The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Moses V Chao - One of the best experts on this subject based on the ideXlab platform.
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TrkA Receptor activation by nerve growth factor induces shedding of the p75 neurotrophin Receptor followed by endosomal γ secretase mediated release of the p75 intracellular domain
Journal of Biological Chemistry, 2007Co-Authors: Soledad Urra, Moses V Chao, Claudia A Escudero, Patricio Ramos, Fernanda Lisbona, Edgardo Allende, Paulina Covarrubias, Jose I Parraguez, Niccolo Zampieri, W AnnaertAbstract: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 γ-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 γ-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for γ-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.
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cell survival through trk neurotrophin Receptors is differentially regulated by ubiquitination
Neuron, 2006Co-Authors: Juan Carlos Arevalo, Zhe Yu Chen, Rithwick Rajagopal, Janelle C Waite, Mercedes Beyna, Moses V ChaoAbstract:Specificity of neurotrophin factor signaling is dictated through the action of Trk Receptor tyrosine kinases. Once activated, Trk Receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk Receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA Receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA Receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB Receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin Receptors are differentially regulated by ubiquitination to modulate the survival of neurons.
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a novel p75 neurotrophin Receptor related protein nrh2 regulates nerve growth factor binding to the TrkA Receptor
The Journal of Neuroscience, 2004Co-Authors: Simon S Murray, Pilar Perez, Barbara L Hempstead, Ramee Lee, Moses V ChaoAbstract:Nerve growth factor (NGF) functions as a ligand for two Receptors, the TrkA tyrosine kinase Receptor and the p75 neurotrophin Receptor (p75NTR). The Ig-like domains of Trk Receptors and the cysteine-rich repeats of p75NTR are involved in binding to the neurotrophins. Recently, a closely related gene to p75NTR called neurotrophin Receptor homolog-2 (NRH2) was identified; however, the function of NRH2 and its relevance to neurotrophin signaling are unclear. NRH2 contains a similar transmembrane and intracellular domain as p75NTR but lacks the characteristic cysteine-rich repeats in the extracellular domain. Here we show that NRH2 is expressed in several neuronal populations that also express p75NTR and Trk Receptors. NRH2 does not bind to NGF; however, coimmunoprecipitation experiments demonstrate that NRH2 is capable of interacting with TrkA Receptors. Coexpression of NRH2 with TrkA Receptors resulted in the formation of high-affinity binding sites for NGF. These results indicate that a transmembrane protein related to p75NTR is capable of modulating Trk Receptor binding properties.
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regulated intramembrane proteolysis of the p75 neurotrophin Receptor modulates its association with the TrkA Receptor
Journal of Biological Chemistry, 2003Co-Authors: Kwangmook Jung, Serena Tan, Natalie Landman, Kseniya Petrova, Simon S Murray, Renee Lewis, Peter K Kim, Dae Sup Kim, Sung Ho Ryu, Moses V ChaoAbstract:The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent γ-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including β-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 γ-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 γ-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid β-peptide 40 (Aβ40) 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 γ-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that γ-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.
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a novel mutation within the extracellular domain of TrkA causes constitutive Receptor activation
Oncogene, 2001Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Blanca Conde, Barbara I Hempstead, Pilar PerezAbstract:The TrkA NGF Receptor extracellular region contains three leucine repeats flanked by cysteine clusters and two immunoglobulin-like domains that are required for specific ligand binding. Deletion of the immunoglobulin-like domains abolishes NGF binding and causes ligand independent activation of the Receptor. Here we report a specific mutation that increases the binding affinity of the TrkA Receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the first Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated Receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant Receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A Receptor in fibroblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk Receptors.
Pilar Perez - One of the best experts on this subject based on the ideXlab platform.
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a novel p75 neurotrophin Receptor related protein nrh2 regulates nerve growth factor binding to the TrkA Receptor
The Journal of Neuroscience, 2004Co-Authors: Simon S Murray, Pilar Perez, Barbara L Hempstead, Ramee Lee, Moses V ChaoAbstract:Nerve growth factor (NGF) functions as a ligand for two Receptors, the TrkA tyrosine kinase Receptor and the p75 neurotrophin Receptor (p75NTR). The Ig-like domains of Trk Receptors and the cysteine-rich repeats of p75NTR are involved in binding to the neurotrophins. Recently, a closely related gene to p75NTR called neurotrophin Receptor homolog-2 (NRH2) was identified; however, the function of NRH2 and its relevance to neurotrophin signaling are unclear. NRH2 contains a similar transmembrane and intracellular domain as p75NTR but lacks the characteristic cysteine-rich repeats in the extracellular domain. Here we show that NRH2 is expressed in several neuronal populations that also express p75NTR and Trk Receptors. NRH2 does not bind to NGF; however, coimmunoprecipitation experiments demonstrate that NRH2 is capable of interacting with TrkA Receptors. Coexpression of NRH2 with TrkA Receptors resulted in the formation of high-affinity binding sites for NGF. These results indicate that a transmembrane protein related to p75NTR is capable of modulating Trk Receptor binding properties.
