Identification of 2-amino-5-(thioaryl)thiazoles as inhibitors of nerve growth factor receptor TrkA

2-Amino-5-(thioaryl)thiazoles are potent inhibitors of TrkA (e.g., 20h, TrkA IC(50)=0.6 nM) that show anti-proliferative effect in cellular assays. A proposed inhibitor binding mode to TrkA active site is consistent with key SAR observations.     

Lysine 63 polyubiquitination of the nerve growth factor receptor TrkA directs internalization and signaling

Nerve growth factor (NGF) binding to p75(NTR) influences TrkA signaling, yet the molecular mechanism is unknown. We observe that NGF stimulates TrkA polyubiquitination, which was attenuated in p75(-/-) mouse brain. TrkA is a substrate of tumor necrosis factor receptor-associated factor 6 (TRAF6), and expression of K63R mutant ubiquitin or an absence of TRAF6 abrogated TrkA polyubiquitination and internalization. NGF stimulated formation of a TrkA/p75(NTR) complex through the p62 scaffold, recruiting the E3/TRAF6 and E2/UbcH7. Peptide targeted to the TRAF6 binding site present in p62 blocked interaction with TRAF6 and inhibited ubiquitination of TrkA, signaling, internalization, and NGF-dependent neurite outgrowth. Mutation of K485 to R blocked TRAF6 and NGF-dependent polyubiquitination of TrkA, resulting in retention of the receptor on the membrane and an absence in activation of specific signaling pathways. These findings reveal that polyubiquitination serves as a common platform for the control of receptor internalization and signaling.     

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF).

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.     

Chagas' disease parasite promotes neuron survival and differentiation through TrkA nerve growth factor receptor

TrkA is a receptor tyrosine kinase activated primarily by nerve growth factor (NGF) to regulate differentiation, survival, and other important functions of neurons. Given the critical role TrkA plays in neural maintenance, it may be that microbial invaders of the nervous system utilize this receptor to reduce tissue damage for maximizing host-parasite equilibrium. Candidate pathogens could be those, like Trypanosoma cruzi, which may produce relatively little brain or nerve damage in long-lasting infections. We show here that T. cruzi, via its neuraminidase, binds TrkA in a NGF-inhibitable manner, induces TrkA autophosphorylation, and, through TrkA-dependent mechanisms, triggers phosphatidylinositol 3-kinase (PI3K)/Akt kinase signaling, cell survival, and neurite outgrowth. Unlike NGF, the neuraminidase does not react with the apoptosis-causing pan-neurotrophin receptor p75NTR. Therefore, these studies identify a novel and unique TrkA ligand in a microbial invader of the nervous system, raising the thus far unsuspected prospect of TrkA underlying clinical progression of an important human infectious disease.     

Mouse natural killer (NK) cells express the nerve growth factor receptor TrkA, which is dynamically regulated

Background

Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75^NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.

Methodology/Principal Findings

We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75^NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.

Conclusions/Significance

We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.     

TrkA amino acids controlling specificity for nerve growth factor

Neurotrophins are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the Trk family. The extracellular portion of the Trk receptors has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major neurotrophin-binding site in the Trk receptors resides in the second immunoglobulin-like domain. Although the individual amino acids in TrkA involved in binding to nerve growth factor (NGF) and those in TrkC involved in binding to neurotrophin-3 have been mapped in this domain, the Trk amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human TrkC second immunoglobulin-like domain, which does not bind nerve growth factor (NGF), were substituted with those from human TrkA. The resulting Trk variant recruited binding of NGF equivalent to TrkA, maintained neurotrophin-3 binding equivalent to TrkC, and also bound brain-derived neurotrophin, although with lower affinity compared with TrkB. This implies that the amino acids in the second immunoglobulin-like domain that determine Trk specificity are distinct for each Trk.     

Critical role for Kalirin in nerve growth factor signaling through TrkA

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

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

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

TrkA cross-linking mimics neuronal responses to nerve growth factor.

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that > or = 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.     

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.     

The Chagas' disease parasite Trypanosoma cruzi exploits nerve growth factor receptor TrkA to infect mammalian hosts

Trypanosoma cruzi, the agent of Chagas' disease, is an obligate intracellular parasite that invades various organs including several cell types in the nervous system that express the Trk receptor tyrosine kinase. Activation of Trk is a major cell-survival and repair mechanism, and parasites could use Trks to invade cells as a strategy to protect their habitat and prolong parasitism of vertebrate hosts. We show that T. cruzi binds to TrkA specifically and activates TrkA-dependent survival mechanisms. This interaction facilitates parasite adherence and promotes efficient invasion of neuronal, epithelial, and phagocytic cells via a process that requires TrkA kinase activity. Diffusible TrkA and TrkA-blocking agents neutralized infection in cellular and animal models of acute Chagas' disease, suggesting cellular receptors as therapeutic targets against parasitic diseases. Thus, TrkA, the nerve growth factor receptor commonly associated with neural survival and protection, may also underlie clinical progression of an important human parasitic disease.     

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

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

mKlf7, a potential transcriptional regulator of TrkA nerve growth factor receptor expression in sensory and sympathetic neurons

Development of the nervous system relies on stringent regulation of genes that are crucial to this process. TrkA, the receptor for nerve growth factor (NGF), is tightly regulated during embryonic development and is essential for the survival and differentiation of neural crest-derived sensory and sympathetic neurons. We have previously identified a mouse TrkA enhancer and have characterized several cis regulatory elements that are important for appropriate TrkA expression in vivo. We now report the cloning of a novel gene encoding a Kruppel-like factor from a mouse dorsal root ganglion expression library. This Kruppel-like factor, named mKlf7, binds specifically to an Ikaros core binding element that is crucial for in vivo TrkA enhancer function. Using in situ hybridization, we demonstrate that mKlf7 is coexpressed with TrkA in sensory and sympathetic neurons during embryogenesis and in adulthood. These data are consistent with the idea that mKlf7 may directly regulate TrkA gene expression in the peripheral nervous system.     

Facilitated intracellular transport of TrkA by an interaction with nerve growth factor

Intracellular transport of neurotrophin receptors together with neurotrophins is one of the key events of neurotrophin signaling for the growth and the survival of neurons. However, the involvement of neurotrophin signaling in the regulation of intracellular transport of neurotrophin receptors has been remained unclear. We visualized the behavior of TrkA, a receptor of nerve growth factor (NGF), by labeling with GFP in PC12 cells. We found remarkable changes of the behavior of TrkA-GFP upon the application of NGF. Before the application, only ~37% of the fluorescent dots of TrkA showed translocations along neurites of PC12 cells. After the application, number of the dots showing the directional movement increased to ~65%. The averaged velocities of the directional movement of TrkA-GFP dots became higher after the application of NGF. We tested the idea whether NGF binding accelerated the translocations of TrkA by simultaneously observing TrkA-GFP and fluorescently labeled NGF, Cy3.5-NGF. The velocity of TrkA-GFP dots associated with Cy3.5-NGF was remarkably higher than that of TrkA-GFP dots without Cy3.5-NGF. On the basis of these observations, we hypothesize that there is a signaling mechanism within a single vesicle that facilitates the intracellular transport of each vesicle containing the activated TrkA.