Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells)

Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.     

Stable Transfection of GM1 Synthase Gene into GM1-Deficient NG108-15 Cells,CR-72 Cells,Rescues the Responsiveness of TRK-Neurotrophin Receptor to Its Ligand,NGF

Previous studies from this laboratory and others have suggested the evidences that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. The present study was aimed to examine whether Trk expressed in cells that are deficient in endogenous GM1 due to the mutation of GM1 synthase gene (NG-CR72 cells) is responsive to its ligand nerve growth factor and how genetic restoration of GM1 synthase gene by a stable transfection of the gene affects the function of the Trk protein. The data clearly showed that (1) confocal lazor microscopic studies disclosed NG-CR72 cells are really deficient in GM1, (2) stable transfection of GM1 synthase cDNA into these cells (NG-CR72G cells) restores the expression of GM1 in the cells, and (3) Trk protein is expressed in NG-CR72 cells but its location seemed not to be on the plasma membrane, whereas we clearly observed that the Trk protein is expressed on the plasma membrane in NG-CR72G cells. (4) NGF did not elicit the autophosphorylation of the Trk protein in GM1 deficient NG-CR72 cells but did elicit the activation of the Trk protein in NG-CR72G cells with an activation of mitogen activated protein kinase. These studies strongly suggested that GM1 is necessary for the normal expression of the Trk protein function and for normal targeting of the Trk protein to the plasma membrane.     

TrkA glycosylation regulates receptor localization and activity

The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-gamma. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity.     

Autocrine regulation of nerve growth factor expression by Trk receptors

Activation of the neurotrophin receptor Trk induces the release of neurotrophins. However, little is known about the ability of released neurotrophins to modulate their own synthesis in an autocrine manner. As a step towards understanding the role of Trk in regulating the synthesis of neurotrophins, we exposed NIH-3T3 cells expressing TrkA or TrkC receptors to their cognate ligands as well as to GM1, a ganglioside that activates TrkA and TrkC by inducing the release of neurotrophin-3. Nerve growth factor and neurotrophin-3 synthesis were then determined by measuring the relative levels of protein and mRNA. TrkA-expressing cells exposed to human recombinant nerve growth factor exhibited higher levels of nerve growth factor mRNA. Human recombinant neurotrophin-3 evoked an increase in nerve growth factor mRNA in both TrkA and TrkC-expressing cells. GM1 elicited a time-dependent increase in nerve growth factor protein and mRNA in NIH-3T3 cells expressing TrkA or TrkC receptor but not in wild-type cells. Surprisingly, GM1 failed to change neurotrophin-3 levels. The ability of GM1 to increase nerve growth factor mRNA levels was blocked by TrkC-IgG but not by TrkB-IgG receptor body. These data suggest that released neurotrophin-3 may activate a positive autocrine loop of nerve growth factor synthesis by Trk activation.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Gangliosides activate Trk receptors by inducing the release of neurotrophins

We used NIH-3T3 fibroblasts expressing the different Trk receptors to examine whether GM1 ganglioside and its semisynthetic derivative LIGA20 activate various neurotrophin receptors. GM1 induced autophosphorylation of TrkC more potently than TrkA or TrkB receptors. In contrast, LIGA20 activated TrkB tyrosine phosphorylation only. Therefore, Scatchard analysis was performed to determine whether GM1 binds to TrkC. GM1 failed to displace neurotrophin-3 binding, suggesting that this ganglioside does not act as a ligand for Trk receptors. In addition, GM1 failed to induce autophosphorylation of a chimeric receptor consisting of the extracellular domain of the tumor necrosis factor receptor and the intracellular domain of TrkA, suggesting that GM1 does not affect the tyrosine kinase domain. We next determined whether GM1 induces the release of neurotrophins from fibroblast cells. GM1 induced a rapid and significant increase in the amount of neurotrophin-3, but not other neurotrophins. This effect was independent of the presence of Trk because K252a did not prevent GM1-mediated release of neurotrophin-3. Moreover, GM1-mediated TrkC autophosphorylation was blocked by TrkC-IgG (but not TrkB-IgG) receptor bodies, further suggesting that GM1 activates TrkC by inducing the release of neurotrophin-3. This hypothesis was also tested in cultured cerebellar granule cells. GM1 induced neurotrophin-3 (but not brain-derived neurotrophic factor or nerve growth factor) release. In contrast, LIGA20 increased the secretion of brain-derived neurotrophic factor. Our data show that gangliosides may activate different Trk receptors by differentially affecting the release of neurotrophins.     

