Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains.

The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.

Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.

Proteolytic Release of the p75NTR Intracellular Domain by ADAM10 Promotes Metastasis and Resistance to Anoikis.

Resistance to anoikis allows cancer cells to survive during systemic circulation; however, the mechanism underlying anoikis resistance remains unclear. Here we show that A disintegrin and metalloprotease 10 (ADAM10)-mediated cleavage of p75 neurotrophin receptor (p75NTR) and subsequent generation of the p75NTR intracellular domain (ICD) endow cancer cells with resistance to anoikis. p75NTR ICD promoted expression of TNF receptor-associated factor 6 (TRAF6), a critical intermediary in p75NTR ICD-mediated signal transduction, at the translational level. Cell detachment-induced activation of EGFR triggered autoubiquitination of TRAF6 by facilitating its dimerization, subsequently activated NFκB, and eventually led to anoikis resistance. ADAM10 and p75NTR ICD also promoted tumor metastasis formation in vivo Together, our findings uncover a previously unknown function for the ADAM10-p75NTR ICD-TRAF6-NFκB axis in preventing anoikis and suggest ADAM10 and p75NTR ICD as potential cancer therapeutic targets.Significance: These findings identify the ADAM10-p75NTR ICD-TRAF6-NFκB signaling axis as a potential candidate for cancer therapy. Cancer Res; 78(9); 2262-76. ©2018 AACR.

Generation of rationally-designed nerve growth factor (NGF) variants with receptor specificity.

Nerve growth factor (NGF) is the prototypic member of the neurotrophin family and binds two receptors, TrkA and the 75 kDa neurotrophin receptor (p75NTR), through which diverse and sometimes opposing effects are mediated. Using the FoldX protein design algorithm, we generated eight NGF variants with different point mutations predicted to have altered binding to TrkA or p75NTR. Of these, the I31R NGF variant exhibited specific binding to p75NTR. The generation of this NGF variant with selective affinity for p75NTR can be used to enhance understanding of neurotrophin receptor imbalance in diseases and identifies a key targetable residue for the development of small molecules to disrupt binding of NGF to TrkA with potential uses in chronic pain.

Nerve Growth Factor Signaling from Membrane Microdomains to the Nucleus: Differential Regulation by Caveolins.

Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.

Structural Characterization of the p75 Neurotrophin Receptor: A Stranger in the TNFR Superfamily.

Although p75 neurotrophin receptor (p75NTR) was the founding member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), it is an atypical TNFRSF protein. p75NTR like TNF-R1 and Fas-R contain an extracellular domain with four cysteine-rich domains (CRD) and a death domain (DD) in the intracellular region. While TNFRSF proteins are activated by trimeric TNFSF ligands, p75NTR forms dimers activated by dimeric neurotrophins that are structurally unrelated to TNFSF proteins. In addition, although p75NTR shares with other members the interaction with the TNF receptor-associated factors to activate the NF-κB and cell death pathways, p75NTR does not interact with the DD-containing proteins FADD, TRADD, or MyD88. By contrast, the DD of p75NTR is able to recruit several protein interactors via a full catalog of DD interactions not described before in the TNFRSF. p75-DD forms homotypic symmetrical DD-DD complexes with itself and with the related p45-DD; forms heterotypic DD-CARD interactions with the RIP2-CARD domain, and forms a new interaction between a DD and RhoGDI. All these features, in addition to its promiscuous interactions with several ligands and coreceptors, its processing by α- and γ-secretases, the dimeric nature of its transmembrane domain and its "special" juxtamembrane region, make p75NTR a truly stranger in the TNFR superfamily. In this chapter, I will summarize the known structural aspects of p75NTR and I will analyze from a structural point of view, the similitudes and differences between p75NTR and the other members of the TNFRSF.

Predicted 3D Model of the Rabies Virus Glycoprotein Trimer.

The RABVG ectodomain is a homotrimer, and trimers are often called spikes. They are responsible for the attachment of the virus through the interaction with nicotinic acetylcholine receptors, neural cell adhesion molecule (NCAM), and the p75 neurotrophin receptor (p75NTR). This makes them relevant in viral pathogenesis. The antigenic structure differs significantly between the trimers and monomers. Surfaces rich in hydrophobic amino acids are important for trimer stabilization in which the C-terminal of the ectodomain plays an important role; to understand these interactions between the G proteins, a mechanistic study of their functions was performed with a molecular model of G protein in its trimeric form. This verified its 3D conformation. The molecular modeling of G protein was performed by a I-TASSER server and was evaluated via a Rachamandran plot and ERRAT program obtained 84.64% and 89.9% of the residues in the favorable regions and overall quality factor, respectively. The molecular dynamics simulations were carried out on RABVG trimer at 310 K. From these theoretical studies, we retrieved the RMSD values from Cα atoms to assess stability. Preliminary model of G protein of rabies virus stable at 12 ns with molecular dynamics was obtained.

