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• W2114816633 abstract "Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTPμ, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTPμ wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTPμ wedge Tat peptide had no effect on PC12 cells but blocked the PTPμ-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTPμ substrate, whereas LAR wedge peptide had no effect. The survival- and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function. Inhibition of protein-tyrosine phosphatases (PTPs) counterbalancing protein-tyrosine kinases (PTKs) offers a strategy for augmenting PTK actions. Conservation of PTP catalytic sites limits development of specific PTP inhibitors. A number of receptor PTPs, including the leukocyte common antigen-related (LAR) receptor and PTPμ, contain a wedge-shaped helix-loop-helix located near the first catalytic domain. Helix-loop-helix domains in other proteins demonstrate homophilic binding and inhibit function; therefore, we tested the hypothesis that LAR wedge domain peptides would exhibit homophilic binding, bind to LAR, and inhibit LAR function. Fluorescent beads coated with LAR or PTPμ wedge peptides demonstrated PTP-specific homophilic binding, and LAR wedge peptide-coated beads precipitated LAR protein. Administration of LAR wedge Tat peptide to PC12 cells resulted in increased proliferation, decreased cell death, increased neurite outgrowth, and augmented Trk PTK-mediated responses to nerve growth factor (NGF), a phenotype matching that found in PC12 cells with reduced LAR levels. PTPμ wedge Tat peptide had no effect on PC12 cells but blocked the PTPμ-dependent phenotype of neurite outgrowth of retinal ganglion neurons on a PTPμ substrate, whereas LAR wedge peptide had no effect. The survival- and neurite-promoting effect of the LAR wedge peptide was blocked by the Trk inhibitor K252a, and reciprocal co-immunoprecipitation demonstrated LAR/TrkA association. The addition of LAR wedge peptide inhibited LAR co-immunoprecipitation with TrkA, augmented NGF-induced activation of TrkA, ERK, and AKT, and in the absence of exogenous NGF, induced activation of TrkA, ERK, and AKT. PTP wedge domain peptides provide a unique PTP inhibition strategy and offer a novel approach for augmenting PTK function. Within intracellular signaling networks, protein-tyrosine kinases (PTKs) 3The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; RPTP, receptor PTP; ERK, extracellular signal-regulated kinase; LAR, leukocyte common antigen; NGF, nerve growth factor; HLH, helix-loop-helix; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HS, horse serum; FBS, fetal bovine serum; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BrdUrd, bromodeoxyuridine; TUNEL, terminal dUTP nick-end labeling; DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor.3The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; RPTP, receptor PTP; ERK, extracellular signal-regulated kinase; LAR, leukocyte common antigen; NGF, nerve growth factor; HLH, helix-loop-helix; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HS, horse serum; FBS, fetal bovine serum; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BrdUrd, bromodeoxyuridine; TUNEL, terminal dUTP nick-end labeling; DAPI, 4′,6-diamidino-2-phenylindole; EGF, epidermal growth factor. can be counterbalanced by protein-tyrosine phosphatases (PTPs) (1Hunter T. Cell. 1996; 80: 225-236Abstract Full Text PDF Scopus (2594) Google Scholar, 2Bixby J.L. Neuroreport. 2000; 11: 5-10Crossref PubMed Scopus (36) Google Scholar, 3Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (463) Google Scholar, 4Ostman A. Bohmer F.-D. Trends Cell Biol. 2001; 11: 258-266Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Trk-family neurotrophin PTK receptors undergo ligand-induced tyrosine transphosphorylation and downstream activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways (5Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1655) Google Scholar, 6Huang E.J. Reichardt L.F. Annu. Rev. Biochem. 