Neurotrophin signal transduction by the Trk receptor

The initial event in the neuronal differentiation of PC12 cells is the binding of the neurotrophin nerve growth factor (NGF) to the Trk receptor. This interaction stimulates the intrinsic tyrosine kinase activity of TRk, initiating a signalling cascade involving the phosphorylation of intracellular proteins on tyrosine, serine, and threonine residues. These signals are then in turn propagated to other messengers, ultimately leading to differentiation, neurotrophin-dependent survival and the loss of proliferative capacity. To transmit NGF signals, NGF-activated Trk rapidly associated with the cytoplasmic proteins, SHC, PI-3 kinase, and PLC-γ1. These proteins are involved in stimulating the formation of various second messenger molecules and activating the Ras signal transduction pathway. Studies with Trk mutants indicate that the acivation of the Ras pathway is necessary for complete differentiation of PC12-derived cells and for the maintenance of the differentiated phenotype. Trk also induces the tyrosine phosphorylation of SNT, a specific target of neurotrophic factor activity in neuronal cells. This review will discuss the potential roles of Trk and the proteins of the Trk signalling pathways in NGF function, and summarize our attempts to understand the mechanisms used by Trk to generate dthe many phenotypic responses of PC12 cells to NGF. 1994 John Wiley & Sons, Inc.     

Human tumorous imaginal disc 1 (TID1) associates with Trk receptor tyrosine kinases and regulates neurite outgrowth in nnr5-TrkA cells

The human tumorous imaginal disc 1 (TID1) proteins including TID1(L) and TID1(S), members of the DnaJ domain protein family, are involved in multiple intracellular signaling pathways such as apoptosis induction, cell proliferation, and survival. Here we report that TID1 associates with the Trk receptor tyrosine kinases and regulates nerve growth factor (NGF)-induced neurite outgrowth in PC12-derived nnr5 cells. Binding assays and transfection studies showed that the carboxyl-terminal end of TID1 (residues 224-429) bound to Trk at the activation loop (Tyr(P)(683)-Tyr(684)(P)(684) in rat TrkA) and that TID1 was tyrosine phosphorylated by Trk both in yeast and in transfected cells. Moreover endogenous TID1 was also tyrosine phosphorylated by and co-immunoprecipitated with Trk in neurotrophin-stimulated primary rat hippocampal neurons. Overexpression studies showed that both TID1(L) and TID1(S) significantly facilitated NGF-induced neurite outgrowth in TrkA-expressing nnr5 cells possibly through a mechanism involving increased activation of mitogen-activated protein kinase. Consistently knockdown of endogenous TID1, mediated with specific short hairpin RNA, significantly reduced NGF-induced neurite growth in nnr5-TrkA cells. These data provide the first evidence that TID1 is a novel intracellular adaptor that interacts with the Trk receptor tyrosine kinases in an activity-dependent manner to facilitate Trk-dependent intracellular signaling.     

A Purine Analog-Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors

Abstract: Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and ∼50–80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.     

Nerve growth factor signaling involves interaction between the Trk A receptor and lysophosphatidate receptor 1 systems: nuclear translocation of the lysophosphatidate receptor 1 and Trk A receptors in pheochromocytoma 12 cells

We report here that the nerve growth factor (NGF) and lysophosphatidate (LPA) receptor signaling systems interact to regulate the p42/p44 MAPK pathway in PC12 cells. This is based upon several lines of evidence. First, the treatment of PC12 cells, which express LPA(1) receptors, with a sub-maximal concentration of LPA and NGF induced synergistic activation of p42/p44 MAPK. Second, the transfection of PC12 cells with LPA(1) receptor anti-sense construct, which reduced the expression of LPA(1), abrogated both LPA- and NGF-stimulated activation of p42/p44 MAPK. Third, the over-expression of recombinant LPA(1) receptor potentiated LPA- and NGF-dependent activation of p42/p44 MAPK. Fourth, the over-expression of C-terminal GRK2 peptide (which sequesters G-protein betagamma subunits) or beta-arrestin I clathrin binding domain (amino acids: 319-418) or pre-treatment of cells with pertussis toxin reduced the LPA- and NGF-dependent stimulation of p42/p44 MAPK. These findings support a model in which the Trk A receptor uses a G-protein-mediated mechanism to regulate the p42/p44 MAPK pathway. Such G-protein-mediated signaling is activated by the LPA(1) receptor as a means of cross-talk regulation with the Trk A receptor. Fifth, the treatment of cells with LPA induced the transactivation of the Trk A receptor. Sixth, LPA and/or NGF stimulated the translocation of tyrosine phosphorylated Trk A receptor and LPA(1) receptor to the nucleus. Taken together, these findings suggest that NGF and LPA exert cross-talk regulation both at the level of p42/p44 MAPK signaling and in the nuclear translocation of LPA(1) and Trk A receptors.     