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a novel mutation within the extracellular domain of TrkA causes constitutive Receptor activation
Oncogene, 2001Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Blanca Conde, Barbara I Hempstead, Pilar PerezAbstract:The TrkA NGF Receptor extracellular region contains three leucine repeats flanked by cysteine clusters and two immunoglobulin-like domains that are required for specific ligand binding. Deletion of the immunoglobulin-like domains abolishes NGF binding and causes ligand independent activation of the Receptor. Here we report a specific mutation that increases the binding affinity of the TrkA Receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the first Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated Receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant Receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A Receptor in fibroblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk Receptors.
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TrkA immunoglobulin like ligand binding domains inhibit spontaneous activation of the Receptor
Molecular and Cellular Biology, 2000Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Barbara L Hempstead, Blanca Conde, Pilar PerezAbstract:The extracellular region of the nerve growth factor (NGF) Receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in Receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent Receptor dimerization, chimeric Receptors were generated by swapping the Ig-like domains of the TrkA Receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant Receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit Receptor dimerization in the absence of NGF.
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ngf binding to the trk tyrosine kinase Receptor requires the extracellular immunoglobulin like domains
Molecular and Cellular Neuroscience, 1995Co-Authors: Pilar Perez, Dionisio Martinzanca, Pedro M Coll, Barbara L Hempstead, Moses V ChaoAbstract:Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk Receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the Receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric Receptors was constructed using cDNA sequences from rat TrkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each Receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of TrkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the TrkA Receptor.
Juan Carlos Arevalo - One of the best experts on this subject based on the ideXlab platform.
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selective and differential interactions of bnn27 a novel c17 spiroepoxy steroid derivative with TrkA Receptors regulating neuronal survival and differentiation
Neuropharmacology, 2016Co-Authors: Iosif Pediaditakis, Juan Carlos Arevalo, Paschalis Efstathopoulos, Kyriakos C Prousis, Maria I Zervou, Vasileia Ismini Alexaki, Vassiliki Nikoletopoulou, Efthymia Karagianni, Constantinos Potamitis, Nektarios TavernarakisAbstract:Nerve growth factor (NGF) holds a pivotal role in brain development and maintenance, been also involved in the pathophysiology of neurodegenerative diseases. Here, we provide evidence that a novel C17-spiroepoxy steroid derivative, BNN27, specifically interacts with and activates the TrkA Receptor of NGF, inducing phosphorylation of TrkA tyrosine residues and down-stream neuronal survival-related kinase signaling. Additionally, BNN27 potentiates the efficacy of low levels of NGF, by facilitating its binding to the TrkA Receptors and differentially inducing fast return of internalized TrkA Receptors into neuronal cell membranes. Furthermore, BNN27 synergizes with NGF in promoting axonal outgrowth, effectively rescues from apoptosis NGF-dependent and TrkA positive sympathetic and sensory neurons, in vitro, ex vivo and in vivo in NGF null mice. Interestingly, BNN27 does not possess the hyperalgesic properties of NGF. BNN27 represents a lead molecule for the development of neuroprotective TrkA Receptor agonists, with potential therapeutic applications in neurodegenerative diseases and in brain trauma.
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cell survival through trk neurotrophin Receptors is differentially regulated by ubiquitination
Neuron, 2006Co-Authors: Juan Carlos Arevalo, Zhe Yu Chen, Rithwick Rajagopal, Janelle C Waite, Mercedes Beyna, Moses V ChaoAbstract:Specificity of neurotrophin factor signaling is dictated through the action of Trk Receptor tyrosine kinases. Once activated, Trk Receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk Receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA Receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA Receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB Receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin Receptors are differentially regulated by ubiquitination to modulate the survival of neurons.
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a novel mutation within the extracellular domain of TrkA causes constitutive Receptor activation
Oncogene, 2001Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Blanca Conde, Barbara I Hempstead, Pilar PerezAbstract:The TrkA NGF Receptor extracellular region contains three leucine repeats flanked by cysteine clusters and two immunoglobulin-like domains that are required for specific ligand binding. Deletion of the immunoglobulin-like domains abolishes NGF binding and causes ligand independent activation of the Receptor. Here we report a specific mutation that increases the binding affinity of the TrkA Receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the first Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated Receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant Receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A Receptor in fibroblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk Receptors.