2003; 72: 609-642Crossref PubMed Scopus (1953) Google Scholar). A fundamental area of Trk signaling that remains to be investigated is the identification of the PTPs that directly or indirectly associate with Trk receptors and regulate their signaling. The leukocyte common antigen-related (LAR) receptor is a prototypical receptor PTP containing tandem catalytic domains (D1 and D2) in its cytoplasmic region with D1, constituting the primary catalytic site, and D2, conferring regulatory function (7Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar). LAR is expressed by neurons and regulates neuronal survival and neurite outgrowth (2Bixby J.L. Neuroreport. 2000; 11: 5-10Crossref PubMed Scopus (36) Google Scholar, 8Zhang J.S. Longo F.M. J. Cell Biol. 1995; 128: 415-431Crossref PubMed Scopus (80) Google Scholar, 9Yeo T.T. Yang T. Massa S.M. Zhang J.S. Honkaniemi J. Butcher L.L. Longo F.M. J. Neurosci. Res. 1997; 47: 348-360Crossref PubMed Scopus (110) Google Scholar, 10Zhang J.S. Honkaniemi J. Yang T. Yeo T.T. Longo F.M. Mol. Cell. Neurosci. 1998; 10: 271-286Crossref Scopus (44) Google Scholar, 11Stoker A.W. Curr. Opin. Neurobiol. 2001; 11: 95-102Crossref PubMed Scopus (57) Google Scholar, 12Xie Y.M. Yeo T.T. Zhang C. Yang T. Tisi M.T. Massa S.M. Longo F.M. J. Neurosci. 2001; 21: 5130-5138Crossref PubMed Google Scholar, 13Van der Zee C.E.E.M. Man T.Y. Van Lieshout E.M.M. Van der Heijden I. Van Bree M. Hendriks W.J.A.J. Eur. J. Neurosci. 2003; 17: 991-1005Crossref PubMed Scopus (38) Google Scholar, 14Johnson K.G. Van Vactor D. Physiol. Rev. 2003; 83: 1-24Crossref PubMed Scopus (192) Google Scholar). The physiological ligand(s) for LAR in mammalian systems is unknown, although a LAR ectodomain isoform binds LAR homophilically and promotes neurite outgrowth (15Yang T. Bernabeu R. Xie Y.-M. Zhang J.S. Massa S.M. Rempel H.C. Longo F.M. J. Neurosci. 2003; 23: 3353-3363Crossref PubMed Google Scholar). The LAR enzymatic substrates within neurons remain to be established. Several lines of evidence point to LAR as a candidate PTP modulating Trk phosphorylation. First, LAR and Trk are co-expressed in multiple neuronal populations (16Longo F.M. Martignetti J.A. LeBeau J.M. Zhang J.S. Barnes J.P. Brosius J. J. Biol. Chem. 1993; 268: 26503-26511Abstract Full Text PDF PubMed Google Scholar); second, LAR associates with caveolin, a component of TrkA signaling complexes (17Bilderback T.R. Gazula V.-R. Lisanti M.P. Dobrowsky R.T. J. Biol. Chem. 1999; 274: 257-263Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 18Caselli A. Mazzinghi B. Camici G. Manao G. Ramponi G. Biochem. Biophys. Res. Commun. 2002; 296: 692-697Crossref PubMed Scopus (60) Google Scholar); finally, antisense-mediated down-regulation of LAR in PC12 cells leads to enhanced nerve growth factor (NGF)/TrkA-induced differentiation (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). The conserved nature of PTP catalytic sites has limited development of PTP-specific catalytic site inhibitors (20Zhang Z.-Y. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 209-234Crossref PubMed Scopus (374) Google Scholar). LAR is one of a subgroup of receptor PTPs (RPTPs), including PTPμ, RPTPα, PTPδ, PTPσ, and CD45, that contain a helix-loop-helix (HLH), wedge-shaped sequence located between the membrane proximal region and the D1 catalytic domain (21Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar, 22Nam H.-J. Poy F. Krueger N.X. Saito H. Frederick C.A. Cell. 1999; 97: 449-457Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Although functions of the LAR HLH wedge domain remain unknown, HLH domains in other types of proteins mediate homophilic or heterophilic binding (23Goldfarb A.N. Lewandowska K. Shoham M. J. Biol. Chem. 1996; 271: 2683-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 24Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Crossref PubMed Scopus (1385) Google Scholar, 25Olson V.A. Wetter J.A. Friesen P.D. J. Virol. 