Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75^NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75^NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75^NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced cell death.     

LINGO-1 Protein Interacts with the p75 Neurotrophin Receptor in Intracellular Membrane Compartments

Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75^NTR·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75^NTR associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75^NTR. Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75^NTR in a manner that is difficult to reconcile with the existence of a LINGO-1·p75^NTR·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75^NTR·NgR complexes in the plasma membrane.     

Death domain of p75 neurotrophin receptor: a structural perspective on an intracellular signalling hub

The death domain (DD) is a globular protein motif with a signature feature of an all‐helical Greek‐key motif. It is a primary mediator of a variety of biological activities, including apoptosis, cell survival and cytoskeletal changes, which are related to many neurodegenerative diseases, neurotrauma, and cancers. DDs exist in a wide range of signalling proteins including p75 neurotrophin receptor (p75^NTR), a member of the tumour necrosis factor receptor superfamily. The specific signalling mediated by p75^NTR in a given cell depends on the type of ligand engaging the extracellular domain and the recruitment of cytosolic interactors to the intracellular domain, especially the DD, of the receptor. In solution, the p75^NTR‐DDs mainly form a symmetric non‐covalent homodimer. In response to extracellular signals, conformational changes in the p75^NTR extracellular domain (ECD) propagate to the p75^NTR‐DD through the disulfide‐bonded transmembrane domain (TMD) and destabilize the p75^NTR‐DD homodimer, leading to protomer separation and exposure of binding sites on the DD surface. In this review, we focus on recent advances in the study of the structural mechanism of p75^NTR‐DD signalling through recruitment of diverse intracellular interactors for the regulation and control of diverse functional outputs.     

NRH2 is a trafficking switch to regulate sortilin localization and permit proneurotrophin‐induced cell death

Proneurotrophins mediate neuronal apoptosis using a dual receptor complex of sortilin and p75^NTR. Although p75^NTR is highly expressed on the plasma membrane and accessible to proneurotrophin ligands, sortilin is primarily localized to intracellular membranes, limiting the formation of a cell surface co‐receptor complex. Here, we show that the mammalian p75^NTR homologue NRH2 critically regulates the expression of sortilin on the neuronal cell surface and promotes p75^NTR and sortilin receptor complex formation, rendering cells responsive to proneurotrophins. This is accomplished by interactions between the cytoplasmic domains of NRH2 and sortilin that impair lysosomal degradation of sortilin. In proneurotrophin‐responsive neurons, acute silencing of endogenous NRH2 significantly reduces cell surface‐expressed sortilin and abolishes proneurotrophin‐induced neuronal death. Thus, these data suggest that NRH2 acts as a trafficking switch to impair lysosomal‐dependant sortilin degradation and to redistribute sortilin to the cell surface, rendering p75^NTR‐expressing cells susceptible to proneurotrophin‐induced death.     

A Role for the p75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress

The p75 neurotrophin receptor (p75^NTR) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75^NTR has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75^NTR signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75^NTR−/− mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75^NTR via a ligand-independent mechanism. Previous studies have established that proteolysis of p75^NTR by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75^NTR-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75^NTR. Pharmacological blockade of p75^NTR proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75^NTR is necessary for oxidant-induced neurodegeneration. In vivo, p75^NTR−/− mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75^NTR, resulting in axonal fragmentation and neuronal death.     

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 Extracellular Domain of Neurotrophin Receptor p75 as a Candidate Biomarker for Amyotrophic Lateral Sclerosis

Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1^G93A mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinson''s disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTR^ECD levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1^G93A mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.     

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 (p75^NTR) 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 p75^NTR 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 p75^NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75^NTR signaling remain poorly understood. Here we report an NMR structure of the p75^NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75^NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75^NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75^NTR 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 p75^NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75^NTR, to propagate downstream signaling in developing neurons.     

Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor

The multifunctional signaling protein p75 neurotrophin receptor (p75^NTR) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75^NTR is required for p75^NTR-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75^NTR or treatment of animals bearing p75^NTR-positive intracranial tumors with clinically applicable γ-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75^NTR was observed in p75^NTR-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75^NTR as a therapeutic target, suggesting that γ-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.     

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co‐receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6‐month‐old swedish‐amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose‐ and time‐dependent manner in SH‐SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75^NTR (P75ECD‐FC), or the antibody against the extracellular domain of p75^NTR, blocked the up‐regulation of sortilin induced by Amyloid‐β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH‐SY5Y via the p75^NTR receptor. Inhibition of ROCK, but not Jun N‐terminal kinase, suppressed constitutive and Aβ₄₂‐induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75^NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

     

p75NTR,but Not proNGF,Is Upregulated Following Status Epilepticus in Mice

ProNGF and p75^NTR are upregulated and induce cell death following status epilepticus (SE) in rats. However, less is known about the proneurotrophin response to SE in mice, a more genetically tractable species where mechanisms can be more readily dissected. We evaluated the temporal- and cell-specific induction of the proneurotrophins and their receptors, including p75^NTR, sortilin, and sorCS2, following mild SE induced with kainic acid (KA) or severe SE induced by pilocarpine. We found that mature NGF, p75^NTR, and proBDNF were upregulated following SE, while proNGF was not altered, indicating potential mechanistic differences between rats and mice. ProBDNF was localized to mossy fibers and microglia following SE. p75^NTR was transiently induced primarily in axons and axon terminals following SE, as well as in neuron and astrocyte cell bodies. ProBDNF and p75^NTR increased independently of cell death and their localization was different depending on the severity of SE. We also examined the expression of proneurotrophin co-receptors, sortilin and sorCS2. Following severe SE, sorCS2, but not sortilin, was elevated in neurons and astrocytes. These data indicate that important differences exist between rat and mouse in the proneurotrophin response following SE. Moreover, the proBDNF and p75^NTR increase after seizures in the absence of significant cell death suggests that proneurotrophin signaling may play other roles following SE.     

