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• W2080306074 abstract "Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments. Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments. nerve growth factor brain-derived neurotrophic factor neurotrophin 3 phospholipase C mitogen-activated protein kinase extracellular signal-regulated kinase The survival and differentiation of neurons in the peripheral nervous system are dependent on neurotrophic factors, which are secreted by the target tissues. The first discovered family member of the closely related neurotrophins is nerve growth factor (NGF)1 (1Levi-Montalcini R. Science. 1987; 237: 1154-1162Crossref PubMed Scopus (2699) Google Scholar). Other members are brain-derived neurotrophic factor (BDNF) (2Leibrock J. Lottspeich F. Hohn A. Hofer M. Hengerer B. Masiakowski P. Thoenen H. Barde Y.A. Nature. 1989; 341: 149-152Crossref PubMed Scopus (1235) Google Scholar, 3Ernfors P. Ibanez C.F. Ebendal T. Olson L. Persson H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5454-5458Crossref PubMed Scopus (608) Google Scholar), neurotrophin 3 (NT-3) (4Hohn A. Leibrock J. Bailey K. Barde Y.A. Nature. 1990; 344: 339-341Crossref PubMed Scopus (945) Google Scholar, 5Jones K.R. Reichardt L.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8060-8064Crossref PubMed Scopus (316) Google Scholar, 6Kaisho Y. Yoshimura K. Nakahama K. FEBS Lett. 1990; 266: 187-191Crossref PubMed Scopus (158) Google Scholar, 7Maisonpierre P.C. Belluscio L. Squinto S. Ip N.Y. Furth M.E. Lindsay R.M. Yancopoulos G.D. Science. 1990; 247: 1446-1451Crossref PubMed Scopus (1118) Google Scholar, 8Rosenthal A. Goeddel D.V. Nguyen T. Lewis M. Shih A. Laramee G.R. Nikolics K. Winslow J.W. Neuron. 1990; 4: 767-773Abstract Full Text PDF PubMed Scopus (449) Google Scholar), and neurotrophin 4/5 (NT-4/5) (9Berkemeier L.R. Winslow J.W. Kaplan D.R. Nikolics K. Goeddel D.V. Rosenthal A. Neuron. 1991; 7: 857-866Abstract Full Text PDF PubMed Scopus (708) Google Scholar, 10Hallbook F. Ibanez C.F. Persson H. Neuron. 1991; 6: 845-858Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 11Ip N.Y. Ibanez C.F. Nye S.H. McClain J. Jones P.F. Gies D.R. Belluscio L. Le Beau M.M. Espinosa R.d. Squinto S.P. Persson H. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3060-3064Crossref PubMed Scopus (553) Google Scholar). The primary event in neurotrophin signaling is the specific activation of receptor tyrosine kinases of the Trk family. Trk receptors have restricted ligand-binding specificities, NGF being a preferred ligand to TrkA, BDNF, and NT-4/5 to TrkB and NT-3 to TrkC (9Berkemeier L.R. Winslow J.W. Kaplan D.R. Nikolics K. Goeddel D.V. Rosenthal A. Neuron. 1991; 7: 857-866Abstract Full Text PDF PubMed Scopus (708) Google Scholar, 11Ip N.Y. Ibanez C.F. Nye S.H. McClain J. Jones P.F. Gies D.R. Belluscio L. Le Beau M.M. Espinosa R.d. Squinto S.P. Persson H. Yancopoulos G.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3060-3064Crossref PubMed Scopus (553) Google Scholar, 12Kaplan D.R. Martin-Zanca D. Parada L.F. Nature. 1991; 350: 158-160Crossref PubMed Scopus (841) Google Scholar, 13Kaplan D.R. Hempstead B.L. Martin-Zanca D. Chao M.V. Parada L.F. Science. 1991; 252: 554-558Crossref PubMed Scopus (1142) Google Scholar, 14Lamballe F. Klein R. Barbacid M. Cell. 1991; 66: 967-979Abstract Full Text PDF PubMed Scopus (920) Google Scholar). NT-3 can activate also TrkA and TrkB, although at much higher neurotrophin concentrations than TrkC (reviewed in Refs. 15Barbacid M. Oncogene. 1993; 8: 2033-2042PubMed Google Scholar, 16Hempstead B.L. Exp. Neurol. 1993; 124: 31-35Crossref PubMed Scopus (8) Google Scholar, 17Dechant G. Rodriguez-Tebar A. Barde Y.A. Prog. Neurobiol. 1994; 42: 347-352Crossref PubMed Scopus (98) Google Scholar). Neurotrophin binding to a specific Trk receptor causes receptor dimerization and the activation of the intrinsic tyrosine kinase domain of Trk. The initial substrate of the Trk kinase is the receptor itself, which results from tyrosine phosphorylation of the other subunit of Trk dimer (18Jing S. Tapley P. Barbacid M. Neuron. 