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.     

Temporal and spatial expression of neurotrophins and their receptors during male germ cell development.

Spermatogenesis is a stepwise cellular differentiation process involving proliferation and commitment to differentiate in spermatogonia, meiosis in spermatocytes, and morphological changes in round spermatids. The whole process is regulated by intercellular communication between the germ cells and the supporting cells. In order to investigate whether neurotrophin family and their receptors contribute to the intercellular communication, we examined the expression of neurotrophins and their receptors in testis during spermatogenesis. One of neurotrophin family, NT-3 was expressed in spermatocytes and spermatogonia while its high affinity receptor, TrkC was found mainly in late spermatids and their low affinity receptor, TrkA in spermatocytes and round spermatids. On the other hand, BDNF immunoreactivity was found in Sertoli cells while its high affinity receptor, TrkB was found in spermatogonia. The temporally and spatially regulated expression of neurotrophins, NT-3 and BDNF, and their receptors, TrkC and TrkB, during male germ cell development suggests that neurotrophins play as the paracrine factors in the intercellular communication between the germ cells and the supporting somatic cells to control germ cell development.     

Overcoming the inhibitors of myelin with a novel neurotrophin strategy

Myelin inhibitors activate a p75(NTR)-dependent signaling cascade in neurons that not only inhibits axonal growth but also prevents neurotrophins (NT) from stimulating growth. Most intriguingly, in addition to Trk receptors, neurotrophins also bind to p75(NTR). We have designed a "mini-neurotrophin" called B(AG) to activate TrkB in the absence of p75(NTR) binding. We find that B(AG) is as effective as the natural TrkB ligands (brain-derived neurotrophic factor (BDNF) and NT-4) at promoting neurite outgrowth from cerebellar neurons. Furthermore, the neurite outgrowth responses stimulated by BDNF and B(AG) are inhibited by a common set of reagents, including the Trk receptor inhibitor K252a, as well as protein kinase A and phosphoinositide 3-kinase inhibitors. However, in contrast to BDNF, B(AG) promotes growth in the presence of a myelin inhibitor or when antibodies directly activate the p75(NTR) inhibitory pathway. On the basis of this observation, we postulated that the binding of BDNF to the p75(NTR) might compromise the ability of BDNF to stimulate neurite outgrowth in an inhibitory environment. To test this, we used NGF, and an NGF-derived peptide, to compete for the BDNF/p75(NTR) interaction; remarkably, in the presence of either agent, BDNF acquired the ability to promote neurite outgrowth in the presence of a myelin inhibitor. The data suggest that in an inhibitory environment, the BDNF/p75(NTR) interaction compromises regeneration. Agents that activate Trk receptors in the absence of p75(NTR) binding, or agents that inhibit neurotrophin/p75(NTR) binding, might therefore be better therapeutic candidates than neurotrophins.     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。     

Functional analysis of mutant neurotrophins deficient in low-affinity binding reveals a role for p75LNGFR in NT-4 signalling.

The neurotrophins mediate their effects through binding to two classes of receptors, a tyrosine kinase receptor, member of the Trk family, and the low-affinity neurotrophin receptor, p75LNGFR, of as yet undefined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-directed mutagenesis, we have identified positively charged surfaces in BDNF, NT-3 and NT-4 that mediate binding to p75LNGFR. Arg31 and His33 in NT-3, and Arg34 and Arg36 in NT-4, located in an exposed hairpin loop, were found to be essential for binding to p75LNGFR. In BDNF, however, positively charged residues critical for p75LNGFR binding (Lys95, Lys96 and Arg97) were found in a spatially close but distinct loop region. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials revealed similar clusters of positively charged residues in each neurotrophin but with differences in their precise spatial locations. Disruption of this positively charged interface abolished binding to p75LNGFR but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity binding in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75LNGFR and TrkB, suggesting a role for p75LNGFR in regulating biological responsiveness to NT-4. These findings reveal a possible mechanism of ligand discrimination by p75LNGFR and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family members.     

TrkB mutant lacking the amino-terminal half of the extracellular portion acts as a functional brain-derived neurotrophic factor receptor.

A series of mutants with deletion in the extracellular portion of TrkB were expressed transiently and stably in mammalian cells to examine the brain-derived neurotrophic factor (BDNF)-binding properties of TrkB. We found that these binding activities were retained by the TrkB deletion mutant (TrkBDelta4) lacking most of the extracellular portion, cysteine-rich cluster 1 and 2, leucine-rich motif and most of the first immunoglobulin-like domain (Ig1). Furthermore, the results of the neurotrophin selectivity, the equilibrium binding constant, auto-phosphorylation and BDNF dependent cell survival indicate that TrkBDelta4 acts as a functional BDNF receptor comparable to wild-type TrkB. Thus, our findings showed that only the carboxyl-terminal half of the extracellular portion of TrkB, which includes the Ig2 domain, is essential for the functional BDNF receptor.     

Neurotrophins and neurotrophin receptors in pulmonary sarcoidosis - granulomas as a source of expression

Background

Pulmonary sarcoidosis is an inflammatory disease, characterized by an accumulation of CD4⁺ lymphocytes and the formation of non-caseating epithelioid cell granulomas in the lungs. The disease either resolves spontaneously or develops into a chronic disease with fibrosis. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been suggested to be important mediators of inflammation and mediate tissue remodelling. In support of this, we have recently reported enhanced NGF levels in the airways of patients with pulmonary sarcoidosis. However, less is known about levels of BDNF and NT-3, and moreover, knowledge in the cellular sources of neurotrophins and the distribution of the corresponding neurotrophin receptors in airway tissue in sarcoidosis is lacking.

