TrkA Mutant Research Articles

In vivo regulation of NGF-mediated functions by Nedd4-2 ubiquitination of TrkA.

Trk neurotrophin receptor ubiquitination in response to ligand activation regulates signaling, trafficking, and degradation of the receptors. However, the in vivo consequences of Trk ubiquitination remain to be addressed. We have developed a mouse model with a mutation in the TrkA neurotrophin receptor (P782S) that results in reduced ubiquitination due to a lack of binding to the E3 ubiquitin ligase, Nedd4-2. In vivo analyses of TrkAP782S indicate that defective ubiquitination of the TrkA mutant results in an altered trafficking and degradation of the receptor that affects the survival of sensory neurons. The dorsal root ganglia from the TrkAP782S knock-in mice display an increased number of neurons expressing CGRP and substance P. Moreover, the mutant mice show enhanced sensitivity to thermal and inflammatory pain. Our results indicate that the ubiquitination of the TrkA neurotrophin receptor plays a critical role in NGF-mediated functions, such as neuronal survival and sensitivity to pain.

Dual Innervation of Neonatal Merkel Cells in Mouse Touch Domes

Merkel cell-neurite complexes are specialized mechanosensory end organs that mediate discriminative touch sensation. It is well established that type I slowly adapting (SAI) mechanoreceptors, which express neural filament heavy chain (NFH), innervate Merkel cells. It was previously shown that neurotrophic factor NT3 and its receptor TrkC play crucial roles in controlling touch dome Merkel cell innervation of NFH+ fibers. In addition, nerve fibers expressing another neurotrophic tyrosine receptor kinase (NTRK), Ret, innervate touch dome Merkel cells as well. However, the relationship between afferents responsive to NT3/TrkC signaling and those expressing Ret is unclear. It is also controversial if these Ret+ fibers belong to the early or late Ret+ DRG neurons, which are defined based on the co-expression and developmental dependence of TrkA. To address these questions, we genetically traced Ret+ and TrkC+ fibers and analyzed their developmental dependence on TrkA. We found that Merkel cells in neonatal mouse touch domes receive innervation of two types of fibers: one group is Ret+, while the other subset expresses TrkC and NFH. In addition, Ret+ fibers depend on TrkA for their survival and normal innervation whereas NFH+ Merkel cell innervating fibers are almost unaltered in TrkA mutant mice, supporting that Ret+ and NFH+/TrkC+ afferents are two distinct groups. Ret signaling, on the other hand, plays a minor role for the innervation of neonatal touch domes. In contrast, Merkel cells in the glabrous skin are mainly contacted by NFH+/TrkC+ afferents. Taken together, our results suggest that neonatal Merkel cells around hair follicles receive dual innervation while Merkel cells in the glabrous skin are mainly innervated by only SAI mechanoreceptors. In addition, our results suggest that neonatal Ret+ Merkel cell innervating fibers most likely belong to the late but not early Ret+ DRG neurons.

P2‐390: Emerging relationship between development and degeneration: TrkA‐APP interaction as a therapeutic target in Alzheimer's disease

It has been proposed that disrupted retrograde transport of NGF-TrkA complexes underlies basal forebrain cholinergic neuron (BFCN) degeneration in Alzheimer's disease (AD). Indeed, targeted BFCN delivery of NGF has been found promising in AD treatment and is currently in a phase II clinical trial. The prevailing view of Alzheimer's disease (AD) is that amyloid-beta (Abeta) causes toxicity through chemical and physical mechanisms. Our data suggest that Abeta functions physiologically as an anti-trophin, and Abeta binding to APP induces APP processing to multiple peptides that mediate physiological neurite retraction and synaptic reorganization (Nat Med 6:397; PNAS 103:7130). An unbiased screen for compounds that block APP caspase cleavage identified ADDN1351, which reduced APP-C31 by 90%. Surprisingly, ADDN1351 proved to be a TrkA inhibitor. We then evaluated the effect of TrkA over-expression on APP-C31 production and cell death induction through transfection, Western blots and MTT assays. We also studied the interaction between APP and TrkA through co-immunoprecipitation and APP-Gal4 transactivation. Furthermore, we treated PDAPP transgenic mice with TrkA inhibitor GW441756 (10mg/kg, 5 days) and assayed the levels of Abeta and sAPPalpha through ELISA and AlphaLISA. TrkA over-expression increased APP-C31 and cell death. APP-C31 cleavage did not occur with the kinase-dead TrkA mutant or in the presence of TrkA inhibitor GW441756, whereas induction was enhanced by NGF. TrkA was shown to interact with APP in co-immunoprecipitation. In APP-Gal4 transactivation assays, TrkA inhibited the transactivation directed by Fe65 or Mint3/YAP by over 90%. Moreover, treatment of PDAPP transgenic mice with GW441756 not only decreased Abeta, but also increased sAPPalpha. These results suggest TrkA inhibition-rather than NGF activation-as a novel therapeutic approach, and raise the possibility that such an approach may counteract the hyperactive signaling resulting from the accumulation of active NGF-TrkA complexes due to reduced retrograde transport. The results also suggest that the optimal therapy for AD may involve both the delivery of NGF or NGF mimetics to basal forebrain cholinergic neuron somata and a TrkA inhibitor that is active more distally.