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TrkA immunoglobulin like ligand binding domains inhibit spontaneous activation of the Receptor
Molecular and Cellular Biology, 2000Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Barbara L Hempstead, Blanca Conde, Pilar PerezAbstract:The extracellular region of the nerve growth factor (NGF) Receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in Receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent Receptor dimerization, chimeric Receptors were generated by swapping the Ig-like domains of the TrkA Receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant Receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit Receptor dimerization in the absence of NGF.
Uri H Saragovi - One of the best experts on this subject based on the ideXlab platform.
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TrkA Receptor hot spots for binding of nt 3 as a heterologous ligand
Journal of Biological Chemistry, 2007Co-Authors: Ljubica Ivanisevic, Uri H Saragovi, Wenhua Zheng, Sang B Woo, Kenneth E NeetAbstract:Neurotrophins signal via Trk tyrosine kinase Receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate Receptors (e.g. NGF binds and activates through TrkA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TrkA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TrkA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TrkA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several Receptor subdomains and hot spots.
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long lasting rescue of age associated deficits in cognition and the cns cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA
The Journal of Neuroscience, 2004Co-Authors: Martin A Bruno, Remi Quirion, Paul B S Clarke, Alicia Seltzer, Kevin Burgess, Claudio A Cuello, Uri H SaragoviAbstract:Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA Receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA Receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor Receptor may have therapeutic potential for neurodegenerative disorders.
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design and solution structure of functional peptide mimetics of nerve growth factor
Journal of Medicinal Chemistry, 2000Co-Authors: Natalia Beglova, Sergei Maliartchouk, Irena Ekiel, Maria Clara Zaccaro, Uri H Saragovi, Kalle GehringAbstract:The C−D loop in nerve growth factor (NGF) is involved in binding to the NGF Receptor, TrkA. It is flexible and adopts several different types conformations in different NGF crystal forms. We have previously shown that a small cyclic peptide derived from the C−D loop of NGF binds to the TrkA Receptor by mimicking the structure of this loop. To understand structure−function relationships in NGF C−D loop mimetics, we have produced a series of peptides predicted to form different types of β-turns. The peptides were tested for their ability to promote cell survival in serum-free medium and to induce TrkA tyrosine phosphorylation. NMR structural studies were used to determined the backbone conformation and the spatial orientation of side chains involved in binding to the TrkA Receptor. Peptides that form type I or type γL-αR β-turns were the most active. The variety of active loop conformations suggests that the mimetics (and NGF) accommodate the binding site on TrkA by an ‘induced fit‘ mechanism. In agreement ...
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solution structure and internal motion of a bioactive peptide derived from nerve growth factor
Journal of Biological Chemistry, 1998Co-Authors: Natalia Beglova, Irena Ekiel, Uri H Saragovi, Lynne Lesauteur, Kalle GehringAbstract:The conformation and internal dynamics of a bioactive cyclic peptide, N-acetyl-YCTDEKQCY, derived from the C-D loop of beta-nerve growth factor (beta-NGF) were analyzed by solution NMR spectroscopy. NMR experimental data were used to calculate an ensemble of peptide structures. All of the structures had a beta-turn at residues Asp4-Gln7 but could be divided into two families according the presence or absence of a hydrogen bond at Gln7. Comparison of the calculated structures with the corresponding C-D loops from the x-ray structures of the NGF revealed striking similarity. The orientation of Glu5, Lys6, and Gln7 side chains in the NGF mimetic was very similar to the C-D loop of NGF. These residues are known to participate in interactions with the TrkA Receptor. Relaxation measurements of the peptidomimetic alpha-carbons at 13C natural abundance and calculated dynamic parameters suggest that the loop region of peptide is well structured but that residues Thr3, Asp4, Glu5, and Lys6 undergo slow conformational exchange. These results suggest that conformational similarity and possibly peptide dynamics are responsible for the bioactivity of the peptide.
