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。     

The rabies virus glycoprotein receptor p75NTR is not essential for rabies virus infection

Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75^NTR), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75^NTR in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75^NTR positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75^NTR-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75^NTR is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75^{NTRExonIV−/−} mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75^NTR binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75^NTR interaction. We conclude that although p75^NTR is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75^NTR interaction is not necessary for RV infection of primary neurons. This means that other receptors are required to mediate RV infection in vivo and in vitro.     

The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors —trkA binds only NGF, the relatedtrkB receptor binds BDNF and NT-3, andtrkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to thetrk gene products.     

The p75 neurotrophin receptor

The low-affinity p75 molecule and trk tyrosine kinases serve as receptors for target-derived neurotrophins. While the mechanism by which receptor tyrosine kinases impart intracellular signaling has become well understood, the precise roles of the p75 receptor are not fully defined. The p75 neurotrophin receptor belongs to a family of transmembrane molecules which also serve as receptors for the tumor necrosis factor family of cytokines. Each receptor shares a common extracellular structure highlighted by conserved cysteine-rich repeats. Because NGF, BDNF, NT-3, and NT-4/5 bind to p75 with similar affinity, p75 may either act as a common subunit in a neurotrophin receptor complex with trk family members, or act by independent mechanisms to mediate biological actions of each neurotrophin. 1994 John Wiley & Sons, Inc.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

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.     

Differential Involvement of Metabotropic and p75 Neurotrophin Receptors in Effects of Nerve Growth Factor and Neurotrophin-3 on Cultured Purkinje Cell Survival

Abstract: We have examined the role of the p75 neurotrophin receptor in survival-promoting effects of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT-3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival-promoting effect of wild-type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT-3 was potentiated by anti-p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF-ACPD and a negative modulatory role of p75 in the action of NT-3. To probe the role of ACPD in the p75-dependent response to NGF, metabotropic receptor subtype-specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down-regulation or blockade of PKC abolished the response to NGF-ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT-3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75-mediated action of NGF. NT-3 acts on Purkinje cells by a different mechanism that is not absolutely p75-dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.     

Effects of castration on the immunoreactivity to NGF, BDNF and their receptors in the pelvic ganglia of the male rat

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and are members of the neurotrophin family, a family of neurotrophic factors that also includes neurotrophin (NT) 3 and NT4/5. Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems. Neurotrophins exert their effects by binding to corresponding receptors which are formed by the tyrosine protein kinases TrkA, TrkB and TrkC, and the low affinity neurotrophic receptor (p75NTR). In the present study, using immunohistochemistry and quantitative analysis, we have investigated immunoreactivity to BDNF, NGF, TrkB, p75NTR and TrkA in the pelvic ganglia of normal and castrated rats. Neurons of the pelvic ganglia expressed both these neurotrophins and their receptors. After castration the immunoreactivity persisted. However, the number of BDNF- and p75NTR-IR cells statistically significant decreased after castration. These results suggest that castration modulates the expression of neurotrophins and their receptors in pelvic autonomic neurons.     

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.     

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.     

The binding epitopes of neurotrophin-3 to its receptors trkC and gp75 and the design of a multifunctional human neurotrophin.

Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin-3 (NT-3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta-strand bundle and in contrast to NGF does not make use of non-conserved loops and the six N-terminal residues. The gp75 epitope is dominated by loop residues and the C-terminus of NT-3. A novel rapid biological screening procedure allowed the identification of NT-3 mutants that are able to signal efficiently through the non-preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT-3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF-, BDNF- and NT-3-dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.     

Expression and characterization of a variant of TACI (CRD2-shortTACIFc) in Pichia pastoris

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. The extracellular domain of a typical TNFR contains multiple copies of CRD, which bind in the monomermonomer interfaces of a trimeric ligand. TACI binds to two ligands, APRIL and BAFF, with high affinity and contains two CRD in its extracellular regions, while BCMA and BR3, contain a single or partial CRD for binding the two ligands. However, TACI can be classified as a single CRD receptor because the amino-terminal CRD1 doesn't contribute to ligand binding. To obtain a new variant of TACI possessing higher affinities for binding, we fused a repeat sequence of CRD2 to the N-terminus of the short form of TACI. The new APRIL antagonist peptide, CRD2-shortTACI-Fc, was designed based on the modeling 3-D complex structure of TACI and APRIL. As expected, the purified recombinant CRD2-shortTACI-Fc fusion protein could bind to APRIL in vitro and demonstrated dose-dependent inhibition of APRIL-induced proliferative activity in Raji cells. We found that CRD2-shortTACI-Fc, has a higher affinity for binding to ligands than short-TACI-Fc, which contains a single CRD2.     

