Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors

Nerve growth factor engages two structurally distinct transmembrane receptors, TrkA and p75, which have been proposed to create a "high-affinity" NGF binding site through formation of a ternary TrkA/NGF/p75 complex. To define a structural basis for the high-affinity site, we have determined the three-dimensional structure of a complete extracellular domain of TrkA complexed with NGF. The complex reveals a crab-shaped homodimeric TrkA structure, but a mechanism for p75 coordination is not obvious. We investigated the heterodimerization of membrane-bound TrkA and p75, on intact mammalian cells, using a beta-gal protein-protein interaction system. We find that NGF dimerizes TrkA and that p75 exists on the cell surface as a preformed oligomer that is not dissociated by NGF. We find no evidence for a direct TrkA/p75 interaction. We propose that TrkA and p75 likely communicate through convergence of downstream signaling pathways and/or shared adaptor molecules, rather than through direct extracellular interactions.     

The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.

Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

Characterization of symmetric complexes of nerve growth factor and the ectodomain of the pan-neurotrophin receptor, p75NTR

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.     

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

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

Changes in the expression of the nerve growth factor receptors TrkA and p75LNGR in the rat thymus with ageing and increased nerve growth factor plasma levels

The nerve growth factor (NGF) receptors p75LNGR and TrkA are expressed by thymic epithelial cells. Presumably, the NGF-TrkA system is involved in the paracrine communication between thymic epithelial cells and thymocytes, whereas the functional role of p75LNGR is still unknown. The thymus of vertebrates undergoes age-related changes that in part depend on hormonal factors. In order to find out whether thymic epithelial cells are responsive to NGF during the whole lifespan of the rat, we studied NGF receptor expression in the thymus from birth to 2 years of age, using immunohistochemistry. Furthermore, to evaluate whether increased plasma levels of NGF affected the ageing process, either NGF or 4-methylcatechol (4MC), an inductor of NGF synthesis, was administered. Both TrkA and p75LNGR were expressed by a subpopulation of thymic epithelial cells during the whole age range studied and their expression peaked at around 3 months. TrkA was primarily found in subcortical and medullary epithelial cells, whereas p75LNGR was seen in a subpopulation of medullary cells. Cortical epithelial cells, neural crest-derived cells, other stromal cells and thymocytes were not immunoreactive for NGF receptors. Neither the administration of NGF nor the increased NGF plasma levels obtained after 4MC treatment seemed to affect the ageing of the thymus as assessed by morphological and immunohistochemical criteria, but this increase in NGF levels did produce a shift in the expression of p75LNGR from epithelial cells to ED1-positive macrophages in animals of 6 months and older. Present results indicate that the expression of p75LNGR and TrkA in the rat thymus undergoes age-dependent changes that parallel those of epithelial cells. NGF could therefore be important for thymus homeostasis, possibly acting on epithelial cells. Nevertheless, NGF did not seem to be able to prevent the involution of this organ, although it produced a switch in the expression of p75LNGR, the significance of which remains to be established.     

An aging pathway controls the TrkA to p75NTR receptor switch and amyloid beta-peptide generation

Aging of the brain is characterized by marked changes in the expression levels of the neurotrophin receptors, TrkA and p75(NTR). An expression pattern in which TrkA predominates in younger animals switches to one in which p75(NTR) predominates in older animals. This TrkA-to-p75(NTR) switch is accompanied by activation of the second messenger ceramide, stabilization of beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1), and increased production of amyloid beta-peptide (Abeta). Here, we show that the insulin-like growth factor-1 receptor (IGF1-R), the common regulator of lifespan and age-related events in many different organisms, is responsible for the TrkA-to-p75(NTR) switch in both human neuroblastoma cell lines and primary neurons from mouse brain. The signaling pathway that controls the level of TrkA and p75(NTR) downstream of the IGF1-R requires IRS2, PIP3/Akt, and is under the control of PTEN and p44, the short isoform of p53. We also show that hyperactivation of IGF1-R signaling in p44 transgenic animals, which show an accelerated form of aging, is characterized by early TrkA-to-p75(NTR) switch and increased production of Abeta in the brain.     

Evidence for the participation of nerve growth factor and its low-affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their common low-affinity receptor p75^{neurotrophin receptor} (p75^NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite-promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell-derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF-mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75^NTR-overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor-overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75^NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high-affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low-affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998. © 1998 Wiley-Liss, Inc.     

