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.
In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.
Regulation of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) by IP3 and Ca2+ allows them to initiate and regeneratively propagate intracellular Ca2+ signals. The distribution and mobility of IP3R determines the spatial organization of these Ca2+ signals. Until now, there has been no systematic comparison of the distribution and mobility of the three mammalian IP3R subtypes in a uniform background. We used confocal microscopy and fluorescence recovery after photobleaching to define these properties for each IP3R subtype expressed heterologously in COS-7 cells. IP3R1 and IP3R3 were uniformly distributed within the membranes of the endoplasmic reticulum (ER), but the distribution of IP3R2 was punctate. The mobile fractions (Mf = 84 ± 2 and 80 ± 2%) and diffusion coefficients (D = 0.018 ± 0.001 and 0.016 ± 0.002 μm2/s) of IP3R1 and IP3R3 were similar. Other ER membrane proteins (ryanodine receptor type 1 and sarco/endoplasmic reticulum Ca2+-ATPase type 1) and a luminal protein (enhanced GFP with a KDEL retrieval sequence) had similar mobile fractions, suggesting that IP3R1 and IP3R3 move freely within an ER that is largely, although not entirely, continuous. IP3R2 was less mobile, but IP3R2 mobility differed between perinuclear (Mf = 47 ± 4% and D = 0.004 ± 0.001 μm2/s) and near-plasma membrane (Mf = 64 ± 6% and D = 0.013 ± 0.004 μm2/s) regions, whereas IP3R3 behaved similarly in both regions. We conclude that IP3R1 and IP3R3 diffuse freely within a largely continuous ER, but IP3R2 is more heterogeneously distributed and less mobile, and its mobility differs between regions of the cell.
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