Methods

The concentrations of NGF, BDNF and NT-3 in bronchoalveolar lavage fluid (BALF) of 41 patients with newly diagnosed pulmonary sarcoidosis and 27 healthy controls were determined with ELISA. The localization of neurotrophins and neurotrophin receptors were examined by immunohistochemistry on transbronchial lung biopsies from sarcoidosis patients.

Results

The sarcoidosis patients showed significantly enhanced NT-3 and NGF levels in BALF, whereas BDNF was undetectable in both patients and controls. NT-3 levels in BALF were found higher in patients with non-Löfgren sarcoidosis as compared to patients with Löfgren''s syndrome, and in more advanced disease stage. Epithelioid cells and multinucleated giant cells within the sarcoid granulomas showed marked immunoreactivity for NGF, BDNF and NT-3. Also, immunoreactivity for the neurotrophin receptor TrkA, TrkB and TrkC, was found within the granulomas. In addition, alveolar macrophages showed positive immunoreactivity for NGF, BDNF and NT-3 as well as for TrkA, TrkB and TrkC.

Conclusions

This study provides evidence of enhanced neurotrophin levels locally within the airways of patients with sarcoidosis. Findings suggest that sarcoid granuloma cells and alveolar macrophages are possible cellular sources of, as well as targets for, neurotrophins in the airways of these patients.     

An immunoglobulin-like domain determines the specificity of neurotrophin receptors.

The neurotrophins influence survival and maintenance of vertebrate neurons in the embryonic, early post-natal and post-developmental stages of the nervous system. Binding of neurotrophins to receptors encoded by the gene family trk initiates signal transduction into the cell. trkA interacts preferably with nerve growth factor (NGF), trkB with brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and trkC with neurotrophin-3 (NT-3). By constructing 17 different chimeras and domain deletions of the human trk receptors and analyzing their binding affinities to the neurotrophins we have shown that an immunoglobulin-like domain located adjacent to the transmembrane domain is the structural element that determines the interaction of neurotrophins with their receptors. Chimeras of trkC where this domain was exchanged for the homologous sequences from trkB or trkA gained high affinity binding to BDNF or NGF respectively, while deletion of this domain in trkC or trkA abolished binding to NT-3 or NGF respectively. This domain alone retained affinities to neurotrophins similar to the full-length receptors and when expressed on NIH 3T3 cells in fusion with the kinase domain showed neurotrophin-dependent activation.     

Specificity in Trk receptor:neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5.

BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease. RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus. CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

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.     

The nerve growth factor receptor: a multicomponent system that mediates the actions of the neurotrophin family of proteins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) are members of a family of structurally related proteins termed neurotrophins that promote the growth and survival of neurons in the central and peripheral nervous systems. Each of these proteins bind to at least two membrane receptors. One is the low affinity nerve growth factor receptor (p75), which binds each member of the neurotrophin family. The other is one of a family of tyrosine kinase receptors —trkA binds only NGF, the relatedtrkB receptor binds BDNF and NT-3, andtrkC binds NT-3 alone. This article reviews kinetic and biochemical information on p75 and its relationship to thetrk gene products.     

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.     

Effects of castration on the immunoreactivity to NGF, BDNF and their receptors in the pelvic ganglia of the male rat

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and are members of the neurotrophin family, a family of neurotrophic factors that also includes neurotrophin (NT) 3 and NT4/5. Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems. Neurotrophins exert their effects by binding to corresponding receptors which are formed by the tyrosine protein kinases TrkA, TrkB and TrkC, and the low affinity neurotrophic receptor (p75NTR). In the present study, using immunohistochemistry and quantitative analysis, we have investigated immunoreactivity to BDNF, NGF, TrkB, p75NTR and TrkA in the pelvic ganglia of normal and castrated rats. Neurons of the pelvic ganglia expressed both these neurotrophins and their receptors. After castration the immunoreactivity persisted. However, the number of BDNF- and p75NTR-IR cells statistically significant decreased after castration. These results suggest that castration modulates the expression of neurotrophins and their receptors in pelvic autonomic neurons.     

p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.

Neurotrophins signal via Trk tyrosine kinase receptors and a common receptor called p75. Nerve growth factor is the cognate ligand for TrkA, brain-derived neurotrophic factor for TrkB, and neurotrophin-3 (NT-3) for TrkC. NT-3 also binds TrkA and TrkB as a heterologous ligand. All neurotrophins bind p75, which regulates ligand affinity and Trk signals. Trk extracellular domain has five subdomains: a leucine-rich motif, two cysteine-rich clusters, and immunoglobulin-like subdomains IgG-C1 and IgG-C2. The IgG-C1 subdomain is surface exposed in the tertiary structure and regulates ligand-independent activation. The IgG-C2 subdomain is less exposed but regulates cognate ligand binding and Trk activation. NT-3 as a heterologous ligand of TrkA and TrkB optimally requires the IgG-C2 but also binds other subdomains of these receptors. When p75 is co-expressed, major changes are observed; NGF-TrkA activation can occur also via the cysteine 1 subdomain, and brain-derived neurotrophic factor-TrkB activation requires the TrkB leucine-rich motif and cysteine 2 subdomains. We propose a two-site model of Trk binding and activation, regulated conformationally by the IgG-C1 subdomain. Moreover, p75 affects Trk subdomain utilization in ligand-dependent activation, possibly by conformational or allosteric control.     

The neurotrophin receptor TrkB is colocalized to mitochondrial membranes

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.     

p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.     

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.