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PubMed2006

P2Y2 and TrkA receptors interact with Src family kinase for neuronal differentiation.

The crosstalk between the P2Y(2) G-protein-coupled receptor (GPCR) with TrkA receptor tyrosine kinase (RTK) is an important mechanism that regulates neuronal differentiation. We show that Src family kinases (SFK) regulate P2Y(2)-TrkA molecular crosstalk. SFK inhibitors block ATPgammaS/P2Y(2)-promoted enhancement of NGF/TrkA signaling and neuronal differentiation in PC12 cells, abrogate the enhancement by ATPgammaS of neurite outgrowth in primary cultures of dorsal root ganglion neurons, and block co-immunoprecipitation of TrkA, P2Y(2) receptors and SFK. These results identify SFK as mediating nucleotide-enhanced neurotrophin-dependent neuronal differentiation and thus, as a key convergence point for interaction between RTKs and GPCRs.

Adenosine Triphosphate↗
PubMed2002

Nerve growth factor signals via preexisting TrkA receptor oligomers.

Nerve growth factor (NGF) promotes neuronal survival and differentiation by activating TrkA receptors. Similar to other receptor tyrosine kinases, ligand-induced dimerization is thought to be required for TrkA receptor activation. To study this process, we expressed TrkA receptors in Xenopus laevis oocytes and analyzed their response to NGF by using a combination of functional, biochemical, and structural approaches. TrkA receptor protein was detected in the membrane fraction of oocytes injected with TrkA receptor cRNA, but not in uninjected or mock-injected oocytes. Application of NGF to TrkA receptor-expressing oocytes promoted tyrosine phosphorylation and activated an oscillating transmembrane inward current, indicating that the TrkA receptors were functional. Freeze-fracture electron microscopic analysis demonstrated novel transmembrane particles in the P-face (protoplasmic face) of oocytes injected with TrkA cRNA, but not in uninjected or mock injected oocytes. Incubating TrkA cRNA-injected oocytes with the transcriptional inhibitor actinomycin D did not prevent the appearance of these P-face particles or electrophysiological responses to NGF, demonstrating that they did not arise from de novo transcription of an endogenous Xenopus oocyte gene. The appearance of these particles in the plasma membrane correlated with responsiveness to NGF as detected by electrophysiological analysis and receptor phosphorylation, indicating that these novel P-face particles were TrkA receptors. The dimensions of these particles (8.6 x 10 nm) were too large to be accounted for by TrkA monomers, suggesting the formation of TrkA receptor oligomers. Application of NGF did not lead to a discernible change in the size or shape of these TrkA receptor particles during an active response. These results indicate that in Xenopus oocytes, NGF activates signaling via pre-formed TrkA receptor oligomers.

Animals↗
PubMed1997

Nerve growth factor induces apoptosis in human medulloblastoma cell lines that express TrkA receptors.

Neurotrophins act through their cognate receptors to promote the differentiation and/or survival of neuronal progenitor cells, immature neurons, and other cells. Here, we examined the effects of nerve growth factor (NGF) and its cognate receptor (Trk or TrkA) on the survival of a common childhood brain tumor, i.e., medulloblastoma, a tumor that resembles CNS neuroepithelial progenitor cells. To do this, we engineered two human medulloblastoma cell lines (i.e., D283MED and DAOY cells) to express human TrkA using a retroviral expression vector. Surprisingly, NGF-treated medulloblastoma cells expressing the TrkA receptor (D283trk and DAOYtrk cells) grown in the presence or absence of serum underwent massive apoptosis, but similar treatment did not induce apoptosis in wild-type uninfected cells, cells expressing an empty vector, or cells expressing the TrkC receptor. Furthermore, D283MED cells engineered to express the human p75 NGF receptor (D283p75) also did not undergo apoptosis. Significantly, NGF-induced apoptosis in D283trk and DAOYtrk cells can be inhibited by anti-NGF antibodies and by K-252a, an inhibitor of TrkA tyrosine phosphorylation and mimicked by high concentrations of NT3. Because NGF treatment primarily eliminated D283trk cells from the S phase of the cell cycle, this form of NGF-mediated apoptosis is cell cycle-dependent. These findings suggest that a NGF/TrkA signal transduction pathway could activate apoptotic cell death programs in CNS neuroepithelial progenitor cells and in childhood brain tumors.