2001; 75: 6042-6051Crossref PubMed Scopus (37) Google Scholar), and in some cases this binding achieves a potent inhibitory effect (26Benezra R. Davis R.L. Lockshon D. Turner D.L. Weintraub H. Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1801) Google Scholar, 27Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Application of synthetic peptides resembling potential protein-protein interaction sites, including the c-Myc HLH domain (28Nieddu E. Melchiori A. Pescarolo M.P. Bagnasco L. Biasotti B. Licheri B. Malacarne D. Tortolina L. Castagnino N. Pasa S. Cimoli G. Avignolo C. Ponassi R. Balbi C. Patrone E. D'Arrigo C. Barboro P. Vasile F. Orecchia P. Carnemolla B. Damonte G. Millo E. Palomba D. Fassina G. Mazzei M. Parodi S. FASEB J. 2005; 19: 632-634Crossref PubMed Scopus (22) Google Scholar), has served as a powerful tool for development of novel strategies to inhibit protein function (29Souroujon M.C. Mochly-Rosen D. Nat. Biotechnol. 1998; 16: 919-924Crossref PubMed Scopus (201) Google Scholar). Despite the absence of knowledge regarding LAR wedge domain binding targets or function, we tested the empiric possibility that a synthetic peptide resembling this domain would demonstrate homophilic binding, bind to full-length LAR, and induce a LAR-deficient phenotype consistent with inhibition of LAR function. We previously established that antisense-induced down-regulation of LAR in PC12 cells induces a phenotype consisting of increased proliferation in serum-containing medium, decreased death, and increased neurite outgrowth in serum-free medium and enhanced neurite outgrowth in response to NGF stimulation (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). We hypothesized that LAR wedge peptide treatment of PC12 cells would induce a similar phenotype including augmented signaling via the TrkA PTK receptor. The finding that LAR wedge peptides are capable of inhibiting LAR function would suggest novel approaches for inhibition of PTP-dependent functions and for augmentation of neurotrophin signaling. Peptides—Peptides containing the residues shown in Fig. 1 were synthesized by Genemed Synthesis Inc. (South San Francisco, CA) in the amide form. Peptides were purified by high performance liquid chromatography, and amino acid content was verified by quantitative amino acid analysis. Peptides were synthesized to include a membrane-penetrant Tat-derived sequence at either the C or N terminus, a well established strategy for promoting cellular uptake of protein or synthetic peptides (30Wadia J.S. Dowdy S.F. Curr. Opin. Biotech. 2002; 13: 52-56Crossref PubMed Scopus (451) Google Scholar). Microsphere Homophilic Binding Assay—Peptides or bovine serum albumin (BSA) in solutions of 300 μg/ml were each linked to microspheres (Polysciences, Warrington, PA) using the manufacturer's recommended protocol. LAR wedge domain (WLAR-Tat) and scrambled LAR wedge (SLAR-Tat) peptides or BSA were linked to Fluoresbrite Carboxylate NYO (red fluorescing) 1.75-μm microspheres. WPTPμ-Tat and SPTPμ-Tat wedge peptides were linked to Fluoresbrite Carboxylate BB (blue fluorescing) 1.75-μm microspheres. After linkage, microspheres were blocked with 0.25 m ethanolamine for 30 min at room temperature and then with BSA (10 mg/ml) in borate buffer for an additional 30 min. After blocking, microspheres were washed 3 times with phosphate-buffered saline, suspended in 50 μl of phosphate-buffered saline, and then incubated in 96-well plates at room temperature for 1 h on a rotary shaker. 10-μl aliquots were removed and examined on microscope slides using fluorescence microscopy at wavelengths of 360 nm (blue) and 590 nm (red). Cell Culture—PC12 cells (provided by William C. Mobley, Stanford University (31Beattie E.C. Zhou J. Grimes M.L. Bunnett N.W. Howe C.L. Mobley W.C. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 389-406Crossref PubMed Google Scholar)) were propagated in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter glucose (Invitrogen) supplemented with 10% heat-inactivated horse serum (HS), 5% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. The PC12 cells used in the present study remain attached to tissue culture plastic as a dispersed monolayer (31Beattie E.C. Zhou J. Grimes M.L. Bunnett N.W. Howe C.L. Mobley W.C. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 389-406Crossref PubMed Google Scholar). Cells were cultured in a humidified incubator with an atmosphere of 5% CO2, 95% air at 37 °C. LAR-deficient PC12 cells stably transfected with the pMEP4b LAR antisense construct regulated by the metallothionine promoter (32Kulas D.T. Goldstein B.J. Mooney R.A. J. Biol. Chem. 1996; 271: 748-754Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and control PC12 cells stably transfected with a pMEP4b null construct have been described previously (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). Pull-down Assays—Pull-down assays were conducted as previously described (15Yang T. Bernabeu R. Xie Y.-M. Zhang J.S. Massa S.M. Rempel H.C. Longo F.M. J. Neurosci. 2003; 23: 3353-3363Crossref PubMed Google Scholar). PC12 cells were grown to 80% confluence in 10% HS and 5% FBS and then lysed at 4 °C in lysis buffer (20 mm Tris-HCl, pH 7.5, 137 mm NaCl, 1% Nonidet P-40, 10% glycerol, 500 μm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin) for 30 min. Lysates were centrifuged at 14,000 × g at 4 °C for 20 min, and the protein concentrations of the supernatant were determined by the BCA method (Pierce). Lysates aliquots of ∼700 μl were incubated for 1 h at room temperature with WLAR-Tat or SLAR-Tat (30 μg) synthesized to include a poly-His tag at the C terminus (WLAR-Tat-H, SLAR-Tat-H). After the addition of 100 μl of Talon Superflow metal affinity resin (Clontech, Palo Alto, CA), lysate mixtures were incubated by end-over-end rotation overnight at 4 °C followed by washing 3 times with lysis buffer. Bound resin was resuspended in 60 μl of 2× NuPAGE lithium dodecyl sulfate protein sample buffer (Invitrogen) followed by boiling for 10 min and electrophoresis through NuPAGE 4–12% Bis-Tris gels (Invitrogen). Gels were either silver-stained or used for transfer to polyvinylidene difluoride membranes for Western blotting. Blots were incubated with polyclonal anti-LAR antibody raised against a synthetic peptide corresponding to the LAR membrane proximal domain (RAALEYLGSFDHYAT) or with polyclonal anti-LAR antibody raised against a synthetic peptide corresponding to the LAR C terminus (RGFYNRPISPDLSYQC). Both antibodies would be expected to identify the LAR ∼85-kDa intracellular subunit. After LAR antibody, blots were re-probed with polyclonal goat antibody against PTPσ (C18 sc-10871) obtained from Santa Cruz Biotechnology. After incubation with horseradish peroxidase-linked secondary antibody, signal was detected using the ECL chemiluminescence system (Amersham Biosciences). Co-immunoprecipitation—PC12 cells (1.0 × 106 cells/well) were seeded in a volume of 2 ml of culture medium containing 10% HS and 5% FBS in 6-well plates precoated with poly-l-lysine and incubated overnight. Cells were then washed 3 times with serum-free DMEM and cultured in 2 ml of DMEM containing 0.5% FBS overnight followed by exposure to serum-free DMEM alone or DMEM containing NGF (0.18 nm) or NGF plus peptide (peptide 4 μm) followed by harvesting at 10 min. Aliquots (700–800 μl; 500 μg of protein) of PC12 cell lysates were prepared as described above. Immunoprecipitations were performed by adding preimmune serum (5 μg), polyclonal anti-TrkA antibody (Cell Signaling, Beverly, MA; 1:250 dilution), or polyclonal anti-LAR antibody directed against the LAR C terminus (5 μg) and incubating overnight at 4 °C. Immune complexes were captured by adding 100 μl of protein A-agarose (Pierce) followed by incubation with end-over-end rotation overnight at 4 °C followed by