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

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

Structural Characterization of the Self-Association of the Death Domain of p75NTR

The neurotrophin receptor p75^NTR conveys multiple signals via its intracellular death domain. However, how the death domain is activated and interacts with downstream adaptors remains unclear. Here, we report two crystal structures of the p75^NTR death domain in the form of a non-covalent asymmetric dimer and a Cys379-Cys379 disulfide bond linked symmetric dimer, respectively. These two dimer arrangements have not previously been observed in other death domain-containing proteins. Further analysis shows that both the Cys379-Cys379 disulfide linked and non-covalent full-length p75^NTR dimers are present on the cell surface. These observations suggest that various oligomers may exist simultaneously on the cell surface, and that p75^NTR activation and signalling may be modulated by neurotrophins or other factors via inducing a shift of the equilibrium between different oligomeric states.     

Towards the PET radiotracer for p75 neurotrophin receptor: [11C]LM11A-24 shows biological activity in vitro,but unfavorable ex vivo and in vivo profile

Mature neurotrophins as well as their pro forms are critically involved in the regulation of neuronal functions. They are signaling through three distinct types of receptors: tropomyosin receptor kinase family (TrkA/B/C), p75 neurotrophin receptor (p75^NTR) and sortilin. Aberrant expression of p75^NTR in the CNS is implicated in a variety of neurodegenerative diseases, including Alzheimer’s disease. The goal of this work was to evaluate one of the very few reported p75^NTR small molecule ligands as a lead compound for development of novel PET radiotracers for in vivo p75^NTR imaging. Here we report that previously described ligand LM11A-24 shows significant inhibition of carbachol-induced persistent firing (PF) of entorhinal cortex (EC) pyramidal neurons in wild-type mice via selective interaction with p75^NTR. Based on this electrophysiological assay, the compound has very high potency with an EC₅₀ <10 nM. We optimized the radiosynthesis of [¹¹C]LM11A-24 as the first attempt to develop PET radioligand for in vivo imaging of p75^NTR. Despite some weak interaction with CNS tissues, the radiolabeled compound showed unfavorable in vivo profile presumably due to high hydrophilicity.     

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR⁺ and p75NTR^? hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR⁺, p75NTR^? and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR⁺ and p75NTR^? hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR⁺ and p75NTR^? hPDLSCs. The results showed that p75NTR⁺ hPDLSCs demonstrated superior osteogenic capacity than p75NTR^? and unsorted hPDLSCs. Differentially expressed genes between p75NTR⁺ and p75NTR^? hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR⁺ hPDLSCs expressed higher ITGA1 levels than p75NTR^? hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR⁺ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR^? hPDLSCs . These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.     

Sortilin participates in light-dependent photoreceptor degeneration in vivo

Both proNGF and the neurotrophin receptor p75 (p75(NTR)) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75(NTR) co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75(NTR), and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75(NTR) and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the "pro" domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75(NTR)/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies.     

Expression and function of Xenopus laevis p75NTR suggest evolution of developmental regulatory mechanisms

Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75^NTR), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75^NTR has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75^NTR imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75^NTR function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75^NTR genes from Xenopus, which we have termed p75^NTRa and p75^NTRb. We then cloned two different cDNAs, both of which encompass the full coding region of p75^NTRa. Early in development both p75^NTRa and p75^NTRb are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75^NTRa expression largely continues to parallel p75^NTR expression in other species. However, Xenopus p75^NTRa is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75^NTR and each of two truncated mutants in developing retina reveal that p75^NTR probably signals for cell survival in this system. This result contrasts with the reported role of p75^NTR in developing retinae of other species, and the possible implications of this difference are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 79–98, 2001     

BMP7‐induced dendritic growth in sympathetic neurons requires p75NTR signaling

Dendritic morphology is a critical determinant of neuronal connectivity, and in postganglionic sympathetic neurons, tonic activity correlates directly with the size of the dendritic arbor. Thus, identifying signaling mechanisms that regulate dendritic arborization of sympathetic neurons is important to understanding how functional neural circuitry is established and maintained in the sympathetic nervous system. Bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, downstream signaling events that link BMP receptor activation to dendritic growth are poorly characterized. We previously reported that BMP7 upregulates p75^NTR mRNA in cultured sympathetic neurons. This receptor is implicated in controlling dendritic growth in central neurons but whether p75^NTR regulates dendritic growth in peripheral neurons is not known. Here, we demonstrate that BMP7 increases p75^NTR protein in cultured sympathetic neurons, and this effect is blocked by pharmacologic inhibition of signaling via BMP type I receptor. BMP7 does not trigger dendritic growth in sympathetic neurons dissociated from superior cervical ganglia (SCG) of p75^NTR nullizygous mice, and overexpression of p75^NTR in p75^NTR?/? neurons is sufficient to cause dendritic growth even in the absence of BMP7. Morphometric analyses of SCG from wild‐type versus p75^NTR nullizygous mice at 3, 6, and 12 to 16 weeks of age indicated that genetic deletion of p75^NTR does not prevent dendritic growth but does stunt dendritic maturation in sympathetic neurons. These data support the hypotheses that p75^NTR is involved in downstream signaling events that mediate BMP7‐induced dendritic growth in sympathetic neurons, and suggest that p75^NTR signaling positively modulates dendritic complexity in sympathetic neurons in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1003–1013, 2016     

The O-glycosylated Stalk Domain Is Required for Apical Sorting of Neurotrophin Receptors in Polarized MDCK Cells

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75^NTR) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75^NTR causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75^NTR does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75^NTR, providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75^NTR is not required for apical sorting of either transmembrane or secreted forms.