The K+ uptake regulator TrkA controls membrane potential, pH homeostasis and multidrug susceptibility in Mycobacterium smegmatis

Rifampicin is an important first-line antibiotic for the treatment of mycobacterial infections. Although most rifampicin-resistant strains arise through mutations in the rpoB gene in bacteria, a proportion of such strains show no rpoB mutations. This suggests that alternative mechanisms are responsible for rifampicin resistance. We have constructed and analysed a library of 11, 000 Mycobacterium smegmatis insertion mutants to find other possible rifampicin-resistance determinants. We found that disruption of trkA, a putative regulator of K(+) uptake, leads to increased rifampicin resistance. Our data indicate that TrkA-mediated K(+) uptake is essential for maintenance of the M. smegmatis growth rate, its pH homeostasis and membrane potential. In addition to increased rifampicin resistance, inactivation of trkA confers resistance to other hydrophobic agents, such as novobiocin, as well as increased susceptibility to isoniazid and positively charged aminoglycosides. Our results suggest that trkA is a general regulator of antibiotic susceptibility, and the changes in the multidrug susceptibility/resistance pattern detected in the trkA mutant are associated with membrane hyperpolarization. This study sheds light on the role of ion transport activity in intrinsic and acquired antibiotic resistance in mycobacteria.

Regulation of Trafficking of Activated TrkA Is Critical for NGF‐Mediated Functions

Upon activation by nerve growth factor (NGF), TrkA is internalized, trafficked and sorted through different endosomal compartments. Proper TrkA trafficking and sorting are crucial events as alteration of these processes hinders NGF-mediated functions. However, it is not fully known which proteins are involved in the trafficking and sorting of TrkA. Here we report that Nedd4-2 regulates the trafficking of TrkA and NGF functions in sensory neurons. Depletion of Nedd4-2 disrupts the correct sorting of activated TrkA at the early and late endosome stages, resulting in an accumulation of TrkA in these compartments and, as a result of the reduced trafficking to the degradative pathway, TrkA is either reverted to the cell surface through the recycling pathway or retrogradely transported to the cell body. In addition, Nedd4-2 depletion enhances TrkA signaling and the survival of NGF-dependent dorsal root ganglion neurons, but not those of brain-derived neurotrophic factor-dependent neurons. Furthermore, neurons from a knock-in mouse expressing a TrkA mutant that does not bind Nedd4-2 protein exhibit increased NGF-mediated signaling and cell survival. Our data indicate that TrkA trafficking and sorting are regulated by Nedd4-2 protein.

Impaired neurotrophin-3 signaling in a TrkAII mutant associated with hereditary polyneuropathy

Mutations of the neurotrophin receptor tyrosine kinase TrkA (NTRK1) cause congenital sensory neuropathy with insensitivity to pain and anhydrosis (CIPA), also called hereditary sensory and autonomous neuropathy type IV (HSAN IV). The neuronal splice variant of TrkA, TrkAII, binds two neurotrophin ligands, nerve growth factor (NGF) and neurotrophin-3 (NT3). Several studies have demonstrated NGF signaling defects in CIPA-associated TrkA mutants. To date, however, no study has examined NT3/TrkA signaling of CIPA mutants. As the interaction of NT3 and TrkA temporally and spatially precedes the interaction of NGF with TrkA, we examined the signaling of NT3 in a CIPA-associated TrkA mutant. Intriguingly, we revealed remarkable defects in NT3-induced ERK1/2 phosphorylation and neurite outgrowth. The impact of our findings is twofold. First, our data call for a re-examination of previously described TrkAII CIPA mutants regarding their NT3 signaling capability. Second, we envision that CIPA/HSAN IV polyneuropathies might fall into two different subgroups: one with diminished NT3/TrkAII signaling, in which axons actually do not reach their targets, and a second group with sufficient NT3/TrkAII signaling but diminished NGF/TrkAII signaling, in which axons do reach their targets, yet degenerate after successful target engagement.