washing 5 times in lysis buffer. Protein was eluted by boiling for 10 min in 100 μl of 2× NuPAGE lithium dodecyl sulfate sample buffer. Aliquots were submitted to NuPAGE 4–12% Bis-Tris gels and transferred to polyvinylidene difluoride membranes. Western blots were incubated with antibody directed against the LAR C terminus or polyclonal anti-TrkA antibody and then incubated with horseradish peroxidase-linked secondary antibodies. Signal was detected using the ECL chemiluminescence system. PC12 Cell Proliferation—PC12 cell proliferation was quantitated using protocols described in Tisi et al. (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). Cells were seeded at a density of 1.5 × 105 cells/well in a total volume of 2 ml/well of DMEM containing 10% HS and 5% FBS in 6-well plates (Corning, Corning, NY) precoated with poly-l-lysine (10 μg/well). Peptides were added at the time of cell seeding. For each of five assays, single or duplicate wells for each peptide condition were assessed. At the indicated times, cells were harvested by adding 0.5 ml of 0.05% trypsin to each well, incubating at 37 °C for 2 min, and collecting the entire cell content of each well. After trituration, aliquots of well mixed cells were counted using a hemocytometer. Intact, round, phase-bright cells that exclude trypan blue were counted, and two aliquots per well were measured and then averaged. A second set of PC12 cell proliferation studies was conducted using BrdUrd incorporation as a measure of proliferation (33Perry T. Lahiri D.K. Chen D. Zhou J. Shaw K.T.Y. Egan J.M. Greig N.H. J. Pharmacol. Exp. Ther. 2002; 300: 958-966Crossref PubMed Scopus (257) Google Scholar). Cells were seeded on poly-l-lysine-coated 12CIR-1D cover glass slips in 24-well plates (Fisher) by adding 0.25 ml of cell suspension (20,000 cells/cm2), 0.25 ml of DMEM containing 10% HS and 5% FBS, and wedge peptide (4 μm) to each well. To label newly synthesized DNA, BrdUrd (10 μm) was added to the culture medium for 5 h before fixing in 4% paraformaldehyde. BrdUrd immunostaining was conducted with mouse anti-BrdUrd antibody (DakoCytomation, Glostrup, Denmark) at 1:200 with fluorescein isothiocyanate donkey anti-mouse (Molecular Probes, Invitrogen) at 1:200 serving as the secondary antibody. Using a fluorescence microscope (Leica DM IRE2), six fields were systematically acquired in each of three assays for each condition. For each image, the number of BrdUrd-positive cells per area was counted in a blinded fashion. PC12 Cell Survival and Neurite Outgrowth—PC12 cell survival and neurite outgrowth were quantitated using protocols described in Tisi et al. (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). 1.5 × 105 cells/well were seeded in a total volume of 2 ml/well of DMEM containing 10% HS and 5% FBS in 6-well plates precoated with poly-l-lysine and allowed to attach for 2 h. After 2 h cells were washed 3 times with serum-free medium, and medium was replaced with serum-free DMEM supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). NGF (Invitrogen) or peptides were added to the same medium at concentrations indicated in figure legends. Cells were cultured for durations of 2 or 7 days. In 7-day cultures, on days 2, 4, and 6, 0.5 ml of medium was removed and replaced with 0.5 ml of fresh medium containing NGF or peptide at the original concentration. Cells were fixed with 4% formaldehyde in phosphate-buffered saline, and cell survival was quantitated under phase contrast microscopy by quantitating in a blinded fashion the number of intact, round, phase-bright cells per area. Prior studies demonstrated that morphological-based assessment of surviving cells provided data equivalent to that provided using Syto-13 and propidium iodide cell death indicators (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). Neurite length quantification was performed in a blinded fashion using NIH Image analysis software. In separate assays, PC12 cell survival