3T3 Cells↗
PubMed2004

A novel p75 neurotrophin receptor-related protein, NRH2, regulates nerve growth factor binding to the TrkA receptor.

Nerve growth factor (NGF) functions as a ligand for two receptors, the TrkA tyrosine kinase receptor and the p75 neurotrophin receptor (p75NTR). The Ig-like domains of Trk receptors and the cysteine-rich repeats of p75NTR are involved in binding to the neurotrophins. Recently, a closely related gene to p75NTR called neurotrophin receptor homolog-2 (NRH2) was identified; however, the function of NRH2 and its relevance to neurotrophin signaling are unclear. NRH2 contains a similar transmembrane and intracellular domain as p75NTR but lacks the characteristic cysteine-rich repeats in the extracellular domain. Here we show that NRH2 is expressed in several neuronal populations that also express p75NTR and Trk receptors. NRH2 does not bind to NGF; however, coimmunoprecipitation experiments demonstrate that NRH2 is capable of interacting with TrkA receptors. Coexpression of NRH2 with TrkA receptors resulted in the formation of high-affinity binding sites for NGF. These results indicate that a transmembrane protein related to p75NTR is capable of modulating Trk receptor binding properties.

Amino Acid Sequence↗
PubMedDate not supplied

NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.

The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.

3T3 Cells↗
PubMed2005

Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight.

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.

Amino Acid Sequence↗
PubMed1994

An alternatively spliced form of the nerve growth factor receptor TrkA confers an enhanced response to neurotrophin 3.

TrkA, a member of the receptor tyrosine kinase family, binds nerve growth factor (NGF) and subsequently activates intracellular signaling pathways. Previous studies have found variable and weak interaction of the TrkA receptor with neurotrophin 3 (NT-3), another member of the NGF family. TrkA is expressed in two splice forms, differing in the presence of an 18-bp exon in the extracellular domain. The biological responses of each isoform of the TrkA receptor were tested after transfection into the cell line PC12nnr5, a variant of PC12 cells lacking functional TrkA protein. NGF was found to activate each form of the receptor comparably. However, the TrkA isoform containing the variable exon showed significantly higher activation by NT-3, which was detected by stimulation of TrkA autophosphorylation, induction of ZIF268 transcription, and cellular differentiation. Function-perturbing antibodies to the p75 low-affinity NGF receptor potentiated the NT-3 responses of both forms of TrkA in the transfected PC12nnr5 cell lines, suggesting that the low-affinity NGF receptor suppresses the ability of TrkA to respond to NT-3.

Alternative Splicing↗
PubMed1999

The Csk homologous kinase associates with TrkA receptors and is involved in neurite outgrowth of PC12 cells.

Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.

Animals↗
PubMed2003

Protective effect of HSV-mediated gene transfer of nerve growth factor in pyridoxine neuropathy demonstrates functional activity of trkA receptors in large sensory neurons of adult animals.

The distinct distribution of trkA receptors on small neurons and trkC receptors on large neurons in the dorsal root ganglion correlates with the dependence of these two classes of neurons on nerve growth factor and neurotrophin-3, respectively, for survival during development. In adult animals, the distribution of high affinity neurotrophin (trk) is complex and overlapping; neurotrophins are not required for cell survival, but may influence cell phenotype and the response to injury. In order to test the functional activity of trkA receptors in the sensory ganglia of adult animals in vivo, we examined the ability of a nerve growth factor-expressing recombinant replication-defective herpes simplex virus-based vector to prevent the selective degeneration of large sensory fibres caused by intoxication with pyridoxine. Transduction of dorsal root ganglion neurons in vivo by subcutaneous inoculation of the nerve growth factor-expressing vector prevented the development of pyridoxine-induced neuropathy measured by electrophysiological, morphological and behavioural measures. These results demonstrate a functional activity of trkA receptors expressed on large neurons in the dorsal root ganglion in mature animals; this observation has important implications for the choice of neurotrophic factors for treatment of peripheral nerve disease.