1992; 9: 1067-1079Abstract Full Text PDF PubMed Scopus (391) Google Scholar). A tyrosine-phosphorylated Trk recruits, phosphorylates, and activates a variety of adapter molecules that initiate downstream signaling cascades. Phosphotyrosine residues of activated Trk-receptors serve as specific recognition sites for effector molecules that contain a Src homology 2 domain. Among the proteins that interact with tyrosine-phosphorylated TrkA are the enzymes phospholipase Cγ (PLCγ) and phosphoinositol 3-kinase and the adapter protein Shc (19Obermeier A. Halfter H. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. EMBO J. 1993; 12: 933-941Crossref PubMed Scopus (138) Google Scholar, 20Obermeier A. Lammers R. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963Abstract Full Text PDF PubMed Google Scholar, 21Loeb D.M. Stephens R.M. Copeland T. Kaplan D.R. Greene L.A. J. Biol. Chem. 1994; 269: 8901-8910Abstract Full Text PDF PubMed Google Scholar, 22Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). Each of these molecules activates distinct signaling pathways that may have different functions. The Ras/mitogen-activated protein kinase (MAPK) pathway initiated by Shc or PLCγ is involved in differentiation, while the phosphoinositol 3-kinase pathway is important for survival. It appears that in response to NGF, MAPKs like extracellular signal-regulated kinase 2 (ERK2), are cooperatively regulated by Shc and PLCγ in TrkA expressing cell lines (21Loeb D.M. Stephens R.M. Copeland T. Kaplan D.R. Greene L.A. J. Biol. Chem. 1994; 269: 8901-8910Abstract Full Text PDF PubMed Google Scholar, 22Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). A major consequence of Ras activation in cells is threonine/tyrosine phosphorylation and activation of ERKs and Ras appears to be an indispensable element in NGF-promoted ERK activation. In NGF-treated PC12 cells, ERK becomes phosphorylated at a threonine and a tyrosine residue (23Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (842) Google Scholar) leading to activation and translocation of ERK to the nucleus (24Chen R.H. Sarnecki C. Blenis J. Mol. Cell. Biol. 1992; 12: 915-927Crossref PubMed Google Scholar). In the nucleus it phosphorylates its target molecules including several transcription factors, as well as another family of kinases, the ribosomal S6 kinases (summarized in Refs. 25Chen R.H. Tung R. Abate C. Blenis J. Biochem. Soc. Trans. 1993; 21: 895-900Crossref PubMed Scopus (27) Google Scholar, 26Marshall C.J. Nature. 1994; 367: 686Crossref PubMed Scopus (7) Google Scholar, 27Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1198) Google Scholar). A second neurotrophin receptor, p75, binds all neurotrophins (28Rodriguez-Tebar A. Dechant G. Barde Y.A. Neuron. 1990; 4: 487-492Abstract Full Text PDF PubMed Scopus (573) Google Scholar, 29Squinto S.P. Stitt T.N. Aldrich T.H. Davis S. Bianco S.M. Radziejewski C. Glass D.J. Masiakowski P. Furth M.E. Valenzuela D.M. DiStefano P.S. Yancopoulos G.D. Cell. 1991; 65: 885-893Abstract Full Text PDF PubMed Scopus (681) Google Scholar, 30Rodriguez-Tebar A. Dechant G. Gotz R. Barde Y.A. EMBO J. 1992; 11: 917-922Crossref PubMed Scopus (385) Google Scholar). It belongs to the tumor necrosis factor family of receptors, which also includes Fas, CD40, CD30, and CD27 (31Rabizadeh S. Oh J. Zhong L.T. Yang J. Bitler C.M. Butcher L.L. Bredesen D.E. Science. 1993; 261: 345-348Crossref PubMed Scopus (749) Google Scholar). The p75 receptor has a number of distinct effects on Trk function. It alters the ligand binding specificity of Trks and enhances the proportion of TrkA receptors binding NGF with high affinity (13Kaplan D.R. Hempstead B.L. Martin-Zanca D. Chao M.V. Parada L.F. Science. 1991; 252: 554-558Crossref PubMed Scopus (1142) Google Scholar, 32Hempstead B.L. Martin-Zanca D. Kaplan D.R. Parada L.F. Chao M.V. Nature. 1991; 350: 678-683Crossref PubMed Scopus (1022) Google Scholar, 33Battleman D.S. Geller A.I. Chao M.V. J. Neurosci. 1993; 13: 941-951Crossref PubMed Google Scholar). Co-expression of TrkA with an excess of p75 dramatically increases the rate of association of NGF with Trk, in effect increasing the binding affinity (34Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar). Another effect of p75 on TrkA is to enhance NGF-induced TrkA tyrosine kinase activity. Introduction of p75 into MAH sympathoprogenitor cells expressing TrkA increases NGF-stimulated TrkA autophosphorylation (35Verdi J.M. Anderson D.J. Neuron. 