The extracellular domain of p75NTR is necessary to inhibit neurotrophin-3 signaling through TrkA

The TrkA receptor is activated primarily by nerve growth factor (NGF), but it can also be activated by high concentrations of neurotrophin 3 (NT-3). The pan-neurotrophin receptor p75(NTR) strongly inhibits activation of TrkA by NT-3 but not by NGF. To examine the role of p75(NTR) in regulating the specificity of TrkA signaling, we expressed both receptors in Xenopus oocytes. Application of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of (45)Ca(2+) by a phospholipase C-gamma-dependent pathway. Coexpression of p75(NTR) with TrkA inhibited (45)Ca(2+) efflux in response to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA increased with increasing expression of p75(NTR). Coexpression of a truncated p75(NTR) receptor lacking all but the first 9 amino acids of the cytoplasmic domain inhibited NT-3 stimulation of (45)Ca(2+) efflux, whereas coexpression of an epidermal growth factor receptor/p75(NTR) chimera (extracellular domain of epidermal growth factor receptor with transmembrane and cytoplasmic domains of p75(NTR)) did not inhibit NT-3 signaling through TrkA. These studies demonstrated that the extracellular domain of p75(NTR) was necessary to inhibit NT-3 signaling through TrkA. Remarkably, p75(NTR) binding to NT-3 was not required to prevent signaling through TrkA, since occupying p75(NTR) with brain-derived neurotrophic factor or anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate (45)Ca(2+) efflux. These data suggested a physical association between TrkA and p75(NTR). Documenting this physical interaction, we showed that p75(NTR) and TrkA could be coimmunoprecipitated from Xenopus oocytes. Our results suggest that the interaction of these two receptors on the cell surface mediated the inhibition of NT-3-activated signaling through TrkA.     

Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells

Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.     

Induction method of tyrosine kinase A-mediated cell death in rat pheochromocytoma

Nerve Growth Factor (NGF) is a neurotrophic factor that prevents apoptosis in neuronal progenitor cells. In rat pheochromocytoma (PC12) cells, tyrosine kinase A receptor (TrkA) mediates neurotrophic or protective effects, while p75 neurotrophin receptor (p75NTR) functions as a death receptor. We have determined whether TrkA mediates any cytotoxic effect. Following serum deprivation, TrkA expression increased 2.2-fold and apoptosis began with expression of Bax proapoptotic protein. Application of NGF halved cell viability but this was reversed by K252a, the TrkA inhibitor. These results confirmed the paradoxical cytotoxic effect of neurotrophic NGF via TrkA in PC12 cells following serum deprivation.     

Nerve growth factor signaling in caveolae-like domains at the plasma membrane

Nerve growth factor (NGF) binding to its receptors TrkA and p75(NTR) enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75(NTR), we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75(NTR) were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75(NTR) was considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCgamma. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75(NTR) signaling are initiated from these membranes.     

Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.     

Proteomic analysis of SP600125‐controlled TrkA‐dependent targets in SK‐N‐MC neuroblastoma cells: Inhibition of TrkA activity by SP600125

The c‐Jun N‐terminal kinase (JNK) is well known to play an important role in cell death signaling of the p75 neurotrophin receptor. However, little has been studied about a role of JNK in the signaling pathways of the tropomyosin‐related kinase A (TrkA) neurotrophin receptor. In this study, we investigated JNK inhibitor SP600125‐controlled TrkA‐dependent targets by proteomic analysis to better understand an involvement of JNK in TrkA‐mediated signaling pathways. PDQuest image analysis and protein identification results showed that hnRNP C1/C2, α‐tubulin, β‐tubulin homolog, actin homolog, and eIF‐5A‐1 protein spots were upregulated by ectopic expression of TrkA, whereas α‐enolase, peroxiredoxin‐6, PROS‐27, HSP70, PP1‐gamma, and PDH E1‐alpha were downregulated by TrkA, and these TrkA‐dependent upregulation and downregulation were significantly suppressed by SP600125. Notably, TrkA largely affected certain PTM(s) but not total protein amounts of the SP600125‐controlled TrkA‐dependent targets. Moreover, SP600125 strongly suppressed TrkA‐mediated tyrosine phosphorylation signaling pathways as well as JNK signaling, indicating that SP600125 could function as a TrkA inhibitor. Taken together, our results suggest that TrkA could play an important role in the cytoskeleton, cell death, cellular processing, and glucose metabolism through activation or inactivation of the SP600125‐controlled TrkA‐dependent targets.     