Death Domain Signaling by Disulfide-Linked Dimers of the p75 Neurotrophin Receptor Mediates Neuronal Death in the CNS.

UNLABELLED: The p75 neurotrophin receptor (p75(NTR)) mediates neuronal death in response to neural insults by activating a caspase apoptotic pathway. The oligomeric state and activation mechanism that enable p75(NTR) to mediate these effects have recently been called into question. Here, we have investigated mutant mice lacking the p75(NTR) death domain (DD) or a highly conserved transmembrane (TM) cysteine residue (Cys(259)) implicated in receptor dimerization and activation. Neuronal death induced by proneurotrophins or epileptic seizures was assessed and compared with responses in p75(NTR) knock-out mice and wild-type animals. Proneurotrophins induced apoptosis of cultured hippocampal and cortical neurons from wild-type mice, but mutant neurons lacking p75(NTR), only the p75(NTR) DD, or just Cys(259) were all equally resistant to proneurotrophin-induced neuronal death. Homo-FRET anisotropy experiments demonstrated that both NGF and proNGF induce conformational changes in p75(NTR) that are dependent on the TM cysteine. In vivo, neuronal death induced by pilocarpine-mediated seizures was significantly reduced in the hippocampus and somatosensory, piriform, and entorhinal cortices of all three strains of p75(NTR) mutant mice. Interestingly, the levels of protection observed in mice lacking the DD or only Cys(259) were identical to those of p75(NTR) knock-out mice even though the Cys(259) mutant differed from the wild-type receptor in only one amino acid residue. We conclude that, both in vitro and in vivo, neuronal death induced by p75(NTR) requires the DD and TM Cys(259), supporting the physiological relevance of DD signaling by disulfide-linked dimers of p75(NTR) in the CNS. SIGNIFICANCE STATEMENT: A detailed understanding of the physiological significance of distinct structural determinants in the p75 neurotrophin receptor (p75(NTR)) is crucial for the identification of suitable drug targets in this receptor. We have tested the relevance of the p75(NTR) death domain (DD) and the highly conserved transmembrane residue Cys(259) for the ability of p75(NTR) to induce apoptosis in neurons of the CNS using gene-targeted mutant mice. The physiological importance of these determinants had been contested in some recent in vitro studies. Our results indicate a requirement for DD signaling by disulfide-linked dimers of p75(NTR) for neuronal death induced by proneurotrophins and epileptic seizures. These new mouse models will be useful for clarifying different aspects of p75(NTR) physiology.

Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation.

The p75 neurotrophin receptor (p75(NTR)) is a multifunctional receptor that participates in many critical processes in the nervous system, ranging from apoptosis to synaptic plasticity and morphological events. It is a member of the tumor necrosis factor receptor (TNFR) superfamily, whose members undergo trimeric oligomerization. Interestingly, p75(NTR) interacts with dimeric ligands (i.e., proneurotrophins or mature neurotrophins), but several of the intracellular adaptors that mediate p75(NTR) signaling are trimeric (i.e., TNFR-associated factor 6 or TRAF6). Consequently, the active receptor signaling unit remains uncertain. To identify the functional receptor complex, we evaluated its oligomerization in vitro and in mice brain tissues using a combination of biochemical techniques. We found that the most abundant homotypic arrangement for p75(NTR) is a trimer and that monomers and trimers coexist at the cell surface. Interestingly, trimers are not required for ligand-independent or ligand-dependent p75(NTR) activation in a growth cone retraction functional assay. However, monomers are capable of inducing acute morphological effects in neurons. We propose that p75(NTR) activation is regulated by its oligomerization status and its levels of expression. These results indicate that the oligomeric state of p75(NTR) confers differential responses and offers an explanation for the diverse and contradictory actions of this receptor in the nervous system. SIGNIFICANCE STATEMENT: The p75 neurotrophin receptor (p75(NTR)) regulates a wide range of cellular functions, including apoptosis, neuronal processes remodeling, and synaptic plasticity. The goal of our work was to inquire whether oligomers of the receptor are required for function. Here we report that p75(NTR) predominantly assembles as a trimer, similar to other tumor necrosis factor receptors. Interestingly, monomers and trimers coexist at the cell surface, but trimers are not required for p75(NTR) activation in a functional assay. However, monomers are capable of inducing acute morphological effects in neurons. Identification of the oligomerization state of p75(NTR) begins to provide insights to the mechanisms of signal initiation of this noncatalytic receptor, as well as to develop therapeutic interventions to diminish its activity.

Silica Nanopills for Targeted Anticancer Drug Delivery.

