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.     

The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor.

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk proto-oncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells, but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.     

脑源性神经营养因子受体trkB在NIH 3T3细胞上的表达

构建了克隆有大鼠脑源性神经营养因子（BDNF）受体trkB全长基因的真核表达载体pcDNA3.1( )-rat trkB.用脂质体介导法将重组载体转入小鼠NIH3T3细胞,在mRNA和蛋白质水平检测到了trkB基因在用G418筛选到的抗性NIH 3T3细胞中的表达,表达的trkB蛋白定位于细胞膜上。BDNF能够剂量依赖性地促进NIH 3T3－trkB细胞的增殖,说明表达的trkB是有功能的。该表达trkB和NIH3T3细胞为研究BDNF的生理功能、活性测定和从噬菌体展示肽库中筛选BDNF肽提供了一个简便的细胞模型。     

N-terminal tyrosine residues within the potassium channel Kir3 modulate GTPase activity of Galphai

trkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brain-derived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPase-activating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTPgammaS also prevented Kir3 deactivation, and the GTPgammaS effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for Gbetagamma but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation.     

Truncated trkB.T1 is dominant negative inhibitor of trkB.TK+-mediated cell survival

Truncated trkB.T1 (T1) neurotrophin receptor inhibits full-length trkB.TK+ (TK+) signaling. At least two possible mechanisms have been proposed for this action: T1 could trap the ligand or function as a dominant negative receptor. To differentiate between these possibilities we have studied survival of serum-deprived PC12-trkB cells stably expressing TK+. PC12-trkB cells were observed to display constitutive trkB kinase activity which leads to survival of a cell subpopulation in the absence of added brain-derived neurotrophic factor (BDNF) and serum. Exogenous BDNF significantly increased cell survival, and this increase was inhibited by BDNF neutralizing antibody. The antibody treatment had no effect on the constitutive TK+ activity. Transfected T1 completely inhibited survival by BDNF or constitutive trkB kinase activity in PC12-trkB cells similarly to tyrosine kinase inhibitor K252a. In addition, T1 coimmunoprecipitated with TK+ and inhibited its autophosphorylation by BDNF. These data suggest that truncated T1 inhibits TK+ signaling by dominant negative action.     

The binding epitopes of neurotrophin-3 to its receptors trkC and gp75 and the design of a multifunctional human neurotrophin.

Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin-3 (NT-3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta-strand bundle and in contrast to NGF does not make use of non-conserved loops and the six N-terminal residues. The gp75 epitope is dominated by loop residues and the C-terminus of NT-3. A novel rapid biological screening procedure allowed the identification of NT-3 mutants that are able to signal efficiently through the non-preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT-3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF-, BDNF- and NT-3-dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.     

trkB-Receptor Activation Contributes to the Kainate-Induced Increase in BDNF mRNA Synthesis

1. The expression of brain-derived neurotrophic factor (BDNF) mRNA is induced by neuronal activity through increased intracellular calcium. As BDNF also increases intracellular calcium levels through trkB activation, we have examined here whether BDNF also regulates the synthesis of its own mRNA.2. Neurotrophin mRNA expression was induced with kainic acid administration in transgenic mice overexpressing the dominant-negative form of BDNF receptor trkB and wild-type littermates.3. Kainate strongly induced BDNF mRNA expression in both genotypes, but the upregulation was significantly lower in transgenic mice.4. These data suggest that the synthesis of BDNF mRNA is at least partly mediated by BDNF release and the activation of trkB receptors. The present findings further suggest that the BDNF signaling system in brain is regulated by positive feedback.     

Truncated TrkB: beyond a dominant negative receptor

BDNF activates trkB receptors to regulate neuronal survival, differentiation, and proliferation. Mutations in the BDNF gene, altered BDNF expression, and altered trkB expression are associated with degenerative and psychiatric disorders. The full-length trkB receptor (trkB.tk(+)) undergoes autophosphorylation to activate intracellular signaling pathways. The truncated trkB receptor (trkB.t1) is abundantly expressed in the brain but lacks the catalytic tyrosine kinase domain. TrkB.t1 is a dominant-negative receptor that inhibits trkB.tk(+) signaling. While this is an important function of trkB.t1, it is only one of its many functions. TrkB.t1 sequesters and translocate BDNF, induces filopodia and neurite outgrowth, stimulates intracellular signaling cascades, regulates Rho GTPase signaling, and modifies cytoskeletal structures. TrkB.t1 is an active signaling molecule with regulatory effects on neurons and astrocytes.     

NMDA receptor activation modulates programmed cell death during early post-natal retinal development: a BDNF-dependent mechanism

Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate-mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate-induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N-methyl-d-aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin-4 (NT-4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT-4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK-801 increased natural cell death of specific cell populations of the post-natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF-dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.     

Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors.

Neurotrophins, such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), are potent regulators of neuronal functions. Here we show that human immune cells also produce NT-3 mRNA, secrete BDNF, and express their specific receptors trkB and trkC. The truncated trkB receptor, usually expressed in sensory neurons of the central nervous system, was also constitutively expressed in unstimulated Th cells. Full-length trkB was detectable in stimulated PBMC, B cell lines, and Th1, but not in Th2 and Th0 cell clones. Clonally restricted expression was also observed for trkC, until now not detected on blood cells. The Th1 cytokine IL-2 stimulated production of trkB mRNA but not of trkC, whereas the Th2 cytokine IL-4 enhanced NT-3 but not BDNF mRNA expression. Microbial Ags, which influence the Th1/Th2 balance, could therefore modulate the neurotrophic system and thereby affect neuronal synaptic activity of the central nervous system.     