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA.     

eIF4A1与TrkA相互作用后抑制TrkA的泛素化

神经生长因子（NGF）结合细胞表面受体p75NTR(p75神经营养素受体)和TrkA（酪氨酸蛋白激酶A）后介导了细胞分化、细胞生存、凋亡、增殖和侵袭等多个重要的生理病理过程. TrKA能与细胞内多个蛋白质相互作用,但是由于NGF信号通路的复杂性,现在仍有必要发现与之相互作用的蛋白质以更准确地了解NGF信号通路. 本研究中我们通过酵母双杂交的方法筛选到了一个新的与TrKA相互作用的蛋白质——真核生物翻译起始因子4A1（eIF4A1）,然后通过谷胱甘肽巯基转移酶融合蛋白沉降实验（GST-pull-down）和免疫共沉淀实验（Co-IP）证明了TrkA和eIF4A1的相互作用. 此外NGF能够增强TrkA和eIF4A1的相互作用. 在鉴定相互作用位点实验中,我们发现eIF4A1的氨基端结构域和TrkA的TK结构域参与了相互作用. TrkA和eIF4A1共定位在细胞膜上. NGF能够引起TrkA与泛素蛋白63位的赖氨酸连接,而eIF4A1与TrkA相互作用后能够抑制TrkA与泛素蛋白63位的赖氨酸连接. 综上,得出结论 eIF4A1通过与TrkA相互作用抑制其泛素化调控NGF信号通路.     

NGF causes TrkA to specifically attract microtubules to lipid rafts

Membrane protein sorting is mediated by interactions between proteins and lipids. One mechanism that contributes to sorting involves patches of lipids, termed lipid rafts, which are different from their surroundings in lipid and protein composition. Although the nerve growth factor (NGF) receptors, TrkA and p75(NTR) collaborate with each other at the plasma membrane to bind NGF, these two receptors are endocytosed separately and activate different cellular responses. We hypothesized that receptor localization in membrane rafts may play a role in endocytic sorting. TrkA and p75(NTR) both reside in detergent-resistant membranes (DRMs), yet they responded differently to a variety of conditions. The ganglioside, GM1, caused increased association of NGF, TrkA, and microtubules with DRMs, but a decrease in p75(NTR). When microtubules were induced to polymerize and attach to DRMs by in vitro reactions, TrkA, but not p75(NTR), was bound to microtubules in DRMs and in a detergent-resistant endosomal fraction. NGF enhanced the interaction between TrkA and microtubules in DRMs, yet tyrosine phosphorylated TrkA was entirely absent in DRMs under conditions where activated TrkA was detected in detergent-sensitive membranes and endosomes. These data indicate that TrkA and p75(NTR) partition into membrane rafts by different mechanisms, and that the fraction of TrkA that associates with DRMs is internalized but does not directly form signaling endosomes. Rather, by attracting microtubules to lipid rafts, TrkA may mediate other processes such as axon guidance.     

GIPC and GAIP form a complex with TrkA: a putative link between G protein and receptor tyrosine kinase pathways

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.     

Functional Expression of Full-Length TrkA in the Prokaryotic Host Magnetospirillum magneticum AMB-1 by Using a Magnetosome Display System

Tropomyosin receptor kinase A (TrkA), a receptor tyrosine kinase, is known to be associated with various diseases. Thus, TrkA has become a major drug-screening target for these diseases. Despite the fact that the production of recombinant proteins by prokaryotic hosts has advantages, such as fast growth and ease of genetic engineering, the efficient production of functional receptor tyrosine kinase by prokaryotic hosts remains a major experimental challenge. Here, we report the functional expression of full-length TrkA on magnetosomes in Magnetospirillum magneticum AMB-1 by using a magnetosome display system. TrkA was fused with the magnetosome-localized protein Mms13 and expressed on magnetosome surfaces. Recombinant TrkA showed both nerve growth factor (NGF)-binding and autophosphorylation activities. TrkA expressed on magnetosomes has the potential to be used, not only for further functional analysis of TrkA, but also for ligand screening.     

Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.     

TrkA induces apoptosis of neuroblastoma cells and does so via a p53-dependent mechanism

Neuroblastoma (NB) is the most frequent solid extracranial tumor in children. Its clinical prognosis correlates with the expression of members of the Trk neurotrophin receptor family, which includes TrkA and TrkB. TrkA expression is associated with favorable prognosis, whereas TrkB expression is associated with poor prognosis. Here we show that TrkA expression induces the apoptosis of NB cells and does so by modulating the levels or activities of a number of proteins involved in regulating cell survival and apoptosis, including p53, Bcl-2, and caspase-3. TrkA increased the expression of p53 target proteins and failed to induce apoptosis in cells where p53 was inactivated by mutation or via expression of dominant inhibitory p53 or E1B55K, indicating that TrkA mediates apoptosis, at least in part, through p53. Treatment with a caspase inhibitor or overexpression of Bcl-X(L) also prevented TrkA from inducing apoptosis. In contrast, elevated expression of TrkA in non-transformed sympathetic neurons resulted in the suppression of p53 levels and enhanced survival. These results identify apoptosis as a novel biological response of TrkA in NB cells and imply that TrkA is a good prognosis marker for NB due in part to its ability to mediate apoptosis when expressed at sufficient levels.     

Nerve Growth Factor Receptor TrkA,a New Receptor in Insulin Signaling Pathway in PC12 Cells

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.     

Syntaxin 8 Modulates the Post-synthetic Trafficking of the TrkA Receptor and Inflammatory Pain Transmission

Nerve growth factor (NGF) promotes the survival, maintenance, and neurite outgrowth of sensory and sympathetic neurons, and the effects are mediated by TrkA receptor signaling. Thus, the cell surface location of the TrkA receptor is crucial for NGF-mediated functions. However, the regulatory mechanism underlying TrkA cell surface levels remains incompletely understood. In this study, we identified syntaxin 8 (STX8), a Q-SNARE protein, as a novel TrkA-binding protein. Overexpression and knockdown studies showed that STX8 facilitates TrkA transport from the Golgi to the plasma membrane and regulates the surface levels of TrkA but not TrkB receptors. Furthermore, STX8 modulates downstream NGF-induced TrkA signaling and, consequently, the survival of NGF-dependent dorsal root ganglia neurons. Finally, knockdown of STX8 in rat dorsal root ganglia by recombinant adeno-associated virus serotype 6-mediated RNA interference led to analgesic effects on formalin-induced inflammatory pain. These findings demonstrate that STX8 is a modulator of TrkA cell surface levels and biological functions.     

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.     

Mitochondrial functional interactions between frataxin and Isu1p, the iron-sulfur cluster scaffold protein, in Saccharomyces cerevisiae

Here we investigated a biological association of constitutively active Src with TrkA in SK-N-MC human neuroblastoma cells. Activation of TrkA and extracellular signal-regulated kinase (ERK) by nerve growth factor (NGF) was inhibited by pretreatment with PP2, an inhibitor of Src family kinases. Moreover, NGF-induced phosphorylation of TrkA and ERK was also attenuated by the transfection with a dominant-negative src construct. On the other hand, the transfection with a constitutively active src construct enhanced these phosphorylations. In addition, we showed that active Src phosphorylates TrkA directly in vitro, and that Src associates with TrkA through Grb2 after NGF stimulation. These results suggest that constitutively active Src that associates with TrkA through Grb2 after NGF stimulation participates in TrkA phosphorylation and in turn enhances the mitogen-activated protein kinase signaling in SK-N-MC cells.