was assessed using the established strategy of combined TUNEL and DAPI staining to quantitate the ratio of apoptotic (TUNEL-positive) over total (DAPI-positive) cells. Cells were seeded on poly-l-lysine-coated 12CIR-1D cover glass in 24-well plates (Fisher) by adding 0.25 ml of cell suspension (30,000 cells/cm2) and 0.25 ml of DMEM containing 10% HS and 5% FBS. After a 2-h attachment period, cells were washed 3 times with serum-free medium, and medium was replaced with serum-free DMEM supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) and containing NGF (5 or 50 ng/ml; 0.18 or 1.8 nm) or peptides (4 μm). During the 7-day culture period, on days 2, 4, and 6, 0.25 ml of medium was removed and replaced with 0.25 ml of fresh medium containing NGF or peptide at the original concentration. After 7 days in culture, cell apoptosis was assessed using the fluorescein-12-dUTP, DeadEnd™ Fluorometric TUNEL system (Promega, Madison, WI) and VECTA-SHIELD® plus DAPI (Vector Labs Burlingame, CA). Samples were analyzed under a fluorescence microscope (Leica DM IRE2) using a standard fluorescence filter set to view green (TUNEL) fluorescence at 520 nm and blue (DAPI) at 460 nm. For each assay, 6 fields were systematically photographed, and DAPI-positive cells were counted as either TUNEL-positive or TUNEL-negative in a blinded fashion. A total of three independent assays were conducted. Bonhoeffer Stripe Assay—Neural retinas were dissected from embryonic day 8 (stage 32) chick embryos, flattened on concanavalin-coated nitrocellulose filters, and cut into 350-μm wide explants perpendicular to the optic fissure. Explants were placed retinal ganglion side down on substrate-coated dishes and grown in 10% fetal bovine serum (Atlas, Fort Collins, CO), 2% chick serum (Sigma), 2 mm l-glutamine (Invitrogen), 2 units/ml penicillin, 2 μg/ml streptomycin, and 5 ng/ml amphotericin in RPMI 1640 (Invitrogen). The substrate lane assay used was a modified version of the Bonhoeffer method (34Vielmetter J. Stolze B. Bonhoeffer F. Stuermer C.A. Exp. Brain Res. 1990; 81: 283-287Crossref PubMed Scopus (107) Google Scholar), performed as previously described (35Burden-Gulley S.M. Ensslen S. Brady-Kalnay S.M. J. Neurosci. 2002; 22: 3615-3627Crossref PubMed Google Scholar). Briefly, tissue culture dishes were coated with nitrocellulose and dried before applying the silicon lane matrix to the dish surface. A PTPμ-Fc chimera, a fusion between the extracellular domain of PTPμ (amino acids 1–621) and the Fc region of immunoglobulin heavy chain (36Rosdahl J.A. Ensslen S.E. Niedenthal J.A. Brady-Kalnay S.M. J. Neurobiol. 2003; 56: 199-208Crossref PubMed Scopus (13) Google Scholar), was used as the first substrate. PTPμ-Fc chimera (80 ng total) containing a small amount of Texas Red-conjugated BSA (for visualization of the lanes; Molecular Probes) was injected into the channels of the matrix, incubated, aspirated, then replaced with a fresh aliquot of the same substrate. All remaining binding sites within the lanes were blocked with BSA (fraction V; Sigma) and rinsed with calcium-magnesium-free phosphate buffer. The matrix was removed, and 875 ng (total) of laminin (Biomedical Technologies Inc., Stoughton, MA) was spread across the lane area and incubated for 30 min. The entire dish was blocked with BSA and then rinsed with RPMI. Explants were cultured for 48 h before imaging. Representative images from a minimum of three separate experiments are shown. Quantitation of stripe assays was performed using a rating scale as previously described (37Walter J. Kern-Veits B. Huf J. Stolze B. Bonhoeffer F. Development. 1987; 101: 685-696Crossref PubMed Google Scholar, 38Ensslen S. Brady-Kalnay S.M. Mol. Cell. Neurosci. 