1994; 13: 1359-1372Abstract Full Text PDF PubMed Scopus (116) Google Scholar) and inhibition of NGF binding to p75 on PC12 cells reduces NGF-mediated TrkA autophosphorylation (36Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). p75 also has a number of Trk-independent effects on cell processes. These include retrograde transport of neurotrophins (37Curtis R. Adryan K.M. Stark J.L. Park J.S. Compton D.L. Weskamp G. Huber L.J. Chao M.V. Jaenisch R. Lee K.F. Lindsay R.M. DiStefano P.S. Neuron. 1995; 14: 1201-1211Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 38von Bartheld C.S. Williams R. Lefcort F. Clary D.O. Reichardt L.F. Bothwell M. J. Neurosci. 1996; 16: 2995-3008Crossref PubMed Google Scholar), the promotion of Schwann cell migration (39Anton E.S. Weskamp G. Reichardt L.F. Matthew W.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2795-2799Crossref PubMed Scopus (306) Google Scholar), and the activation of the transcription factor NF-κB (40Carter B.D. Kaltschmidt C. Kaltschmidt B. Offenhauser N. Bohm- Matthaei R. Baeuerle P.A. Barde Y.A. Science. 1996; 272: 542-545Crossref PubMed Scopus (614) Google Scholar). In the presence of NGF, p75 can also either rescue cells from apoptosis or trigger the apoptotic cascade (31Rabizadeh S. Oh J. Zhong L.T. Yang J. Bitler C.M. Butcher L.L. Bredesen D.E. Science. 1993; 261: 345-348Crossref PubMed Scopus (749) Google Scholar, 41Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (719) Google Scholar, 42Frade J.M. Rodriguez-Tebar A. Barde Y.A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (668) Google Scholar). The latter may be due to elevated sphingomyelin hydrolysis leading to increased intracellular ceramide level (43Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (548) Google Scholar). Although the collaboration of p75 and TrkA has been characterized by several groups, the influence of p75 on TrkB and TrkC mediated signaling is more poorly understood. A truncated form of p75 lacking most of the cytoplasmic domain can collaborate with TrkA and TrkB, but not with TrkC, in a mouse fibroblast cell line expressing endogenous NGF and BDNF but not NT-3 (44Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. Neuron. 1994; 13: 187-201Abstract Full Text PDF PubMed Scopus (261) Google Scholar). p75 is also needed for neural differentiation in avian neural crest cultures expressing tyrosine kinase-deficient TrkC (45Hapner S.J. Boeshore K.L. Large T.H. Lefcort F. Dev. Biol. 1998; 201: 90-100Crossref PubMed Scopus (80) Google Scholar). Recently Bibel et al. (46Bibel M. Hoppe E. Barde Y.A. EMBO J. 1999; 18: 616-622Crossref PubMed Scopus (372) Google Scholar) using co-immunoprecipitation reported the physical interaction of p75 with all Trk receptors having a hemagglutinin epitope at their N-terminal end. They also found that the transient expression of p75 in a fibroblast cell line stably expressing TrkB reduced the TrkB autophosphorylation induced by NT-4/5 and NT-3, but not by BDNF. In light of these observations we have studied the function of p75 on TrkB and TrkC mediated signaling in a mouse fibroblast cell line stably expressing both p75 and TrkB or TrkC receptors as well as in PC12 cells stably transfected with TrkB and revealed a dual function of p75 in Trk-mediated neurotrophin signaling. The G418-resistant MG87 mouse fibroblast cell lines stably transfected with rat TrkB or TrkC cDNA were from Regeneron Pharmaceuticals, Inc.. These cell lines were stably transfected with 9 μg of rat p75 cDNA and 1 μg of histidinol selection plasmid, and selected for 10 days with 500 μg/ml G418 (Life Technologies, Inc.) and 2.5 mm histidinol (Sigma). To confirm the expression of p75 in the TrkB and TrkC lines, clones were lysed with RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS) supplemented with protease inhibitors (CompleteTM, Roche Molecular Biochemicals) and subjected to a Western blot analysis. Blots were probed with rabbit anti-p75 (Promega) and rabbit anti-pan-Trk (Santa Cruz Biotechnology) antibodies. Horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma) was used as a secondary antibody. The expression of Trk and p75 was detected with the enhanced chemiluminescence reagent (DuPont) and by exposure to x-ray film (X-Omat AR, Kodak). Selected clones were tested by enzyme-linked immunosorbent assay (47Krüttgen A. Moller J.C. Heymach Jr., J.V. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9614-9619Crossref PubMed Scopus (106) Google Scholar) and Northern blot analysis (data not shown) to exclude the endogenous expression of NGF, BDNF, NT-3, and NT-4/5, which could potentially interfere with the results. Cells were maintained in Dulbecco's modified Eagle's medium (Cellgro) containing 200 μg/ml G418 (Life Technologies, Inc.), 1 mm histidinol (Sigma), antibiotics (penicillin, 100 units/ml; streptomycin, 100 μg/ml, Life Technologies, Inc.), and 10% bovine calf serum (HyClone) in 5% CO2 at 37 °C. The PC12 cell line was stably transfected with TrkB cDNA as described above using the neomycin selection plasmid and maintained in the media noted above except that 6% of bovine calf serum, 6% of equine serum (HyClone), and 200 μg/ml G418 were used. 5 × 105 cells/well were plated in 6-well plates 1 day prior to neurotrophin treatment. Cells were starved in Dulbecco's modified Eagle's medium supplemented with antibiotics and 0.1% of bovine serum albumin, fraction V, (Amersham Pharmacia Biotech) in the presence or absence of protein A purified REX antibody (20 μg/ml) (48Weskamp G. Reichardt L.F. Neuron. 1991; 6: 649-663Abstract Full Text PDF PubMed Scopus (248) Google Scholar), a generous gift from Dr. Louis F. Reichardt) or NGF (1 μg/ml) for 30 min at 37 °C and treated with 0–100 ng/ml NGF (2.5 S, Harlan Bioproducts for Science, Inc.), BDNF, NT-3, or NT-4/5 (Regeneron Pharmaceuticals, Inc.) for 5 (Trk) or 7 (ERK2) min under the same conditions. Thereafter, cells were washed once with ice-cold phosphate-buffered saline, lysed with RIPA buffer supplemented with 1 mm sodium orthovanadate (Sigma) and protease inhibitors at 4 °C for 20 min, centrifuged, and the supernatants transferred to a fresh tube. Protein concentration was measured using bicinchoninic acid protein assay (Pierce) according to the manufacturer's instructions. For Trk phosphorylation assays, equal amounts of protein were immunoprecipitated with pan-Trk antibody and protein A-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C. Immunocomplexes were washed three times with ice-cold RIPA buffer and resuspended in 30 μl of Laemmli sample buffer (49Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207479) Google Scholar). For ERK2 assays, equal amount of whole cell lysates were analyzed. After boiling for 5 min, proteins were separated by 7.5 (Trk) or 12% (ERK2) SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting. Blots were probed with mouse monoclonal anti-phosphotyrosine 4G10 antibody (Upstate Biotechnology) or mouse monoclonal anti-pERK antibody (Santa Cruz Biotechnology), the secondary antibody being horseradish peroxidase-conjugated donkey anti-mouse antibody (Pierce). The phosphorylation of Trk and ERK2 was detected with enhanced chemiluminescence reagents (NEN Life Science Products Inc.) and by exposure to x-ray film. The results were analyzed by densitometry. In fibroblast cell lines transfected with TrkB or TrkC cognate neurotrophins are known to induce the autophosphorylation of the Trk receptors with BDNF, NT-3, and NT-4/5 being preferred ligands for TrkB and NT-3 for TrkC (50Ip N.Y. Stitt T.N. Tapley P. Klein R. Glass D.J. Fandl J. Greene L.A. Barbacid M. Yancopoulos G.D. Neuron. 1993; 10: 137-149Abstract Full Text PDF PubMed Scopus (480) Google Scholar). To determine which ligands are able to induce the tyrosine autophosphorylation of TrkB and TrkC in the stably transfected MG87 cell lines expressing either TrkB or TrkC, the lines were exposed to the four neurotrophins in the presence or absence of stably transfected p75. As expected, TrkB was phosphorylated in response to 100 ng/ml BDNF, NT-3, and NT-4/5 both with and without p75 expression (Fig. 1, a andb). In the presence of p75 the tyrosine phosphorylation level of TrkB was similar in response to all three neurotrophins but in the absence of p75 the phosphorylation level was lower in response to NT-3 compared with BDNF and NT-4/5. There are two potential explanations for this observation. Either p75 increases TrkB phosphorylation in response to NT-3 or p75 decreases TrkB phosphorylation in response to BDNF and NT-4/5. The data in the next section show that the latter explanation is correct. TrkC was uniquely phosphorylated in response to NT-3 only in the absence of p75 (Fig.1 C). Interestingly, a slight tyrosine phosphorylation of TrkC was observed in response to BDNF and NT-4/5 when p75 was coexpressed (Fig. 1 d). p75 has been reported to enhance TrkA mediated signaling by increasing the tyrosine autophosphorylation of TrkA in PC12 and MAH cells (35Verdi J.M. Anderson D.J. Neuron. 