Relation of potassium uptake to proton transport and activity of hydrogenases in Escherichia coli grown at low pH

Escherichia coli grown under anaerobic conditions in acidic medium (pH 5.5) upon hyperosmotic stress accumulates potassium ions mainly through the Kup system, the functioning of which is associated with proton efflux decrease. It was shown that H + secretion but not glucose-induced K + uptake was inhibited by N,N'-dicyclohexylcarbodiimide (DCC). The inhibitory effect of DCC on the H + efflux was stronger in the trkA mutant with defective potassium transport. The K + and H + fluxes depended on the extent of hyperosmotic stress in the absence or presence of DCC. The decrease in external oxidation/reduction potential and H 2 liberation insen- sitive to DCC were recorded. It was found that the atpD mutant with nonfunctional F 0 F 1 -ATPase produced a sub- stantial amount of H 2 , while in the hyc mutant (but not the hyf mutant defective in hydrogenases 3 (Hyd-3) and 4 (Hyd-4)) the H 2 production was significantly suppressed. At the same time, the rate of K + uptake was mark- edly lower in hyfR and hyfB-R but not in hycE or hyfA-B mutants; H + transport was lowered and sensitive to DCC in hyf but not in hyc mutants. The results point to the relationship of K + uptake with the Hyd-4 activity. Novel options of the expression of some hyf genes in E. coli grown at pH 5.5 are proposed. It is possible that the hyfB-R genes expressed under acidic conditions or their gene products interact with the gene coding for the Kup protein or directly with the Kup system.

Interaction of Mint2 with TrkA Is Involved in Regulation of Nerve Growth Factor-induced Neurite Outgrowth

TrkA receptor signaling is essential for nerve growth factor (NGF)-induced survival and differentiation of sensory neurons. To identify possible effectors or regulators of TrkA signaling, yeast two-hybrid screening was performed using the intracellular domain of TrkA as bait. We identified muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation assays showed that endogenous TrkA interacted with Mint2 in rat tissue homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2 overexpression inhibited NGF-induced neurite outgrowth in both PC12 and cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2 was found to promote the retention of TrkA in the Golgi apparatus and inhibit its surface sorting. Taken together, our data provide evidence that Mint2 is a novel TrkA-regulating protein that affects NGF-induced neurite outgrowth, possibly through a mechanism involving retention of TrkA in the Golgi apparatus.

A SAGE-based screen for genes expressed in sub-populations of neurons in the mouse dorsal root ganglion

BackgroundThe different sensory modalities temperature, pain, touch and muscle proprioception are carried by somatosensory neurons of the dorsal root ganglia. Study of this system is hampered by the lack of molecular markers for many of these neuronal sub-types. In order to detect genes expressed in sub-populations of somatosensory neurons, gene profiling was carried out on wild-type and TrkA mutant neonatal dorsal root ganglia (DRG) using SAGE (serial analysis of gene expression) methodology. Thermo-nociceptors constitute up to 80 % of the neurons in the DRG. In TrkA mutant DRGs, the nociceptor sub-class of sensory neurons is lost due to absence of nerve growth factor survival signaling through its receptor TrkA. Thus, comparison of wild-type and TrkA mutants allows the identification of transcripts preferentially expressed in the nociceptor or mechano-proprioceptor subclasses, respectively.ResultsOur comparison revealed 240 genes differentially expressed between the two tissues (P < 0.01). Some of these genes, CGRP, Scn10a are known markers of sensory neuron sub-types. Several potential markers of sub-populations, Dok4, Crip2 and Grik1/GluR5 were further analyzed by quantitative RT-PCR and double labeling with TrkA,-B,-C, c-ret, parvalbumin and isolectin B4, known markers of DRG neuron sub-types. Expression of Grik1/GluR5 was restricted to the isolectin B4+ nociceptive population, while Dok4 and Crip2 had broader expression profiles. Crip2 expression was however excluded from the proprioceptor sub-population.ConclusionWe have identified and characterized the detailed expression patterns of three genes in the developing DRG, placing them in the context of the known major neuronal sub-types defined by molecular markers. Further analysis of differentially expressed genes in this tissue promises to extend our knowledge of the molecular diversity of different cell types and forms the basis for understanding their particular functional specificities.

TrkA‐mediated Activation of the Rit GTPase

Members of the Ras family of small GTP binding proteins (Ras GTPases) control numerous cellular processes including cell growth and differentiation. Rit (Ras-like protein in tissues) is a novel member of Ras GTPase superfamily that that is widely expressed in both the developing and adult nervous system and has been shown to play critical roles in controlling neuronal differentiation and promoting pro-survival signaling pathways. In pheochromocytoma 6 cells (PC6), nerve growth factor stimulation activates Rit, and RNAi-mediated silencing of Rit disrupts NGF-mediated stimulation of both ERK and p38 MAP kinase pathways, implicating Rit signaling in NGF-mediated neuronal differentiation. In the present study, we report the characterization of the molecular mechanism of linking NGF stimulation to Rit activation. Activation of the TrkA receptor tyrosine kinase is responsible for NGF-mediated Rit activation, and analysis of a series of TrkA mutant receptors has identified the phospho-tyrosine residue responsible for coupling TrkA to Rit activation. Ongoing studies are characterizing the role of both signaling adapter proteins and Rit guanine nucleotide exchange proteins in this process. Collectively, our studies suggest that Rit represents a regulator of neurotrophin signaling, required to couple NGF stimulation to neuronal development and survival. (Source of research NIH NINDS)

NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels

Nociceptors, or pain-sensitive receptors, are unique among sensory receptors in that their sensitivity is increased by noxious stimulation. This process, called sensitization or hyperalgesia, is mediated by a variety of proinflammatory factors, including bradykinin, ATP and NGF, which cause sensitization to noxious heat stimuli by enhancing the membrane current carried by the heat- and capsaicin-gated ion channel, TRPV1. Several different mechanisms for sensitization of TRPV1 have been proposed. Here we show that NGF, acting on the TrkA receptor, activates a signalling pathway in which PI3 kinase plays a crucial early role, with Src kinase as the downstream element which binds to and phosphorylates TRPV1. Phosphorylation of TRPV1 at a single tyrosine residue, Y200, followed by insertion of TRPV1 channels into the surface membrane, explains most of the rapid sensitizing actions of NGF.

A double cysteine trkA mutant exhibiting reduced NGF binding and delayed Erk signaling.

The NGF receptor trkA is a tyrosine kinase receptor comprising an extracellular domain with a ligand-binding site, a transmembrane-spanning domain (TMD), and an intracellular domain composed of a juxtamembrane region (JMR), a tyrosine kinase domain, and a short carboxy-terminal tail. Nerve growth factor (NGF) binds and activates this receptor, leading to phosphorylation of signaling substrates involved in neuronal proliferation, differentiation, and survival. Human trkA contains one cysteine residue in the TMD (C423) and another, separated by 12 residues, in the JMR (C436). We hypothesized that the removal of one or both of the cysteines would affect NGF-induced signaling of the trkA receptor. Here we show that NGF induces rapid receptor autophosphorylation in a wild-type, trkA-expressing clone (WT11), in a single cysteine trkA mutants (C423T or C436A), but lower autophosphorylation activity in a double-cysteine trkA mutant (C423T/C436A). WT11 and SM cells had similar binding affinity, but that of DM cells was lower, according to the NGF radioreceptor assay. NGF-induced Erk phosphorylation was rapid in WT11 and C423T cells, but delayed in C436A and C423T/C436A cells. NGF induced [3H]thymidine incorporation into WT11 and SM cells, but had no effect on DM cells. However, basic fibroblast growth factor (bFGF) induced rapid phosphorylation of Erk1/2, and [3H]thymidine incorporation in NIH3T3, WT11, single mutant (SM), and double mutant (DM) cells, suggesting that the impaired NGF-induced Erk phosphorylation and thymidine incorporation observed in DM cells are due to the double-cysteine mutations in the trkA receptor. Cumulatively, our findings support a model in which Cys436 of the trkA is responsible for the rapid transfer of the transmembrane occupancy signal to the SHC adaptor protein for activation of the Ras-Erk pathway and DNA synthesis.

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor.

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.

Corneal innervation and sensitivity to noxious stimuli in trkA knockout mice.

Most primary sensory neurones depend on neurotrophins for survival. Mutant mice in which TrkA, the high-affinity receptor for nerve growth factor (NGF), has been inactivated lack nociceptive neurones in sensory ganglia and do not respond to noxious stimuli. The cornea of the eye is innervated by trigeminal neurones that are activated by noxious mechanical, thermal and chemical stimuli. In the human cornea, these stimuli evoke only sensations of pain. We have analysed the innervation pattern and the response to noxious stimulation of the cornea of trkA (-/-) mutant mice. Corneal nerves were stained with the gold chloride impregnation method. Corneal sensitivity to noxious stimuli was assessed by counting blinking movements evoked by von Frey hairs, topical application of saline at different temperatures and application of acetic acid and capsaicin at different concentrations. In the cornea of trkA (-/-) mutant animals, we observed a drastic reduction in the number of nerve trunks and branches in the corneal stroma. Furthermore, quantitative analysis of the number of thin nerve terminals revealed a marked decrease in the corneal epithelium of trkA (-/-) mice when compared to those present in wild type and trkA (+/-) animals. The blinking response of trkA (-/-) mice to mechanical, thermal and chemical noxious stimuli was also significantly reduced. These results indicate that the population of corneal sensory neurones is markedly depleted in trkA (-/-) mutant mice. However, a small portion of corneal sensory neurones survive in these mice suggesting that they may be NGF independent. On the basis of our results, we propose that these surviving cells are polymodal nociceptive neurones, sensitive to mechanical stimulation, noxious heat and acid.