2004; 25: 558-571Crossref PubMed Scopus (22) Google Scholar). Neurites that show no preference for either substrate are rated 0, and neurites that grow exclusively on one substrate are rated 3. A rating of 2 indicates that most of the neurites grow on the laminin lanes with an occasional neurite crossing over PTPμ lanes, whereas a rating of 1 is given when there is a significant amount of neurite crossing but a tendency to fasciculate on laminin. Data from a minimum of three experiments were combined (with a minimum sample size of nine explants per condition) to determine the average degree of avoidance for each condition, then analyzed with Fisher's PLSD (Statview 4.51; Abacus Concepts, Inc., Calabasas, CA) at a 95% confidence level. TrkA Activation in PC12 LAR Antisense Cells—PC12 cells stably transfected with LAR antisense construct or null vector control construct have been described previously (19Tisi M.A. Xie Y. Yeo T.T. Longo F.M. J. Neurobiol. 2000; 43: 477-486Crossref Scopus (40) Google Scholar). In LAR antisense-expressing cells (clone LAS-1) LAR protein levels are reduced to ∼35% of levels present in null-transfected control cells (clone LC-2). To assess the effects of LAR deficiency on TrkA activation, 1.0 × 106 cells per well were seeded in a total volume of 2 ml of culture medium containing 5% FBS and 10% HS in 6-well plates precoated with poly-l-lysine and incubated until cells reached ∼80% confluence. Cells were washed 3 times with serum-free DMEM and cultured in 2 ml of DMEM containing 0.5% FBS overnight at 37 °C. Cells were then cultured in serum-free DMEM for 4 h followed by exposure to either serum-free DMEM alone or DMEM containing NGF or peptide for 10- or 30-min durations followed by harvesting in lysis buffer. Western Blot Analysis—Aliquots of PC12 cell lysates containing 15 μg of protein were mixed with NuPAGE lithium dodecyl sulfate protein sample buffer and boiled for 10 min. Samples were then run on NuPAGE 4–12% Bis-Tris gels. Proteins were electrophoretically transferred from gels to polyvinylidene difluoride membranes (Amersham Biosciences) for 1 h at 30 V. Filters were blocked in blocking buffer consisting of 5% nonfat dry milk (Bio-Rad) in TBST (20 mm Tris-HCl, pH 7.5, 137 mm, 0.2% Tween 20) for 1 h at room temperature. After 1 h of blocking, membranes were incubated overnight at 4 °C with one of the following antibodies (all from Cell Signaling): polyclonal anti-phospho-TrkY490, monoclonal anti-phospho-ERKT202/Y204, or monoclonal anti-phospho-AKTS473. Blots were reprobed with polyclonal antibody recognizing total TrkA protein, total ERK1/2 protein, or total AKT protein. Polyclonal goat antibody against PTPσ (C19 sc-10871) was obtained from Santa Cruz Biotechnology. One Western blot was performed in each assay for each cell extract derived from each culture condition. In studies of TrkA activation described in Fig. 7D, duplicate Western blots were performed in two of the five assays conducted, and their resulting values were averaged. Wedge Domain Peptides—The crystal structure of the LAR D1 and D2 catalytic domains and the location of the D1 wedge domain are illustrated in Fig. 1, A and B. Peptides containing a 24-residue segment corresponding to the LAR wedge HLH domain were synthesized. Inclusion of an 11-residue Tat-derived protein transduction domain provides a well established method for delivery of peptides into cells (30Wadia J.S. Dowdy S.F. Curr. Opin. Biotech. 2002; 13: 52-56Crossref PubMed Scopus (451) Google Scholar). As demonstrated in Fig. 1C, four peptides containing the following LAR residues were synthesized, (i) residues containing the wedge sequence alone (WLAR), (ii) the wedge sequence with the Tat domain linked to the C terminus (WLAR-Tat), (iii) the wedge sequence with the Tat domain linked to the N terminus (Tat-WLAR), and (iv) the wedge sequence randomly scrambled with the Tat domain linked to the C terminus (SLAR-Tat). LAR and PTPμ are both members of the type II RPTP subfamily (39Brady-Kalnay S.M. Cell Adhesion: Frontiers in Molecular Biology. 2001; 39 (Oxford University Press, Oxford, UK): 217-258Google Scholar, 40" @default.
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