1994; 13: 1359-1372Abstract Full Text PDF PubMed Scopus (116) Google Scholar, 36Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). Recently, p75 was observed to reduce the tyrosine autophosphorylation of TrkB in response to NT-3 and NT-4/5 in the A293 cell line stably transfected with TrkB and transiently with p75 (46Bibel M. Hoppe E. Barde Y.A. EMBO J. 1999; 18: 616-622Crossref PubMed Scopus (372) Google Scholar). We examined the influence of p75 on TrkB and TrkC mediated signaling with the Trk tyrosine phosphorylation assay in stably transfected MG87 cells. The neurotrophin binding to p75 was blocked with either the REX antibody (48Weskamp G. Reichardt L.F. Neuron. 1991; 6: 649-663Abstract Full Text PDF PubMed Scopus (248) Google Scholar) or an excess of NGF. The positive influence of REX and nonspecific rabbit IgG antibodies on TrkB and TrkC phosphorylation was excluded using Western blotting (data not shown). The blocking of BDNF binding to p75 with the REX antibody caused an increased tyrosine autophosphorylation of TrkB over a wide range of BDNF concentrations suggesting an inhibitory effect of p75 on BDNF-induced TrkB mediated signaling (Fig.2 a). Since NGF is known to bind to p75 but not to TrkB or TrkC, and it was not able to induce TrkB or TrkC phosphorylation in the MG87 cell lines (Fig. 1) we used its p75 blocking capacity in BDNF treatment using a TrkB/p75 expressing cell line. Blocking p75 with NGF gave similar results as with the REX antibody blocking (Fig. 2 b). The blocking of NT-4/5 binding to p75 with NGF caused a reduced TrkB tyrosine phosphorylation, which was more prominent at lower neurotrophin concentrations but was not observed at high NT-4/5 concentration (100 ng/ml, Fig. 2 c). Interestingly, p75 had no influence on NT-3 induced TrkB tyrosine phosphorylation (Fig.2 d). In contrast to the TrkB expressing cell line, TrkC in the TrkC expressing line was significantly tyrosine autophosphorylated only in response to NT-3 (Fig. 1, c and d). Therefore, the influence of p75 on TrkC phosphorylation was examined using only NT-3 treatment. The inhibition of NT-3 binding to p75 with REX antibody did not have any influence on TrkC phosphorylation over a wide range of NT-3 concentrations (Fig. 3). Based on these observations we suggest that p75 does not play a role in TrkB or TrkC tyrosine autophosphorylation in response to NT-3. All three Trk receptors are expressed at the highest levels in the nervous system (14Lamballe F. Klein R. Barbacid M. Cell. 1991; 66: 967-979Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 51Klein R. Parada L.F. Coulier F. Barbacid M. EMBO J. 1989; 8: 3701-3709Crossref PubMed Scopus (484) Google Scholar, 52Martin-Zanca D. Barbacid M. Parada L.F. Genes Dev. 1990; 4: 683-694Crossref PubMed Scopus (289) Google Scholar), whereas the expression of p75 is not so highly restricted (53Lomen-Hoerth C. Shooter E.M. J. Neurochem. 1995; 64: 1780-1789Crossref PubMed Scopus (143) Google Scholar). Therefore, we wanted to confirm our results in a more neuronal-like environment and for that purpose PC12 cells stably transfected with TrkB cDNA were used. TrkA was not phosphorylated in response to BDNF, NT-3, or NT-4/5 in PC12 cells (Ref. 47Krüttgen A. Moller J.C. Heymach Jr., J.V. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9614-9619Crossref PubMed Scopus (106) Google Scholar and data not shown). The phosphorylation status of TrkB was determined in the presence or absence of REX antibody in BDNF and NT-4/5 treatments. In both instances the blocking of neurotrophin binding to endogenously expressed p75 increased the autophosphorylation of TrkB (Fig.4), which is consistent with the results obtained from stably transfected MG87 fibroblast cell lines. We also tested the influence of p75 on TrkA phosphorylation mediated by NGF treatment. We were able to use the same PC12/TrkB cell line because PC12 cells express endogenous TrkA in addition to p75. As previously reported (36Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar), the