Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent gamma-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for gamma-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that gamma-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid beta-peptide 40 (Abeta40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that gamma-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that gamma-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.     

Association between Presenilin-1 and TRAF6 modulates regulated intramembrane proteolysis of the p75NTR neurotrophin receptor

The p75 neurotrophin receptor (p75^NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners including the tumour necrosis factor receptor-associated factor 6 (TRAF6) E3 ubiquitin ligase to produce cellular responses. Recently, p75^NTR was also shown to undergo presenilin-dependent, γ-secretase-mediated regulated intramembrane proteolysis. In this study, we report the characterization of a highly conserved TRAF6-binding site (PxExxAr/Ac) in presenilin-1 (PS1) that mediates nerve growth factor (NGF)-induced association between PS1 and TRAF6. We demonstrate that disruption of this interaction between PS1 and TRAF6 inhibits TRAF6 autoubiquitination and γ-secretase cleavage of p75^NTR. Additionally, we show that PS1-deficiency antagonizes NGF-induced I-κB degradation. Finally, we also show that p75^NTR is a substrate for TRAF6-mediated ubiquitination and that TRAF6 E3 ligase activity is required for regulated intramembrane proteolysis of p75^NTR. In summary, our data suggest that an NGF-induced association between PS1 and TRAF6 influences regulated intramembrane proteolysis of p75^NTR.     

c-Cbl binds to tyrosine-phosphorylated neurotrophin receptor p75 and induces its ubiquitination

The p75 neurotrophin receptor (p75NTR) has dual functions in cell survival and cell death but its intracellular signalling pathways are not understood. Here we describe that in rat brain and in pervanadate-stimulated PCNA and HEK293 cells p75NTR is phosphorylated at a single tyrosine residue within the cytosolic C-terminus. Phosphorylated tyrosine 308 constitutes a binding site for the ubiquitin ligase c-Cbl. This interaction is a prerequisite for ubiquitination of p75NTR. Our data suggest a c-Cbl-dependent ubiquitination of p75NTR involved in the regulation of p75NTR signalling.     

The p75 neurotrophin receptor

The pan neurotrophin receptor (p75(NTR)) is best known for mediating neural cell death during development as well as in the adult following injury, the latter making it a target for the treatment of neurodegenerative disease. Although p75(NTR) has been studied for over 30 years, a number of recent discoveries have changed our understanding of its regulation. Here we provide a brief overview of the p75(NTR) protein, its post-translational modifications, and the phenotype of p75(NTR)-deficient mice as a starting point for researchers unfamiliar with this complex receptor. The accepted mechanisms underlying the ability of p75(NTR) to regulate cell death as well as a number of other neural functions, most notably neuronal differentiation, neurite outgrowth and synaptic plasticity, are also summarised.     

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.     

Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells

Background

Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Results

Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.

Conclusion

The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells.     

Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAG's receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.     

Inhibition of neurotrophin receptor p75 intramembran proteolysis by gamma-secretase inhibitor reduces medulloblastoma spinal metastasis

Medulloblastoma (MB) is the most devastating and common pediatric brain tumor. Tumor cells invading into surrounding tissue and disseminating through cerebrospinal fluid make treatment extremely difficult. Identifying the mechanisms of MB cells is therefore imperative for the development of novel treatments. A research group demonstrated recently that the multifunctional signaling protein neurotrophin receptor p75(NTR) is a central regulator for glioma invasion. γ-secretase mediated processing of the p75(NTR) is a major contributor to the highly invasive nature of malignant gliomas. In this study we examine the p75(NTR) expression and processing in medulloblastoma cells. Results show that p75(NTR) is a critical regulator of medulloblastoma spinal metastasis. γ-secretase inhibitor, which blocks p75(NTR) proteolytic processing, significantly abrogates p75(NTR) induced medulloblastoma migration and invasion in vitro and in vivo. This data suggests that p75(NTR) is also an important therapeutic target for MB. γ-secretase inhibitor may be a potentially effective clinical application for the treatment of medulloblastoma spinal metastasis.     

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.     