Animals↗
PubMed1999

Heterologous upregulation of nerve growth factor-TrkA receptors in PC12 cells by pituitary adenylate cyclase-activating polypeptide (PACAP).

The capacity for the neurotrophic factor PACAP38 to regulate expression of nerve growth factor (NGF)-trkA receptors in PC12 cells has been examined. Treatment of PC12 cells with 5 nM PACAP38 for 48 h elicited a 2.5-fold increase in 125I-NGF binding sites. FACS and Western analysis of trkA receptor protein indicate an abundance of receptors. The PACAP38-selective antagonist PACAP 6-38 blocked trkA receptor upregulation elicited by PACAP38. The expression of epidermal growth factor receptors was not affected by PACAP38 suggesting that upregulation of trkA represents a selective effect of this neurotrophic peptide. Similarly, expression of the pan-neurotrophin binding receptor p75 was not altered by PACAP38 treatment. In addition to effects on trkA observed in wild-type PC12 cells, PACAP38 stimulated an increase in the level of expressed human trkA receptors stably transfected into PC12 cells. PACAP38 provoked an increase in basal and NGF-stimulated phosphorylation of trkA. Enhanced phosphorylation of trkA was detected as early as 6 h following addition of PACAP38 and was maximal at 48 h. Increased incorporation of phosphate occurs on both serine and tyrosine residues of trkA. These results suggest that PACAP38 is able to promote upregulation of trkA receptors, an event associated with elevated serine/tyrosine phosphorylation of trkA.

Animals↗
PubMed2003

Interactions between p75 and TrkA receptors in differentiation and vulnerability of SN56 cholinergic cells to beta-amyloid.

NGF modifies cholinergic neurons through its low-p75 and high affinity-TrkA receptors. Native p75(+)TrkA(-) and trkA-transfected p75(+)TrkA(+) SN56 hybrid cholinergic septal cells were used here to discriminate effects mediated by each receptor. In TrkA(-) cells, NGF (100 ng/ml) affected neither choline acetyltransferase nor morphology but depressed pyruvate dehydrogenase activity by about 30%. Aged 25-35 beta-amyloid (1 microM) caused no changes in choline acetyltransferase and pyruvate dehydrogenase activities in nondifferentiated and differentiated TrkA(-) cells. On the contrary, in nondiferentiated TrkA(+) NGF brought about a 2.5-fold increase of choline acetyltransferase. In differentiated TrkA(+) cells, beta-amyloid resulted in no change in PDH but 65% suppression of choline acetyltransferase activity and reduction of their extensions. Thus, activation of TrkA receptors may overcome p75 receptor-mediated inhibitory effects on pyruvate dehydrogenase expression in cholinergic cells. On the other hand, it would make expression of choline acetyltransferase and cell differentiation more susceptible to suppressory effects of beta-amyloid.

Acetylcholine↗
PubMed2003

Characterization of nerve growth factors (NGFs) from snake venoms by use of a novel, quantitative bioassay utilizing pheochromocytoma (PC12) cells overexpressing human trkA receptors.

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays. The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin. These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80-90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF. The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.

Animals↗
PubMed1998

p75 and TrkA receptor signaling independently regulate amyloid precursor protein mRNA expression, isoform composition, and protein secretion in PC12 cells.

The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose-dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain-derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain-derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2-ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR-activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross-linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors--in contrast to p75NTR-associated signaling--stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.

Amyloid beta-Protein Precursor↗
PubMed2005

Selective small molecule peptidomimetic ligands of TrkC and TrkA receptors afford discrete or complete neurotrophic activities.