Multifunctional SiO2 microtubes for targeted drug delivery are produced with precise control over shape and size by combining lithography and electrochemical etching. The hollow core is loaded with a lipophilic anticancer drug generating nanopills and an antibody is conjugated to the external surface for cancer cell targeting. Results demonstrate selective killing of neuroblastoma cells that express the cognate receptor.

p75NTR ectodomain is a physiological neuroprotective molecule against amyloid-beta toxicity in the brain of Alzheimer's disease.

In Alzheimer's disease (AD), neurodegenerative signals such as amyloid-beta (Aß) and the precursors of neurotrophins, outbalance neurotrophic signals, causing synaptic dysfunction and neurodegeneration. The neurotrophin receptor p75 (p75NTR) is a receptor of Aß and mediates Aß-induced neurodegenerative signals. The shedding of its ectodomain from the cell surface is physiologically regulated; however, the function of the diffusible p75NTR ectodomain (p75ECD) after shedding remains largely not known. Here, we show that p75ECD levels in cerebrospinal fluid and in the brains of Alzheimer's patients and amyloid-beta precursor protein (APP)/PS1 transgenic mice were significantly reduced, due to inhibition of the sheddase-tumor necrosis factor-alpha-converting enzyme by Aß. Restoration of p75ECD to the normal level by brain delivery of the gene encoding human p75ECD before or after Aß deposition in the brain of APP/PS1 mice reversed the behavioral deficits and AD-type pathologies, such as Aß deposit, apoptotic events, neuroinflammation, Tau phosphorylation and loss of dendritic spine, neuronal structures and synaptic proteins. Furthermore, p75ECD can also reduce amyloidogenesis by suppressing ß-secretase expression and activities. Our data demonstrate that p75ECD is a physiologically neuroprotective molecule against Aß toxicity and would be a novel therapeutic target and biomarker for AD.

Association of p75(NTR) and α9ß1 integrin modulates NGF-dependent cellular responses.

Direct interaction of α9ß1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75(NTR) in α9ß1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75(NTR) on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75(NTR) complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including ß1 were not found in complex with p75(NTR). FRET analysis indicated that p75(NTR) and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9ß1 integrin with another ligand, VCAM-1 was not modulated by the p75(NTR). α9/p75(NTR) complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

Heterodimerization of p45-p75 modulates p75 signaling: structural basis and mechanism of action.

The p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily, is required as a co-receptor for the Nogo receptor (NgR) to mediate the activity of myelin-associated inhibitors such as Nogo, MAG, and OMgp. p45/NRH2/PLAIDD is a p75 homologue and contains a death domain (DD). Here we report that p45 markedly interferes with the function of p75 as a co-receptor for NgR. P45 forms heterodimers with p75 and thereby blocks RhoA activation and inhibition of neurite outgrowth induced by myelin-associated inhibitors. p45 binds p75 through both its transmembrane (TM) domain and DD. To understand the underlying mechanisms, we have determined the three-dimensional NMR solution structure of the intracellular domain of p45 and characterized its interaction with p75. We have identified the residues involved in such interaction by NMR and co-immunoprecipitation. The DD of p45 binds the DD of p75 by electrostatic interactions. In addition, previous reports suggested that Cys257 in the p75 TM domain is required for signaling. We found that the interaction of the cysteine 58 of p45 with the cysteine 257 of p75 within the TM domain is necessary for p45-p75 heterodimerization. These results suggest a mechanism involving both the TM domain and the DD of p45 to regulate p75-mediated signaling.

Efficacious anticancer drug delivery mediated by a pH-sensitive self-assembly of a conserved tripeptide derived from tyrosine kinase NGF receptor.

We present herein a short tripeptide sequence (Lys-Phe-Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self-assembles in water and shows a reversible and concentration-dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH-sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug-sensitive and drug-resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence-activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.

Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α.

Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75(NTR)) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75(NTR) directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75(NTR) stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75(NTR) dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75(NTR-/-) mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75(NTR) constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.

Identification of a linear epitope in sortilin that partakes in pro-neurotrophin binding.

Sortilin acts as a cell surface receptor for pro-neurotrophins (pro-NT) that upon complex formation with the p75 neurotrophin receptor (p75(NTR)) is able to signal neuronal cell death. Here we screened a sortilin peptide library comprising 16-mer overlapping sequences for binding of the pro-domains of nerve growth factor and brain-derived neurotrophic factor. We find that a linear surface-exposed sequence, (163)RIFRSSDFAKNF(174), constitutes an important pro-NT binding epitope in sortilin. Systematic mutational analysis revealed residues Arg(163), Phe(165), Arg(166), and Phe(170) to be critical for the interaction. Expression of a sortilin mutant in which these four amino acids were substituted by alanines disrupted pro-NT binding without affecting receptor heterodimerization with p75(NTR) or binding of ligands that selectively engages the centrally located tunnel in the beta-propeller of sortilin. We furthermore demonstrate that a peptide comprising the ligand-binding epitope can prevent pro-NT-induced apoptosis in RN22 schwannoma cells.