Neurite outgrowth is enhanced by laminin-mediated down-regulation of the low affinity neurotrophin receptor, p75NTR

Laminin (LN), an extracellular matrix component, is a key factor in promoting axonal regeneration, coordinately regulating growth in conjunction with trophic signals provided by the neurotrophins, including nerve growth factor (NGF). This study investigated potential interactions between the LN and NGF-mediated signaling pathways in PC12 cells and primary neurons. Neurite outgrowth stimulated by NGF was enhanced on a LN substrate. Western blot analysis of pertinent signal transduction components revealed both enhanced phosphorylation of early signaling intermediates upon co-stimulation, and a LN-induced down-regulation of p75NTR which could be prevented by the addition of integrin inhibitory arginine-glycine-aspartate (RGD) peptides. This p75NTR down-regulation was associated with a LN-mediated up-regulation of PTEN and resulted in a decrease in Rho activity. Studies using over-expression or siRNA-mediated knock-down of PTEN demonstrate a consistent inverse relationship with p75NTR, and the over-expression of p75NTR impaired neurite outgrowth on a LN substrate, as well as resulting in sustained activation of Rho which is inhibitory to neurite outgrowth. p75NTR is documented for its role in the transduction of inhibitory myelin-derived signals, and our results point to extracellular matrix regulation of p75NTR as a potential mechanism to ameliorate inhibitory signaling leading to optimized neurite outgrowth.     

p75受体信号转导途径的研究进展

神经生长因子 (ｎｅｒｖｅｇｒｏｗｔｈｆａｃｔｏｒ,ＮＧＦ)通过与两种受体结合而发挥作用。一种是高亲和性受体ＴｒｋＡ受体 ,它是由原癌基因Ｔｒｋ编码的蛋白质酪氨酸激酶 ;另一种是低亲和性受体ｐ75,它以相同的亲和力与神经营养素 (ｎｅｕｒｏｔｒｏｐｈｉｎ ,ＮＴ)家族的各成员结合 ,因此被称为神经营养素受体ｐ75(ｐ75ＮＴＲ)。ＮＧＦ与ＴｒｋＡ受体结合后发挥其促进神经细胞生长和存活的作用 ,而近年来发现ｐ75受体在特定条件下却能够诱导某些神经细胞和胶质细胞的凋亡 ,由于ｐ75ＮＴＲ的这一作用 ,它受到越来越多的关注。1 .ｐ75Ｎ…     

p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.     

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.     

Toll-like receptors stimulate regulated intramembrane proteolysis of the CSF-1 receptor through Erk activation

The CSF-1 receptor is a protein-tyrosine kinase that regulates the renewal, differentiation and activation of monocytes and macrophages. We have recently shown that the CSF-1 receptor undergoes regulated intramembrane proteolysis, or RIPping. Here, we report that RIPping can be observed in response to pathogen-associated molecules, which act through Toll-like receptors (TLRs). TLR-induced CSF-1 receptor RIPping is largely independent of protein kinase C, while maximal RIPping depends on Erk activation. Our studies show that CSF-1 receptor RIPping can be activated by various intracellular signal transduction pathways and that RIPping is likely to play an important role during macrophage activation.     

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.     

Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.     

Proteasome inhibition induces neurite outgrowth through posttranslational modification of TrkA receptor

The ubiquitin-proteasome pathway regulates many biological processes, including protein degradation, receptor endocytosis, protein sorting, subnuclear trafficking and neuronal differentiation. While proteasome inhibition is known to induce neurite outgrowth, the signaling mechanisms that mediate these effects have not been defined. In this study, we investigated the underlying mechanisms that link proteasome inhibition with neurite generation. We found that the proteasome inhibitors, MG132 and lactacystin, induced neurite outgrowth and also activated extracellular signal-regulated kinase/mitogen activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways. These proteasome inhibitors also induced phosphorylation and ubiquitination of TrkA receptors, indicating that proteasome inhibition activates the major pathways of TrkA signaling. However, in contrast to nerve growth factor stimulation, which induces internalization of surface TrkA receptors, proteasome inhibitor-induced neurite outgrowth did not require TrkA receptor internalization. These results indicate that the ubiquitin-proteasome system regulates neurite formation through posttranslational modification of TrkA receptors.     

The p75 neurotrophin receptor interacts with multiple MAGE proteins

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.