blocking of NGF binding to p75 with REX caused a reduced autophosphorylation of TrkA at relatively low concentrations of NGF (Fig. 4). To analyze downstream effects of p75 on TrkB and TrkC mediated signaling, we determined the tyrosine phosphorylation status of ERK2 in MG87 cells stably expressing both p75 and either TrkB or TrkC. Cells were treated with BDNF, NT-3, or NT-4/5 for 7 min and whole cell lysates were analyzed by Western blotting using phopho-ERK specific antibody. In the TrkB expressing cell line, BDNF and NT-4/5 were able to induce the phosphorylation of ERK2 to a similar extent (Fig.5 a). On the other hand, NT-3 although able to induce ERK2 phosphorylation, was less effective than BDNF and NT-4/5, even though the level of TrkB autophosphorylation is the same in the presence of p75 in response to all three neurotrophins (Fig. 1 b). More surprisingly, the addition of NGF to abolish the binding of other neurotrophins to p75 had no effect on the level of ERK2 phosphorylation induced by these three neurotrophins (Fig.5 a). This suggests that the observed reduction in TrkB autophosphorylation caused by p75 does not affect the Ras/MAPK signaling pathway. In the TrkC expressing cell line only NT-3 was able to increase the phosphorylation of ERK2 (Fig. 5 b). Again, the blocking of p75 had no influence on ERK2 phosphorylation in this cell line. The neurotrophins are distinguished by receptor-signaling systems of two different transmembrane proteins, the Trks and p75. Although at least some of the biological actions of a neurotrophin are mediated solely by its interaction with a Trk receptor p75 has been shown to collaborate with Trks to modify their activation. In this work we found that in MG87 fibroblasts p75 decreases the autophosphorylation of TrkB when induced by BDNF or NT-4/5 whereas p75 has no influence on NT-3-induced TrkB or TrkC activation. The collaboration of p75 with BDNF- or NT-4/5-induced TrkB and TrkC is, therefore, the opposite of what has been reported with NGF-induced TrkA. For example, coexpression of p75 with TrkA in a sympathoadrenal cell line (MAH) led to an 8-fold increase in tyrosine autophosphorylation of TrkA (35Verdi J.M. Anderson D.J. Neuron. 1994; 13: 1359-1372Abstract Full Text PDF PubMed Scopus (116) Google Scholar). Consistent with this result is the blocking of NGF binding to p75 in rat primary sympathetic neurons (54Lachance C. Belliveau D.J. Barker P.A. Neuroscience. 1997; 81: 861-871Crossref PubMed Scopus (27) Google Scholar) and in PC12 cells caused a reduced TrkA phosphorylation and also a decrease in the induction of the expression level of an immediate early gene, c-fos (36Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). Additionally, BDNF binding to p75 in PC12 cells was shown to result in a reduced TrkA signaling (55MacPhee I.J. Barker P.A. J. Biol. Chem. 1997; 272: 23547-23551Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In contrast, transient expression of p75 in A293 cells stably transfected with TrkB has no effect on TrkB phosphorylation induced by BDNF, whereas NT-3 and NT-4/5 caused reduced TrkB phosphorylation (46Bibel M. Hoppe E. Barde Y.A. EMBO J. 1999; 18: 616-622Crossref PubMed Scopus (372) Google Scholar). Only the result with NT-4/5 agrees with the data we have obtained. Possible reasons behind these different results may be differences in the transfection status of p75 (transient versus stable) and/or in the cell lines used in the experiments. It should be noted that we were able to obtain the same results in PC12 cells that we obtained in MG87 cells, i.e. suppression of BDNF and NT-4/5-induced activation of TrkB by p75 and, as a control, p75 enhanced activation of TrkA by NGF at lower concentrations. In this MG87 fibroblast cell line NT-3 was the dominant ligand for TrkC and p75 had no effect on the activation of TrkC by NT-3. What was noticeable about the introduction of p75 into TrkC expressing cell line was a relaxation in the absolute specificity of TrkC for NT-3. In the presence of p75 both BDNF and NT-4/5 activated TrkC to a slight extent. The possibility of TrkB contamination in this cell line was excluded by Western blotting using a TrkB specific antibody (TRB (48Weskamp G. Reichardt L.F. Neuron. 