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reduction in p140 TrkA Receptor protein within the nucleus basalis and cortex in alzheimer s disease
Experimental Neurology, 1997Co-Authors: Elliott J Mufson, Natalie Lavine, Syed Jaffar, Jeffrey H Kordower, Remi Quirion, Uri H SaragoviAbstract:It has been hypothesized that the diminished transport of nerve growth factor (NGF) seen within cholinergic basal forebrain (CBF) neurons in Alzheimer's disease (AD) results from a defect in the expression of its high-affinity TrkA Receptor. The present study used an anti-human TrkA-specific monoclonal antibody (mAb 5C3) that recognizes the NGF docking site, combined with quantitative optical densitometry, to evaluate whether expression of the TrkA protein is altered within the nucleus basalis and its cortical projection sites in AD. In normal aged humans, TrkA immunoreactivity revealed a continuum of positive neurons extending throughout all CBF subfields. In addition, TrkA-positive neurons were scattered throughout the olfactory tubercle and striatum. These regions also displayed intense TrkA neuropil staining. Although fewer in total number, remaining CBF perikarya in AD displayed a significant decrease in TrkA levels relative to aged controls. Biochemical analysis revealed a significant reduction in TrkA protein within both the nucleus basalis and the frontal cortex in AD relative to aged controls. In contrast, TrkA levels in the caudate nucleus were unaffected. The decrease in TrkA protein in conjunction with our recent observations that the message for TrkA is reduced within individual CBF neurons in AD supports the concept that defects in the production and/or utilization of the TrkA Receptor may be a key event mediating degeneration of NGF-responsive CBF neurons in this disease.
Dionisio Martinzanca - One of the best experts on this subject based on the ideXlab platform.
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a novel mutation within the extracellular domain of TrkA causes constitutive Receptor activation
Oncogene, 2001Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Blanca Conde, Barbara I Hempstead, Pilar PerezAbstract:The TrkA NGF Receptor extracellular region contains three leucine repeats flanked by cysteine clusters and two immunoglobulin-like domains that are required for specific ligand binding. Deletion of the immunoglobulin-like domains abolishes NGF binding and causes ligand independent activation of the Receptor. Here we report a specific mutation that increases the binding affinity of the TrkA Receptor for NGF. A change of proline 203 to alanine (P203A) in the linker region between the leucine repeats and the first Ig-like domain increased NGF binding by decreasing the ligand rate of dissociation. This mutated Receptor was appropriately expressed on the cell surface and promoted ligand-independent neurite outgrowth in PC12nnr5 cells. The mutant Receptor was capable of spontaneous dimerization and was constitutively phosphorylated in the absence of ligand. Moreover, expression of TrkA-P203A Receptor in fibroblasts induced DNA synthesis and transformation and generated tumours in nude mice. These data suggest that domains outside of the immunoglobulin-like structure contribute to ligand binding and constitutive activation of Trk Receptors.
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TrkA immunoglobulin like ligand binding domains inhibit spontaneous activation of the Receptor
Molecular and Cellular Biology, 2000Co-Authors: Juan Carlos Arevalo, Moses V Chao, Dionisio Martinzanca, Barbara L Hempstead, Blanca Conde, Pilar PerezAbstract:The extracellular region of the nerve growth factor (NGF) Receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in Receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent Receptor dimerization, chimeric Receptors were generated by swapping the Ig-like domains of the TrkA Receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant Receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit Receptor dimerization in the absence of NGF.
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TrkA Receptor ectodomain cleavage generates a tyrosine phosphorylated cell associated fragment
Journal of Cell Biology, 1996Co-Authors: Noemi Cabrera, Elena Diazrodriguez, Elena Becker, Dionisio Martinzanca, Atanasio PandiellaAbstract:The extracellular domain of several membrane-anchored proteins can be released as a soluble fragment by the action of a cell surface endoproteolytic system. This cleavage results in the generation of a soluble and a cell-bound fragment. In the case of proteins with signaling capability, such as tyrosine kinase Receptors, the cleavage process may have an effect on the kinase activity of the cell-bound Receptor fragment. By using several cell lines that express the TrkA neurotrophin Receptor, we show that this Receptor tyrosine kinase is cleaved by a proteolytic system that mimics the one that acts at the cell surface. TrkA cleavage is regulated by protein kinase C and several Receptor agonists (including the TrkA ligand NGF), occurs at the ectodomain in a membrane-proximal region, and is independent of lysosomal function. TrkA cleavage results in the generation of a cell-associated fragment that is phosphorylated on tyrosine residues. Tyrosine phosphorylation of this fragment is not detected in TrkA mutants devoid of kinase activity, suggesting that phosphorylation requires an intact TrkA kinase domain, and is not due to activation of an intermediate intracellular tyrosine kinase. The increased phosphotyrosine content of the cell-bound fragment may thus reflect higher catalytic activity of the truncated fragment. We postulate that cleavage of Receptor tyrosine kinases by this naturally occurring cellular mechanism may represent an additional mean for the regulation of Receptor activity.
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ngf binding to the trk tyrosine kinase Receptor requires the extracellular immunoglobulin like domains
Molecular and Cellular Neuroscience, 1995Co-Authors: Pilar Perez, Dionisio Martinzanca, Pedro M Coll, Barbara L Hempstead, Moses V ChaoAbstract:Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk Receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the Receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric Receptors was constructed using cDNA sequences from rat TrkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each Receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of TrkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the TrkA Receptor.
