone levels, state of invasion and metastasis of tumor, development and progression of tumor stem cells of breast cancer, as well as signal pathways including Ras/ERK and PI3K/Akt/mTOR. Gene knockout or gene silencing expression helps inhibit tumor growth, irreversibly hindering the ability of the tumor to regain stem cells and disturb the signal pathways of the invasion and metastasis of breast cancer. Recent studies have shown that SHP2 may help in bringing anticancer drugs to a higher level. This article concentrates on the research progress in relationship of SHP2 with invasion and metastasis of breast cancer.

Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.

Receptor protein tyrosine phosphatase beta/zeta is a functional binding partner for vascular endothelial growth factor.

BackgroundReceptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPβ/ζ interacts with ανβ3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, β3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated β3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανβ3 integrin association and subsequent signaling. In the present work, we studied whether RPTPβ/ζ mediates angiogenic actions of VEGF.MethodsHuman umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different β3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPβ/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 μm pores.ResultsRPTPβ/ζ mediates VEGF165-induced c-Src-dependent β3 Tyr773 phosphorylation, which is required for VEGFR2-ανβ3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPβ/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPβ/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect.ConclusionsThese data identify RPTPβ/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0287-3) contains supplementary material, which is available to authorized users.

Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli

Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis–Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0.

Natural antisense RNAs are involved in the regulation of CD45 expression in autoimmune diseases.

CD45 is a transmembrane protein tyrosine phosphatase that is specifically expressed in hematopoietic cells and can initiate signal transduction via the dephosphorylation of tyrosine. Alternatively spliced transcript variants of this gene encode distinct isoforms, which indicate different functional states of CD45. Among these variants, CD45RO, which contains neither exon 4, 5, or 6, is over-expressed in lymphocytes in autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and type I diabetes. The CD45 RO serves as a marker of the immune response activity and lymphocyte development. Previous studies have indicated that exon splicing is generally correlated with local hypermethylated DNA and acetylated histone modification, while autoimmune diseases are commonly associated with global hypomethylation and histone deacetylation in lymphocytes. Thus, the question arises of how exons 4, 5, and 6 of CD45RO are excluded under the status of global DNA hypomethylation and histone deacetylation in these autoimmune diseases. On the basis of the analyses of the context sequence of CD45 and its natural antisense RNA in GenBank, we proposed that the long noncoding RNA encoded by the natural antisense gene of CD45 contributes to the expressional regulation of the CD45RO splicing variant via recruitment of DNA methyltransferase and histone modification modulators specific to the sense gene CD45; thus, it is associated with the over-expression of CD45RO and the functional regulation of lymphocytes in the pathogenic development of autoimmune diseases.

SHP-2 Regulates Growth Factor Dependent Vascular Signalling and Function

Cellular responses to the environment are mediated by intracellular signalling pathways monitoring several essential cellular processes, such as proliferation, migration, differentiation and survival. Cellular dysfunction is caused by dysregulation of intracelleular signalling pathways and may ultimately result in pathophysiological conditions. The non- transmembrane protein tyrosine phosphatase SHP-2 has been shown to be important for the control of cellular behaviour. It influences the activity of several growth factor and cytokine dependent signalling pathways by association with growth factor receptors, cell surface adhesion molecules and adaptor molecules such as Gab-1, Grb2 and IRS-1. Upon FGF-2, EGF and insulin stimulation SHP-2 regulates MAPK pathway activation. In addition, SHP-2 is involved in the regulation of cell survival by influencing the PI3-K/Akt pathway upon EGF, IGF and PDGF stimulation. Due to these properties, SHP-2 function has recently gained more interest in vascular processes, such as in the differentiation of cardiac progenitor cells and angiogenic events. Indeed, SHP-2 was shown to positively regulate endothelial cell motility and angiogenesis in vitro and in vivo as well as controlling intracellular pH of endothelial and vascular smooth muscle cells. On the other hand, SHP-2 was also demonstrated to be responsible for down regulation of VEGF receptor 2 activation upon dopamin and collagen stimulation. Finally, mutations in the Ptpn11 gene (encoding SHP-2) underlie the developmental disorders Noonan syndrome and Leopard syndrome characterized by congenital heart disease and hematologic abnormalities. Different mutations in this gene also result in myeloid and lymphoid malignancies. This article summarizes the role of SHP-2 in signalling pathways relevant for vascular biology and associated disorders.

Micron-scale exclusion of CD45 enhances activation of TCR zeta associated protein of 70 kDa tyrosine kinase. (P1192)

Abstract CD45, a transmembrane protein tyrosine phosphatase, is critical for activation of T cells. CD45 is known to modulate T cell signaling by dephosphorylating the inhibitory Tyr 505 of Lck, but at the same time it has been argued that exclusion of CD45 from the vicinity of the TCR is necessary for activation of T cells. While many data support the notion that CD45 exclusion is necessary for triggering of TCR signaling, there is no evidence that CD45 exclusion is sufficient. In this work, we designed substrates to test if local exclusion of CD45 can trigger TCR without aid of TCR ligation. To test this hypothesis, mouse CD4 T cells were seeded onto surfaces micropatterned with an antibody targeting the extracellular domain of CD45. These surfaces were created by first defining 1- or 2-µm diameter features of PLL-g-PEG onto glass coverslips, followed by adsorption of anti-CD45 to the remaining regions. These substrates depleted CD45 from the PLL-g-PEG patterned regions and enhanced production of pZap70 over levels observed on uniformly coated anti-CD45 surfaces as determined by total internal reflection fluorescence. Surprisingly, the areas of higher pZap70 were not localized in the specific regions of CD45 exclusion, but were concentrated in the center of the interface, approximating early stages of signaling in a T cell immunological synapse. These results demonstrate that local control over CD45 distribution can alter T cell signaling, independent of TCR-MHC recognition.

A sulfated carbohydrate epitope inhibits axon regeneration after injury

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.

Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors invitro and their recovery in transplanted recipients invivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Loss of the Protein‐Tyrosine Phosphatase DEP‐1/PTPRJ Drives Meningioma Cell Motility

DEP-1/PTPRJ is a transmembrane protein-tyrosine phosphatase which has been proposed as a suppressor of epithelial tumors. We have found loss of heterozygosity (LOH) of the PTPRJ gene and loss of DEP-1 protein expression in a subset of human meningiomas. RNAi-mediated suppression of DEP-1 in DEP-1 positive meningioma cell lines caused enhanced motility and colony formation in semi-solid media. Cells devoid of DEP-1 exhibited enhanced signaling of endogenous platelet-derived growth factor (PDGF) receptors, and reduced paxillin phosphorylation upon seeding. Moreover, DEP-1 loss caused diminished adhesion to different matrices, and impaired cell spreading. DEP-1-deficient meningioma cells exhibited invasive growth in an orthotopic xenotransplantation model in nude mice, indicating that elevated motility translates into a biological phenotype in vivo. We propose that negative regulation of PDGF receptor signaling and positive regulation of adhesion signaling by DEP-1 cooperate in inhibition of meningioma cell motility, and possibly tumor invasiveness. These phenotypes of DEP-1 loss reveal functions of DEP-1 in adherent cells, and may be more generally relevant for tumorigenesis.