1991; 6: 649-663Abstract Full Text PDF PubMed Scopus (248) Google Scholar), data not shown). There are at least two general mechanisms whereby the two neurotrophin receptors can collaborate at the level of the receptors. In the first, p75 acts to bind and pass on a neurotrophin such as NGF to TrkA. If the transfer reaction is as rapid as the initial binding to p75 then the relatively slow on rate of NGF binding to TrkA is converted to a more rapid on rate, increasing the affinity of the p75·TrkA complex for NGF (34Mahadeo D. Kaplan L. Chao M.V. Hempstead B.L. J. Biol. Chem. 1994; 269: 6884-6891Abstract Full Text PDF PubMed Google Scholar, 36Barker P.A. Shooter E.M. Neuron. 1994; 13: 203-215Abstract Full Text PDF PubMed Scopus (369) Google Scholar). The existence of a high molar ratio of p75 to TrkA argues in favor of this mechanism. In the second, changes in conformation in one or both receptors are thought to regulate the affinity of neurotrophin binding. A model based on the allosteric properties of the p75 receptor has been described in detail by Bothwell (56Bothwell M. Annu. Rev. Neurosci. 1995; 18: 223-253Crossref PubMed Scopus (765) Google Scholar). The finding that p75 interacts directly with a Trk (46Bibel M. Hoppe E. Barde Y.A. EMBO J. 1999; 18: 616-622Crossref PubMed Scopus (372) Google Scholar) suggests that the second model may be correct. Binding of a neurotrophin to p75 can now be thought of as directly affecting the conformation and, therefore, the affinity of a Trk receptor and this type of regulation could work to increase or decrease Trk affinity. Alternatively, or in addition, p75 and the Trks could indirectly interact to modify Trk phosphorylation through their signaling cascade. For example, elevation of ceramide, one of the signaling molecules activated by p75 (43Dobrowsky R.T. Werner M.H. Castellino A.M. Chao M.V. Hannun Y.A. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (548) Google Scholar), has been shown to locally inhibit sympathetic axonal growth (57de Chaves E.I.P. Bussiere M. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 3028-3035Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) while McPhee and Barker (55MacPhee I.J. Barker P.A. J. Biol. Chem. 1997; 272: 23547-23551Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) have identified a ceramide activated serine-threonine kinase that could potentially down-regulate TrkA-mediated growth signals through TrkA phosphorylation. Interestingly, truncated p75 lacking most, but not all, of the intracellular domain of the receptor still has the ability to enhance both TrkA and TrkB phosphorylation in the MG87 cell line that expresses NGF and BDNF (44Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. Neuron. 1994; 13: 187-201Abstract Full Text PDF PubMed Scopus (261) Google Scholar). These results suggest that changes in receptor-binding site conformation that occur in the absence of the intracellular domain may be of primary importance. The truncated p75 had no effect on TrkC phosphorylation in the absence of exogenously added NT-3. Since these cells do not express endogenous NT-3, it is possible that the autocrine expression of a neurotrophin (NGF and BDNF in this instance) can affect p75/Trk interaction. The inability of p75 to affect exogenous NT-3 binding to TrkB and TrkC could arise for at least two reasons. First, NT-3 binding to TrkB, TrkC, and/or p75 causes no change in the conformation of the complex. If this is correct then TrkB and TrkC behave differently in this regard than TrkA. This difference has been noted before. There are differences in the neurotrophin-binding domains of the Trks and these differences distinguish TrkA from TrkB and TrkC but not the latter two from each other (58Ultsch M.H. Wiesmann C. Simmons L.C. Henrich J. Yang M. Reilly D. Bass S.H. de Vos A.M. J. Mol. Biol. 1999; 290: 149-159Crossref PubMed Scopus (147) Google Scholar). Furthermore, Canossa et al. (59Canossa M. Twiss J.L. Verity A.N. Shooter E.M. EMBO J. 1996; 15: 3369-3376Crossref PubMed Scopus (48) Google Scholar) found that TrkB could transphosphorylate TrkC and vice versa, in COS cells but that TrkA did not take part in this phenomenon. Perhaps the failure of p75 to modify NT-3 binding to TrkB and TrkC fit into the same pattern. The other explanation for the lack of effect of p75 on NT-3-induced TrkB or TrkC phosphorylation is that there is no interaction between the signaling mechanisms of the receptors. Important aspects of the p75/Trk interaction are their physiological implications. The ability of p75 to enhance TrkA activation demonstrated in a number of in vitro experiments as described above is reflected in the properties of sensory neurons from p75 knock-out mice. There neurons show a reduced sensitivity to low concentrations of NGF compared with wild type mice (60Lee K.F. Davies A.M. Jaenisch R. Development. 