We designed a minilibrary of 55 small molecule peptidomimetics based on beta-turns of the neurotrophin growth factor polypeptides neurotrophin-3 (NT-3) and nerve growth factor (NGF). Direct binding, binding competition, and biological screens identified agonistic ligands of the ectodomain of the neurotrophin receptors TrkC and TrkA. Agonism is intrinsic to the peptidomimetic ligand (in the absence of neurotrophins), and/or can also be detected as potentiation of neurotrophin action. Remarkably, some peptidomimetics afford both neurotrophic activities of cell survival and neuronal differentiation, while others afford discrete signals leading to either survival or differentiation. The high rate of hits identified suggests that focused minilibraries may be desirable for developing bioactive ligands of cell surface receptors. Small, selective, proteolytically stable ligands with defined biological activity may have therapeutic potential.

Animals↗
PubMed2003

Establishment and characterization of pheochromocytoma tumor models expressing different levels of trkA receptors.

To date experimental in vivo pheochromocytoma (PC) models have not been available. A major in vitro PC model consists of PC12 cells that respond to nerve growth factor (NGF) by differentiation, mediated by the trkA receptor. We report the establishment of PC12 tumor models expressing low and high levels of trkA receptor in CD1 nude mice. The tumors are characterized by their responsiveness to NGF, karyotype, presence of enolase, and chromaffin granules, as well as dopamine release. These novel PC models facilitate research on the role of the trkA receptor in cancer and the development of trkA-selective anti-cancer agents.

Animals↗
PubMed2002

A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors.

Nerve growth factor (NGF) is a neurotrophin required for differentiation, development, and survival of the sympathetic nervous system, with many of its biological effects being mediated via trkA receptors. There is a need for a standard quantitative bioassay for NGF, to be used in basic research and in pharmaceutical studies. The objective of the present research was to develop a selective, quantitative, and reliable bioassay for NGF, using a morphological criterion: neurite cell outgrowth. In addition, we aimed to apply the aforementioned bioassay to measure NGF administered to mice. Pheochromocytoma PC12 cell variants including wild-type cultures, and a trkA-overexpressing stable transfectant PC12-6.24-I, PC12nnr5, and PC12EN lacking trkA receptors, were used. Dose-response curves were generated with NGF beta-subunit (2.5S) purified from mouse submaxillary glands. Our results demonstrated that the bioassay was sensitive to 0.3-20 ng/mL, and selective, as neurite outgrowth was not seen by any other growth factor other than NGF. In addition, variant clones PC12nnr5 and PC12EN, lacking trkA receptors, did not respond to NGF. The bioassay detected NGF in serum of mice injected with NGF. This novel developed bioassay can serve as a model system for various neuroscience purposes.

Animals↗
PubMed1997

Reduction in p140-TrkA receptor protein within the nucleus basalis and cortex in Alzheimer's disease.

It has been hypothesized that the diminished transport of nerve growth factor (NGF) seen within cholinergic basal forebrain (CBF) neurons in Alzheimer's disease (AD) results from a defect in the expression of its high-affinity trkA receptor. The present study used an anti-human trkA-specific monoclonal antibody (mAb 5C3) that recognizes the NGF docking site, combined with quantitative optical densitometry, to evaluate whether expression of the trkA protein is altered within the nucleus basalis and its cortical projection sites in AD. In normal aged humans, trkA immunoreactivity revealed a continuum of positive neurons extending throughout all CBF subfields. In addition, trkA-positive neurons were scattered throughout the olfactory tubercle and striatum. These regions also displayed intense trkA neuropil staining. Although fewer in total number, remaining CBF perikarya in AD displayed a significant decrease in trkA levels relative to aged controls. Biochemical analysis revealed a significant reduction in trkA protein within both the nucleus basalis and the frontal cortex in AD relative to aged controls. In contrast, trkA levels in the caudate nucleus were unaffected. The decrease in trkA protein in conjunction with our recent observations that the message for trkA is reduced within individual CBF neurons in AD supports the concept that defects in the production and/or utilization of the trkA receptor may be a key event mediating degeneration of NGF-responsive CBF neurons in this disease.

Aged↗