Differential response of neuronal cells to a fusion protein of ciliary neurotrophic factor/soluble CNTF-receptor and leukemia inhibitory factor.

Ciliary neurotrophic factor (CNTF) displays neurotrophic activities on motor neurons and neural cell populations both in vivo and in vitro. On target cells lacking intrinsic expression of specific receptor alpha subunits cytokines of the IL-6 family only act in the presence of their specific agonistic soluble receptors. Here, we report the construction and expression of a CNTF/soluble CNTF-receptor (sCNTF-R) fusion protein (Hyper-CNTF) with enhanced biological activity on cells expressing gp130 and leukemia inhibitory factor receptor (LIF-R), but not membrane-bound CNTF-R. At the cDNA level, the C-terminus of the extracellular domain of human CNTF-R (amino acids 1-346) was linked via a single glycine residue to the N-terminus of human CNTF (amino acids 1-186). Recombinant Hyper-CNTF protein was expressed in COS-7 cells. Hyper-CNTF efficiently induced dose-dependent STAT3 phosphorylation and proliferation of BAF-3 cells stably transfected with gp130 and LIF-R cDNAs. While on BAF3/gp130/LIF-R cells, Hyper-CNTF and LIF exhibited similar biological responses, the activity of Hyper-CNTF on pheochromocytoma cells (PC12 cells) was quite distinct from that of LIF. In contrast to LIF, Hyper-CNTF stimulated neurite outgrowth of PC12 cells in a time- and dose-dependent manner correlating with the ability to phosphorylate MAP kinases. These data indicate that although LIF and Hyper-CNTF use the same heterodimeric receptor complex of gp130 and LIFR, only Hyper-CNTF induces neuronal differentiation. The therapeutic potential of Hyper-CNTF as a superagonistic neurotrophin is discussed.     

Evidence that truncated TrkB isoform, TrkB-Shc can regulate phosphorylated TrkB protein levels

Tropomyosin receptor kinase B (TrkB) is best known as the receptor for brain-derived neurotrophic factor (BDNF). In humans, three major isoforms of TrkB, the full-length receptor (TrkB-TK+) and two C-terminal truncated receptors (TrkB-TK- and TrkB-Shc) are expressed in various tissues. In comparison to TrkB-TK+ and TrkB-TK-, TrkB-Shc is less well characterized. In this study, we analyzed the biological function of the TrkB-Shc receptor in response to exogenous BDNF treatment. In experiments transiently overexpressing TrkB-Shc in CHOK1 cells, we found that TrkB-Shc protein levels were rapidly decreased when cells were exposed to exogenous BDNF. When we assessed the functional impact of TrkB-Shc on TrkB-TK+ activity, we found that phosphorylated TrkB-TK+ protein levels were significantly decreased in the presence of TrkB-Shc and moreso following BDNF exposure. Interestingly, while the reduction of phosphorylated TrkB-TK+ protein was more pronounced in the presence of TrkB-Shc following BDNF exposure, the stability of TrkB-Shc protein itself was increased. Our findings suggest that cells may increase TrkB-Shc protein levels in response to exogenous BDNF exposure to regulate TrkB-TK+ activity by increasing degradation of activated receptor complexes as a means to prevent overactivation or inappropriate temporal and spatial activation of BDNF/TrkB-TK+ signaling.     

Expression of neurotrophins and Trk receptors in the developing,adult, and regenerating avian cochlea

We studied the expression of neurotrophins and their Trk receptors in the chicken cochlea. Based on in situ hybridization, brain-derived neurotrophic factor (BDNF) is the major neurotrophin there, in contrast to the mammalian cochlea, where neurotrophin-3 (NT-3) predominates. NT-3 mRNA labeling was weak and found only during a short time period in the early cochles. During embryogenesis, BDNF mRNA was first seen in early differentiating hair cells. Afferent cochlear neurons expressed trkB mRNA from the early stages of gangliogenesis onward. In accordance, in vitro, BDNF promoted survival of dissociated neurons and stimulated neuritogenesis from ganglionic explants. High levels of BDNF mRNA in hair cells and trkB mRNA in cochlear neurons persisted in the mature cochlea. In addition, mRNA for the truncated TrkB receptor was expressed in nonneuronal cells, specifically in supporting cells, located adjacent to the site of BDNF synthesis and nerve endings. Following acoustic trauma, regenerated hair cells acquired BDNF mRNA expression at early stages of differentiation. Truncated trkB mRNA was lost from supporting cells that regenerated into hair cells. High levels of BDNF mRNA persisted in surviving hair cells and trkB mRNA in cochlear neurons after noise exposure. These results suggest that in the avian cochlea, peripheral target-derived BDNF contributes to the onset and maintenance of hearing function by supporting neuronal survival and regulating the (re)innervation process. Truncated TrkB receptors may regulate the BDNF concentration available to neurites, and they might have an important role during reinnervation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 1019–1033, 1997