1994; 120: 1027-1033Crossref PubMed Google Scholar). It appears, therefore, that these neurons rely on the enhanced activation of TrkA by p75 to respond to the low levels of NGF present at certain stages of development. Although the binding of BDNF to p75 has been shown, for example, to inhibit sympathetic axonal growth in the pineal gland, this phenomenon is mediated directly through p75 signaling (61Kohn J. Aloyz R.S. Toma J.G. Haak-Frendscho M. Miller F.D. J. Neurosci. 1999; 19: 5393-5408Crossref PubMed Google Scholar, 62Yamashita T. Tucker K.L. Barde Y-A. Neuron. 1999; 24: 585-593Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar) rather than by inhibition of TrkA signaling. As far as we are aware, potential systems in which the physiological consequences of the inhibition of TrkB and TrkC signaling through p75 have not been studied. As one attempt to determine if the reduced activation of TrkB and TrkC are due to changes in the signaling components of the receptors we analyzed the phosphorylation status of ERK2, one of the commonest signaling intermediates, after neurotrophin treatment. We found that its phosphorylation status was not affected by blocking neurotrophin binding to p75. Lachange et al. (54Lachance C. Belliveau D.J. Barker P.A. Neuroscience. 1997; 81: 861-871Crossref PubMed Scopus (27) Google Scholar) also noted, in results consistent with these observations, that p75 reduced TrkA phosphorylation but had no effect on NGF-mediated survival in sympathetic neurons. The phosphorylation of tyrosine 490 (Tyr490) in TrkA activates the Ras/MAPK pathways by forming a recognition site for the Shc adaptor protein (20Obermeier A. Lammers R. Wiesmuller K.H. Jung G. Schlessinger J. Ullrich A. J. Biol. Chem. 1993; 268: 22963Abstract Full Text PDF PubMed Google Scholar, 22Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). In addition to this, the binding of PLCγ to the activated TrkA at Tyr785 is sufficient to induce ERK activity in response to NGF (22Stephens R.M. Loeb D.M. Copeland T.D. Pawson T. Greene L.A. Kaplan D.R. Neuron. 1994; 12: 691-705Abstract Full Text PDF PubMed Scopus (471) Google Scholar). Our observation that the ERK2 phosphorylation level is similar despite the blocking of the neurotrophin binding to p75 suggests that, at least, Shc or PLCγ is activated in response to BDNF and NT-4/5 even though the total tyrosine phosphorylation of TrkB is reduced by p75. Most likely, the reduced TrkB tyrosine phosphorylation does not influence the Ras/MAPK signaling pathway but uses another cascade in the signal transduction. Also the invariable phosphorylation status of TrkB, TrkC, and ERK2 after NT-3 treatment suggest that under these circumstances either p75 does not play a role in NT-3-mediated Trk activation or that its influence is mediated by a Trk tyrosine phosphorylation independent pathway. It appears that the role of p75 in neurotrophin activation is far more complex than was initially assumed. The presence of p75 can act in a positive (binding of NGF to TrkA) or negative (binding of BDNF or NT-4/5 to TrkB) fashion or have no effect at all (binding of NT-3 to TrkB or TrkC) in Trk mediated activation. How p75 affects the downstream signaling from the Trks is part of the puzzle that is actively under investigation. We thank Dr. Jose M. Cosgaya for critical reading of the manuscript. We also thank Dr. George Yancopoulos from Regeneron Pharmaceuticals, Inc. for supplying purified NT-3, NT-4/5, and BDNF, and Dr. Louis F. Reichardt (University of California, San Francisco) for providing REX and RTB antibodies." @default.
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• W2080306074 title "p75 Reduces TrkB Tyrosine Autophosphorylation in Response to Brain-derived Neurotrophic Factor and Neurotrophin 4/5" @default.
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