Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 ras

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors, TrkB and TrkC. In contrast to BDNF, NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLCγ were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons. Special issue dedicated to Dr. Hans Thoenen.     

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC₅₀ value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100–150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.     

NT-3 modulates BDNF and proBDNF levels in naïve and kindled rat hippocampus

Both mature and precursor forms of neurotrophins regulate nerve development, survival and plasticity. Brain-derived neurotrophic factor (BDNF) synthesis and secretion in turn are regulated by neuronal activity, such as epilepsy. Further, neurotrophins themselves are regulated by neurotrophin levels. Neurotrophin-3 (NT-3) and BDNF in particular can be co-expressed and each can regulate the levels of the other. This regulation is thought to be mediated through receptor tyrosine kinase (Trk) activity. It is not known whether this neurotrophin-neurotrophin interaction occurs in hippocampal tissue in vivo, or how it is influenced by neuronal activation. In this study, we explored the reciprocal influences of intraventricular infusions of NT-3 and BDNF in na?ve and kindled hippocampi of rats using Western blotting. We confirm that hippocampal kindling resulted in a significant increase in levels of BDNF both in cytochrome C (control) infused and NT-3 infused kindled rats. However, NT-3 infusion significantly reduced BDNF levels in both kindled and non-kindled hippocampi compared to their cytochrome C infused counterparts. These results are consistent with our earlier studies demonstrating lowered levels of TrkA and TrkC (NGF modulates BDNF levels via TrkA) following chronic NT-3 infusion. Although kindling led to an increase in BDNF, this was not accompanied by any detectable change in the levels of proBDNF. However, there was a significant increase in proBDNF following NT-3 infusions, suggesting NT-3 may reduce proBDNF processing. In contrast, neither NT-3 nor proNT-3 levels were affected by kindling or chronic BDNF infusions, consistent with down-regulation of TrkB by chronic BDNF infusion. Thus, modulation of BDNF by NT-3, likely mediated by Trk receptors, occurs in na?ve and kindled adult rat hippocampus.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR.     

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.     

Neurotrophin-3 and Brain-Derived Neurotrophic Factor Activate Multiple Signal Transduction Events but Are Not Survival Factors for Hippocampal Pyramidal Neurons

Abstract: Expression of the neurotrophin-3 (NT-3) receptor (TrkC) and the effects of NT-3 on signal transduction were investigated in highly enriched populations of embryonic rat hippocampal pyramidal neurons grown in bilaminar cultures. PCR analysis revealed that the predominant trkC isoform is K1, which lacks an insert in the kinase domain. Polyclonal TrkC-specific antibodies stained >90% of the neurons and revealed a single ～145-kDa protein in immunoblots of extracts from adult hippocampus and pyramidal neuron cultures. Addition of NT-3 (50 mg/ml) to these cultures induced the tyrosine phosphorylation of TrkC but not TrkB, as determined by anti-phosphotyrosine staining of immunoprecipitates; thus, all the effects of NT-3 are mediated through TrkC. NT-3 also increased the tyrosine phosphorylation of 42-, 44-, 49-, 55-, 95-, and 145-kDa proteins; the pattern induced by brain-derived neurotrophic factor (BDNF) was similar but not identical to that induced by NT-3, suggesting that subtle differences may exist in signaling by TrkB and TrkC receptors. Immunoprecipitation of p21^ras from ³²P-prelabeled cells showed that NT-3 increased the level of the GTP-bound form of the protein threefold over the control within 5 min. Mitogen-activated protein (MAP) kinase activity was maximally elevated by NT-3 within 2 min and then returned slowly toward baseline over the next 60 min. Tyrosine phosphorylation of phospholipase C-γ increased rapidly after NT-3, suggesting that this enzyme becomes activated. Consistent with this, the neurotrophin rapidly increased protein kinase C activity as well as intracellular Ca²⁺ levels. The effects of both NT-3 and BDNF on Ca²⁺ levels were attenuated in Ca²⁺-free medium, suggesting that both neurotrophins increase Ca²⁺ flux across the plasma membrane as well as release from internal stores. NT-3 also increased c-Fos expression in >80% of the cells; the effect peaked at 30 minand declined to baseline by 120 min. Despite the activation of ras-MAP kinase and phosphoinositide signaling pathways, neither NT-3 nor BDNF alone or in combination could sustain hippocampal pyramidal neurons deprived of glial support. We conclude that in this system NT-3 and BDNF do not appear to be acting as classical “neurotrophic” factors and that activation of the MAP kinase pathway is insufficient for the promotion of neuronal survival.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