Structure-based analysis of the herpes simplex virus glycoprotein D binding site present on herpesvirus entry mediator HveA (HVEM)

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.     

TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.     

Low-affinity nerve-growth factor receptor (P75NTR) can serve as a receptor for rabies virus.

A random-primed cDNA expression library constructed from the mRNA of neuroblastoma cells (NG108) was used to clone a specific rabies virus (RV) receptor. A soluble form of the RV glycoprotein (Gs) was utilized as a ligand to detect positive cells. We identified the murine low-affinity nerve-growth factor receptor, p75NTR. BSR cells stably expressing p75NTR were able to bind Gs and G-expressing lepidopteran cells. The ability of the RV glycoprotein to bind p75NTR was dependent on the presence of a lysine and arginine in positions 330 and 333 respectively of antigenic site III, which is known to control virus penetration into motor and sensory neurons of adult mice. P75NTR-expressing BSR cells were permissive for a non-adapted fox RV isolate (street virus) and nerve growth factor (NGF) decreased this infection. In infected cells, p75NTR associates with the RV glycoprotein and could be precipitated with anti-G monoclonal antibodies. Therefore, p75NTR is a receptor for street RV.     

Functional analysis of mutant neurotrophins deficient in low-affinity binding reveals a role for p75LNGFR in NT-4 signalling.

The neurotrophins mediate their effects through binding to two classes of receptors, a tyrosine kinase receptor, member of the Trk family, and the low-affinity neurotrophin receptor, p75LNGFR, of as yet undefined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-directed mutagenesis, we have identified positively charged surfaces in BDNF, NT-3 and NT-4 that mediate binding to p75LNGFR. Arg31 and His33 in NT-3, and Arg34 and Arg36 in NT-4, located in an exposed hairpin loop, were found to be essential for binding to p75LNGFR. In BDNF, however, positively charged residues critical for p75LNGFR binding (Lys95, Lys96 and Arg97) were found in a spatially close but distinct loop region. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials revealed similar clusters of positively charged residues in each neurotrophin but with differences in their precise spatial locations. Disruption of this positively charged interface abolished binding to p75LNGFR but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity binding in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75LNGFR and TrkB, suggesting a role for p75LNGFR in regulating biological responsiveness to NT-4. These findings reveal a possible mechanism of ligand discrimination by p75LNGFR and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family members.     

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

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.     

Dorsal root ganglion neurons expressing trk are selectively sensitive to NGF deprivation in utero.

In utero immune deprivation of the neurotrophic molecule nerve growth factor (NGF) results in the death of most, but not all, mammalian dorsal root ganglion (DRG) neurons. The recent identification of trk, trkB, and trkC as the putative high affinity receptors for NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively, has allowed an examination of whether their expression by DRG neurons correlates with differential sensitivity to immune deprivation of NGF. In situ hybridization demonstrates that virtually all neurons expressing trk are lost during in utero NGF deprivation. Most, if not all, neurons expressing trkB and trkC survive this treatment. In contrast, the low affinity NGF receptor, p75NGFR, is expressed in both NGF deprivation-resistant and -sensitive neurons. These experiments show that DRG neurons expressing trk require NGF for survival. Furthermore, at least some of the DRG neurons that do not require NGF express the high affinity receptor for another neurotrophin. Finally, these experiments provide evidence that trk, and not p75NGFR, is the primary effector of NGF action in vivo.     

Polyubiquitination of the neurotrophin receptor p75 directs neuronal cell survival

Specific binding of nerve growth factor (NGF) to p75 neurotrophin receptor (p75(NTR)) leads to p75(NTR) polyubiquitination and its subsequent interaction with TRAF6 resulting in neuronal cell survival. However, when the binding of NGF to p75(NTR) was blocked with p75 antiserum, p75(NTR) polyubiquitination and neuronal cell survival were impaired. Results showed that tyrosine phosphorylation of p75(NTR) increased the polyubiquitination of p75(NTR) and contributed to the observed apparent neuroprotective effects. Similar to p75(NTR) polyubiquitination, interaction of TRAF6 with p75(NTR) was NGF/tyrosine phosphorylation dependent suggesting that TRAF6 might function as an E3 ubiquitin ligase. In sum, the results show that specific binding of NGF to p75(NTR) mediates neuronal cell survival.