Cardiovascular effects of nerve growth factor analytical review. Part 2

In the second part of the review discusses the features of the influence ofNGFon the functional activity of the cardiovascular system, as well as signaling pathways by which activated NGF TrkA and p75(ntr) receptors regulate the functional state of endothelial and vascular smooth muscle cells and cardiomyocytes. In addition, the review observes the theoretical perspectives of agonists and antagonists of TrkA and p75(ntr) receptors for the treatment of various diseases of the heart and blood vessels.     

Cardiovascular effects of the nerve growth factor: An analytical review. Part I: NGF-mediated intracellular signaling pathways

Traditionally, the nerve growth factor (NGF) is considered to be a chemoattractant participating in the regulation of cell proliferation, differentiation, and neuron myelination. However, the currently available data suggest that the physiological role of NGF in the body is much wider. The features of NGF influence on the functional activity of the cardiovascular system, signaling pathways by which activated NGF TrkA and p75^ntr receptors regulate the functional state of endothelial and vascular smooth muscle cells and cardiomyocytes are discussed. In addition, the theoretical prospects of agonists and antagonists of TrkA and p75ntr receptors for the treatment of heart and vascular disorders are considered.     

Structural model for p75(NTR)-TrkA intracellular domain interaction: a combined FRET and bioinformatics study

Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75(NTR) and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75(NTR) complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by F?rster resonance energy transfer that p75(NTR) and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75(NTR), we modeled the heterodimerization of TrkA and p75(NTR) by docking methods and molecular dynamics. These models, together with the results obtained by F?rster resonance energy transfer, provide structural insights into the receptors' physical association.     

Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation

The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75–TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.     

Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF) receptors, preventing neuronal apoptosis

The neurosteroid dehydroepiandrosterone (DHEA), produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR) membrane receptors of neurotrophin nerve growth factor (NGF), acting as a neurotrophic factor: (1) the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2) [(3)H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR) receptors (K(D): 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively); (3) immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR) receptors; (4) DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR) receptors; and (5) DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.     

Characterization of symmetric complexes of nerve growth factor and the ectodomain of the pan-neurotrophin receptor, p75NTR

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.     

Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors

Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.     

NGF activates the phosphorylation of MAP1B by GSK3beta through the TrkA receptor and not the p75(NTR) receptor

We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75NTR but not TrkA receptors, does not stimulate GSK3beta phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3beta phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75NTR and also fail to show NGF-dependent GSK3beta phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3beta phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3beta phosphorylation of MAP1B. These results suggest that the activation of GSK3beta by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75NTR receptors.     

Structural Model for p75–TrkA Intracellular Domain Interaction: A Combined FRET and Bioinformatics Study

Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75^NTR and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75^NTR complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by Förster resonance energy transfer that p75^NTR and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75^NTR, we modeled the heterodimerization of TrkA and p75^NTR by docking methods and molecular dynamics. These models, together with the results obtained by Förster resonance energy transfer, provide structural insights into the receptors' physical association.     

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.     

神经生长因子及其受体相关信号传导通路的研究进展

神经生长因子是神经营养因子家族成员之一,对不同时期神经元的存活、分化、生长及损伤后的修复和再生都有着十分重要的作用。不仅在神经系统中,随着人类的其他正常和肿瘤组织中同样也检测得到了NGF,神经生长因子在各方面的应用也得到了重视并均已得到了证实。NGF功能的发挥离不开与其受体的结合,根据NGF表面糖蛋白与凝集素结合能力的不同,其受体可被分为高亲和力受体酪氨酸激酶A和低亲和力受体p75。Trk A与NGF结合后所介导的信号通路主要有:1MAPK通路;2PLC-γ通路;3PI3K/PKB通路。而p75与NGF结合介导的信号传导通路主要包括:1NF-κB通路;2JNK-p53-Bax凋亡通路;3神经酰胺通路。Trk A一般介导的是正性信号,如促进神经细胞生长、维持神经细胞的存活等;而p75既可促进神经细胞存活,也可诱导神经细胞凋亡,但以后者为主。当Trk A与p75同时表达时,Trk A可抑制p75诱导细胞凋亡,使受损神经细胞大量增殖,所以其生物学总效应是促进神经细胞的生长和存活。     

Changes in the expression of the nerve growth factor receptors TrkA and p75LNGR in the rat thymus with ageing and increased nerve growth factor plasma levels

The nerve growth factor (NGF) receptors p75LNGR and TrkA are expressed by thymic epithelial cells. Presumably, the NGF-TrkA system is involved in the paracrine communication between thymic epithelial cells and thymocytes, whereas the functional role of p75LNGR is still unknown. The thymus of vertebrates undergoes age-related changes that in part depend on hormonal factors. In order to find out whether thymic epithelial cells are responsive to NGF during the whole lifespan of the rat, we studied NGF receptor expression in the thymus from birth to 2 years of age, using immunohistochemistry. Furthermore, to evaluate whether increased plasma levels of NGF affected the ageing process, either NGF or 4-methylcatechol (4MC), an inductor of NGF synthesis, was administered. Both TrkA and p75LNGR were expressed by a subpopulation of thymic epithelial cells during the whole age range studied and their expression peaked at around 3 months. TrkA was primarily found in subcortical and medullary epithelial cells, whereas p75LNGR was seen in a subpopulation of medullary cells. Cortical epithelial cells, neural crest-derived cells, other stromal cells and thymocytes were not immunoreactive for NGF receptors. Neither the administration of NGF nor the increased NGF plasma levels obtained after 4MC treatment seemed to affect the ageing of the thymus as assessed by morphological and immunohistochemical criteria, but this increase in NGF levels did produce a shift in the expression of p75LNGR from epithelial cells to ED1-positive macrophages in animals of 6 months and older. Present results indicate that the expression of p75LNGR and TrkA in the rat thymus undergoes age-dependent changes that parallel those of epithelial cells. NGF could therefore be important for thymus homeostasis, possibly acting on epithelial cells. Nevertheless, NGF did not seem to be able to prevent the involution of this organ, although it produced a switch in the expression of p75LNGR, the significance of which remains to be established.     

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.     

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.     

p75 and TrkA Receptor Signaling Independently Regulate Amyloid Precursor Protein mRNA Expression, Isoform Composition, and Protein Secretion in PC12 Cells

Abstract: The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75^NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75^NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75^NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75^NTR and TrkA receptors, binding of NGF to the p75^NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75^NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors—in contrast to p75^NTR-associated signaling—stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.     

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75(NTR). During retinal injury, endogenous NGF, TrkA, and p75(NTR) are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75(NTR) affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75(NTR) agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75(NTR) action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α(2)-macroglobulin. Antagonists of p75(NTR) inhibit TNF-α and α(2)-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.     

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.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.