Biochemical Characterization of Intracellular Membranes Bearing Trk Neurotrophin Receptors

Neurotrophin receptor trafficking plays an important role in directing cellular communication in developing as well as mature neurons. However, little is known about the requirements for intracellular localization of the neurotrophin receptors in neurons. To isolate the subcellular membrane compartments containing the Trk neurotrophin receptor, we performed biochemical subcellular fractionation experiments using primary cortical neurons and rat PC12 pheochromocytoma cells. By differential centrifugation and density gradient centrifugation, we have isolated Trk-bearing compartments, suggesting distinct membranous localization of Trk receptors. A number of Trk-interacting proteins, such as GIPC and dynein light chain Tctex-1 were found in these fractions. Additionally, membranes enriched in phosphorylated activated forms of Trk receptors were found upon ligand treatment in primary neurons and PC12 cells. Interestingly, density gradient centrifugation experiments showed that Trk receptors from PC12 cells are present in heavy membrane fractions, while Trk from primary neurons are fractionated in lighter membrane fractions. These results suggest that the intracellular membrane localization of Trk can differ according to cell type. Taken together, these biochemical approaches allowed separation of distinct Trk-bearing membrane pools, which may be involved in different functions of neurotrophin receptor signaling and trafficking.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling

Neurotrophin-induced Trk tyrosine kinase receptor activation and neuronal cell survival responses have been reported to be under the control of a membrane associated sialidase. Here, we identify an unprecedented membrane sialidase mechanism initiated by nerve growth factor (NGF) binding to TrkA to potentiate GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live primary neurons and TrkA- and TrkB-expressing cell lines. Central to this process is that Neu1/MMP-9 complex is bound to TrkA on the cell surface of naïve primary neurons and TrkA-expressing cells. Tamiflu completely blocks this sialidase activity in live TrkA-PC12 cells treated with NGF with an IC₅₀ of 3.876 μM with subsequent inhibition of Trk activation in primary neurons and neurite outgrowth in TrkA-PC12 cells. Our findings uncover a Neu1 and MMP-9 cross-talk on the cell surface that is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and cellular signaling.     

Neurotrophin and Trk receptor-like immunoreactivity in the frog gastrointestinal tract

Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the neurotrophin family, which is involved in the differentiation, growth, repair, plasticity and maintenance of many neuronal populations. They act through three tyrosin-kinase (Trk) specific receptors: NGF bind to TrkA, BDNF to TrkB and NT3 to TrkC. Despite increasing evidence regarding the presence of neurotrophin and their receptors in many vertebrate species, in amphibians there are very few data concerning them. Thus, the aim of this study was to extend the investigation to the presence of both neurotrophins and their Trk receptors in the gut of an anuran amphibian, Rana temporaria. In the frog gut NT-3- like immunoreactivity (IR) was observed in both the nervous system and endocrine cells of the stomach and intestine, while NGF-like IR was observed only in the enteric nervous system, and BDNF-like IR in the intestinal endocrine cells. TrkA- and TrkB-like IR was detected in both neurons and endocrine cells of the intestine, while TrkC-like IR was observed only in intestinal neurons. No Trk IR was detected in the stomach. The occurrence of the IR to neurotrophins and their receptors in the gut of the frog further confirms the well-conserved presence of this family of growth factors and Trk receptors during the evolution of vertebrates and suggests their complex involvement in the biology of the gastrointestinal neuro-endocrine system.     

p48 Ebp1 acts as a downstream mediator of Trk signaling in neurons, contributing neuronal differentiation

Two Ebp1 isoproteins, p48 and p42, regulate cell survival and differentiation distinctively. Here we show that p48 is the major isoform in hippocampal neurons and is localized throughout the entire neuron. Notably, reduction of p48 Ebp1 expression inhibited BDNF-mediated neurite outgrowth in hippocampal neurons. The p48 protein acts as a downstream effector of the Trk receptor, which mediates the functions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in hippocampal cells. Trk receptor activation by both NGF and BDNF induced phosphorylation of Ebp1 at the S360 upon the activation of protein kinase Cδ (PKCδ) and triggered dissociation of p48 from retinoblastoma (Rb). Although both NGF and BDNF activate mitogen-activated protein kinase (MAPK; extracellular signal-related kinase (ERK)) as well as phosphatidylinositide 3-kinase (PI3K)/Akt, their activation is regulated in different time-frame upon growth factor specificity, especially, eliciting PKCδ mediated p48 S360 phosphorylation. Thus, p48 Ebp1 contributes to neuronal cell differentiation and growth factor specificity through the activation of PKCδ, acting as a crucial downstream effector of neurotrophin signaling.     

Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors

We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have a functional impact on NGF-Trk-mediated intracellular signal transduction pathway.     

Glycosphingolipid-enriched signaling domain in mouse neuroblastoma Neuro2a cells. Mechanism of ganglioside-dependent neuritogenesis.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.     

GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.     

The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.

Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.     

Neuroprotection by NGF and BDNF Against Neurotoxin-Exerted Apoptotic Death in Neural Stem Cells Are Mediated Through Trk Receptors,Activating PI3-Kinase and MAPK Pathways

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75^NTR) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75^NTR did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors. An erratum to this article can be found at