Programmed cell death in the developing somites is promoted by nerve growth factor via its p75(NTR) receptor

Neurotrophins control neuron number during development by promoting the generation and survival of neurons and by regulating programmed neuronal death. In the latter case, the cell death induced by nerve growth factor (NGF) in the developing chick retina is mediated by p75(NTR), the common neurotrophin receptor (J. M. Frade, A. Rodriguez-Tebar, and Y.-A. Barde, 1996, Nature 383, 166-168). Here we show that NGF also induces the programmed death of paraxial mesoderm cells in the developing somites. Both NGF and p75(NTR) are expressed in the somites of chick embryos at the time and the place of programmed cell death. Moreover, neutralizing the activity of endogenous NGF with a specific blocking antibody, or antagonizing NGF binding to p75(NTR) by the application of human NT-4/5, reduces the levels of apoptotic cell death in both the sclerotome and the dermamyotome by about 50 and 70%, respectively. Previous data have shown that Sonic hedgehog is necessary for the survival of differentiated somite cells. Consistent with this, Sonic hedgehog induces a decrease of NGF mRNA in somite explant cultures, thus showing the antagonistic effect of NGF and Sonic hedgehog with respect to somite cell survival. The regulation of programmed cell death by NGF/p75(NTR) in a mesoderm-derived tissue demonstrates the capacity of neurotrophins and their receptors to influence critical developmental processes both within and outside of the nervous system.     

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

Identification and structure of the nerve growth factor binding site on TrkA

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.     

Association of a purine-analogue-sensitive protein kinase activity with p75 nerve growth factor receptors.

Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.     

Critical role for Kalirin in nerve growth factor signaling through TrkA

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.     

Evidence for a role of the nerve growth factor receptor TrkA in tyrosine phosphorylation and processing of beta-APP

The cytoplasmic tail of the beta-amyloid precursor protein (APP) contains a Y(682)ENPTY(687) sequence through which APP associates with phosphotyrosine binding (PTB) domain containing proteins in a tyrosine phosphorylation-independent manner. We have recently found that tyrosine phosphorylation of APP-Y(682) promotes docking of Shc proteins that modulate growth factor signaling to the ERK and PI3K/Akt pathways. We have also shown that APP is phosphorylated on Y(682) in cells that overexpress a constitutively active form of the tyrosine kinase abl. Here we present evidence that the nerve growth factor receptor TrkA may also promote phosphorylation of APP. Overexpression of TrkA, but not of mutated, kinase inactive TrkA resulted in tyrosine phosphorylation of APP. Site-directed mutagenesis studies showed that TrkA overexpression was associated with phosphorylation of APP-Y(682). Moreover, overexpression of TrkA also affected APP processing reducing the generation of the APP intracellular domain (AID). Thus, tyrosine phosphorylation of APP may functionally link APP processing and neurotrophic signaling to intracellular pathways associated with cellular differentiation and survival.     

Nerve growth factor stimulates MAPK via the low affinity receptor p75(LNTR)

Apart from its high affinity receptor TrkA, nerve growth factor (NGF) can also stimulate the low affinity receptor p75(LNTR) and induce a Trk-independent signaling cascade. We examined the possible involvement of mitogen-activated protein kinase (MAPK) in this signaling pathway in neuronal cultures of the cerebellum of P2-aged rats and PCNA cells; both cell types express p75(LNTR) but not TrkA. We found a fast and transient phosphorylation of p42- and p44-MAPK after stimulation with NGF or C(2)-ceramide which proved to be sensitive to inhibition of MAPK kinase and protein kinase A (PKA). As stimulation with NGF also activated p21Ras it can be concluded that at least part of the observed MAPK activation was effected via p21Ras and via PKA.     

Properties of the nerve growth factor receptor. Relationship between receptor structure and affinity

Microsomal membranes from A875 human melanoma cells contain nerve growth factor receptors (NGF-receptors) which appear to belong to a single class with homogeneous binding properties, as determined by Scatchard plots. NGF-receptors in these membrane preparations are also uniformly highly sensitive to tryptic proteolysis, and 125I-NGF bound to NGF-receptor in these membranes is rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, analysis of 125I-NGF dissociation kinetics indicated that two classes of NGF-receptor were present in these membranes. Thus, NGF-receptors can express either high or low affinity trypsin-sensitive states in addition to the high affinity trypsin resistant NGF-receptor state described previously (Buxser, S. E., Kelleher, D. J., Watson, L., Puma, P., and Johnson, G. L. (1983) J. Biol. Chem. 258, 3741-3749). The high affinity trypsin-sensitive and low affinity trypsin-sensitive states correlate with 200- and 90-kDa 125I-NGF X NGF-receptor complexes observed in photoaffinity cross-linking experiments. The absence of differences in peptide maps generated from the two sizes of NGF-receptor proteins together with structural and binding data strongly indicates that the 200-kDa NGF-receptor protein is a complex, probably a dimer, consisting of two 80-kDa NGF-receptor proteins associated with a single beta-NGF dimeric molecule. A model is proposed which relates structural states of NGF-receptors with specific receptor binding properties. The model provides an alternative explanation for binding phenomena previously attributed to negative cooperativity.