Efficacy of a murine-p75-saporin immunotoxin for selective lesions of basal forebrain cholinergic neurons in mice.

Selective lesioning of cholinergic neurons in the basal forebrain provides a tool for examining the functional significance of cholinergic loss, which is associated with a number of developmental and neurodegenerative disorders. A new version of an immunotoxin (murine-p75NTR-saporin) was used to produce a selective loss of cholinergic neurons in the adult basal forebrain of the mouse. This new version of the toxin is significantly more potent and selective than a previously developed version. C57Bl/6J mice (n=30) were given 1 microL of either saline or murine-p75NTR-saporin (0.65 microg/microL or 1.3 microg/microL) into the lateral ventricles, and then sacrificed 10-12 days post-surgery for histological analysis. In contrast to results from the previous version of the toxin, survival of the toxin-treated mice was 100% at both doses. A complete loss of cholinergic neurons was seen in the medial septum (MS) with both doses, while a dose-dependent loss of cholinergic neurons was observed in the nucleus basalis magnocellularis (nBM). The lesions were associated with locomotor hypoactivity and anxiolytic-type behavioral effects. These studies describe the efficacy and selectivity of this new version of murine-p75NTR-saporin, which may be used to provide insight into functional deficits that result from the loss of cholinergic neurons in the mouse basal forebrain.

Intrinsic structural disorder of mouse proNGF.

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown. Aiming to gain insights into the specific molecular interactions that govern rm-proNGF biological activities versus those of its mature counterpart, a structural study by synchrotron small angle X-ray scattering (SAXS) in solution was carried out. The different binding properties of the two proteins were investigated by surface plasmon resonance (SPR) using, as structural probes, a panel of anti-NGF antibodies and the soluble forms of TrkA and p75(NTR) receptors. SAXS measurements revealed the rm-proNGF to be dimeric and anisometric, with the propeptide domain being intrinsically unstructured. Ab initio reconstructions assuming twofold symmetry generated two types of structural models, a globular "crab-like" and an elongated shape that resulted in equally good fits of the scattering data. A novel method accounting for possible coexistence of different conformations contributing to the experimental scattering pattern, with no symmetry constraints, suggests the "crab-like" to be a more likely proNGF conformation. To exploit the potential of chemical stabilizers affecting the existing conformational protein populations, SAXS data were also collected in the presence of ammonium sulphate. An increase of the proNGF compactness was observed. SPR data pinpoints that the propeptide of proNGF may act as an intrinsically unstructured protein domain, characterized by a molecular promiscuity in the interaction/binding to multiple partners (TrkA and p75(NTR) receptors and a panel of neutralizing anti-NGF antibodies) depending on the physiological conditions of the cell. These data provide a first insight into the structural basis for the selectivity of mouse short proNGF, versus NGF, towards its binding partners.

Human autoantibodies specific for neurotrophin receptors TrkA, TrkB, and TrkC protect against lethal Trypanosoma cruzi infection in mice.

Patients with Chagas' disease remain asymptomatic for many years, presumably by keeping the etiological agent Trypanosoma cruzi in check through protective immunity against. Recently, we found that T. cruzi uses TrkA, a receptor tyrosine kinase responsive to neurotrophin nerve growth factor in vertebrate nervous systems, to invade cells. We also found that TrkA, TrkB, and TrkC, but not T. cruzi, are targets of specific autoantibodies present in the sera of patients with chronic Chagas' disease. Here we show that TrkA-, TrkB-, and TrkC-specific autoantibodies isolated from the sera of four individuals with chronic indeterminate (asymptomatic) Chagas' disease potently blocked invasion of Trk-bearing neuronal PC12 cells, neuroglial astrocytes, enteroglial cells, and Schwann cells and Trk-expressing non-neural smooth muscle and dendritic cells. However, these autoantibodies did not inhibit T. cruzi invasion of mutant PC12 cells lacking TrkA or of normal cells lacking Trk receptors, suggesting that autoantibodies interfered with parasite/Trk cross talk to access the intracellular milieu. Passive immunization of susceptible and resistant mouse strains with very small doses of these autoantibodies reduced parasitemia and transferred resistance to an otherwise lethal trypanosome infection. Hence, this exquisitely sensitive and unique regulatory immunity against the host (instead of parasite) could benefit infected individuals by blocking cellular invasion of the obligatory intracellular pathogen, resulting in attenuation of tissue infection and clinical manifestations. Such action is contrary to the horror autotoxicus frequently associated with microbe-related autoimmune responses.

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.