A novel role for BDNF-TrkB in the regulation of chemotherapy resistance in head and neck squamous cell carcinoma

Mechanisms of resistance for HNSCC to cisplatin (CDDP), the foundational chemotherapeutic agent in the treatment of this disease, remain poorly understood. We previously demonstrated that cisplatin resistance (CR) can be overcome by targeting Trk receptor. In the current study, we explored the potential mechanistic role of the BDNF-TrkB signaling system in the development of CDDP resistance in HNSCC. Utilizing an in vitro system of acquired CR, we confirmed a substantial up-regulation of both BDNF and TrkB at the protein and mRNA levels in CR cells, suggesting an autocrine pathway dysregulation in this system. Exogenous BDNF stimulation led to an enhanced expression of the drug-resistance and anti-apoptotic proteins MDR1 and XiAP, respectively, in a dose-dependently manner, demonstrating a key role for BDNF-TrkB signaling in modulating the response to cytotoxic agents. In addition, modulation of TrkB expression induced an enhanced sensitivity of cells to CDDP in HNSCC. Moreover, genetic suppression of TrkB resulted in changes in expression of Bim, XiAP, and MDR1 contributing to HNSCC survival. To elucidate intracellular signaling pathways responsible for mechanisms underlying BDNF/TrkB induced CDDP-resistance, we analyzed expression levels of these molecules following inhibition of Akt. Inhibition of Akt eliminated BDNF effect on MDR1 and Bim expression in OSC-19P cells as well as modulated expressions of MDR1, Bim, and XiAP in OSC-19CR cells. These results suggest BDNF/TrkB system plays critical roles in CDDP-resistance development by utilizing Akt-dependent signaling pathways.     

Experience‐dependent regulation of TrkB isoforms in rodent visual cortex

Within primary visual cortex (V1), brain‐derived neurotrophic factor (BDNF) signaling through its high‐affinity receptor TrkB is important for normal development and experience‐dependent plasticity. TrkB is expressed in several alternatively spliced isoforms, including full‐length TrkB (TrkB.FL), and several truncated isoforms (TrkB.T1, TrkB.T2, and TrkB.T4) that lack the intracellular tyrosine kinase domain. These isoforms are important components of BDNF signaling, yet little is known about the developmental or experience‐dependent regulation of their expression. Using immunohistochemistry, we found TrkB.FL and TrkB.T1 expressed in interneurons and pyramidal neurons within V1, but not in cortical astrocytes. We used real‐time PCR to quantify the changes in mRNA expression of BDNF, the four TrkB isoforms, and the low‐affinity receptor P75^NTR during normal development, and in response to visual deprivation at two different ages. BDNF expression increased between postnatal days 10 (P10) and P30, and was rapidly down‐regulated by 3 days of visual deprivation during both the pre‐critical period (P14‐P17) and the critical period (P18‐P21). Over the same developmental period, expression of each TrkB isoform was regulated independently; TrkB.T1 increased, TrkB.FL and TrkB.T2 decreased, and TrkB.T4 showed transient changes. Neither brief visual deprivation nor prolonged dark‐rearing induced changes in either TrkB.FL or TrkB.T1 expression. However, TrkB.T4 expression was reduced by brief visual deprivation, whereas TrkB.T4, TrkB.T2 and P75^NTR were up‐regulated by prolonged dark‐rearing into the critical period. Our data indicate that TrkB isoform expression can be selectively regulated by visual experience, and may contribute to experience‐dependent cortical plasticity. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.     

BDNF‐mediated migration of cardiac microvascular endothelial cells is impaired during ageing

This study indicates that brain‐derived neurotrophic factor (BDNF) can promote young cardiac microvascular endothelial cells (CMECs) to migrate via the activation of the BDNF‐TrkB‐FL‐PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction (MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only one of its truncated isoforms, TrkB‐T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology.     

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.