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.     

TrkA glycosylation regulates receptor localization and activity

The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-gamma. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity.     

Molecular kinetics of nerve growth factor receptor trafficking and activation

A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of approximately 24 +/- 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is approximately 138 +/- 4 min and is shortened to 86 +/- 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140(TrkA) from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75(NTR). An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75(NTR)-modulated decrease in TrkA transit time at the cell surface.     

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.     

Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.     

A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway

Rap1 transduces nerve growth factor (NGF)/tyrosine receptor kinase A (TrkA) signaling in early endosomes, leading to sustained activation of the p44/p42 mitogen-activated protein kinases (MAPK1/2). However, the mechanisms by which NGF, TrkA and Rap1 are trafficked to early endosomes are poorly defined. We investigated trafficking and signaling of NGF, TrkA and Rap1 in PC12 cells and in cultured rat dorsal root ganglion (DRG) neurons. Herein, we show a role for both microtubule- and dynein-based transport in NGF signaling through MAPK1/2. NGF treatment resulted in trafficking of NGF, TrkA and Rap1 to early endosomes in the perinuclear region of PC12 cells where sustained activation of MAPK1/2 was observed. Disruption of microtubules with nocodazole in PC12 cells had no effect on the activation of TrkA and Ras. However, it disrupted intracellular trafficking of TrkA and Rap1. Moreover, NGF-induced activation of Rap1 and sustained activation of MAPK1/2 were markedly suppressed. Inhibition of dynein activity through overexpression of dynamitin (p50) blocked trafficking of Rap1 and the sustained phase of MAPK1/2 activation in PC12 cells. Remarkably, even in the continued presence of NGF, mature DRG neurons that overexpressed p50 became atrophic and most (>80%) developing DRG neurons died. Dynein- and microtubule-based transport is thus necessary for TrkA signaling to Rap1 and MAPK1/2.     

SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocytoma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase Cgamma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kinase cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-Bbeta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF. TrkA bound to glutathione S-transferase fusion proteins containing SH2-Bbeta, and NGF stimulation dramatically increased that binding. In contrast, NGF was unable to stimulate the association of TrkA with a glutathione S-transferase fusion protein containing a mutant SH2-Bbeta(R555E) with a defective SH2 domain. When overexpressed in PC12 cells, SH2-Bbeta co-immunoprecipitated with TrkA in response to NGF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-Bbeta as well as exogenously expressed GFP-SH2-Bbeta but not GFP-SH2-Bbeta(R555E). Overexpression of SH2-Bbeta(R555E) blocked NGF-induced neurite outgrowth of PC12 cells, whereas overexpression of wild type SH2-Bbeta enhanced NGF-induced neurite outgrowth. Overexpression of either wild type or mutant SH2-Bbeta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholipase Cgamma in response to NGF or NGF-induced activation of ERK1/2, suggesting that SH2-Bbeta may initiate a previously unknown pathway(s) that is essential for NGF-induced neurite outgrowth. Taken together, these data indicate that SH2-Bbeta is a novel signaling molecule required for NGF-induced neuronal differentiation.     

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

NGF activates the phosphorylation of MAP1B by GSK3beta through the TrkA receptor and not the p75(NTR) receptor

We have recently shown that nerve growth factor (NGF) induces the phosphorylation of the microtubule-associated protein 1B (MAP1B) by activating the serine/threonine kinase glycogen synthase kinase 3beta (GSK3beta) in a spatio-temporal pattern in PC12 cells that correlates tightly with neurite growth. PC12 cells express two types of membrane receptor for NGF: TrkA receptors and p75NTR receptors, and it was not clear from our studies which receptor was responsible. We show here that brain-derived neurotrophic factor, which activates p75NTR but not TrkA receptors, does not stimulate GSK3beta phosphorylation of MAP1B in PC12 cells. Similarly, NGF fails to activate GSK3beta phosphorylation of MAP1B in PC12 cells that lack TrkA receptors but express p75NTR receptors (PC12 nnr). Chick ciliary ganglion neurons in culture lack TrkA receptors but express p75NTR and also fail to show NGF-dependent GSK3beta phosphorylation of MAP1B, whereas in rat superior cervical ganglion neurons in culture, NGF activation of TrkA receptors elicits GSK3beta phosphorylation of MAP1B. Finally, inhibition of TrkA receptor tyrosine kinase activity in PC12 cells and superior cervical ganglion neurons with K252a potently and dose-dependently inhibits neurite elongation while concomitantly blocking GSK3beta phosphorylation of MAP1B. These results suggest that the activation of GSK3beta by NGF is mediated through the TrkA tyrosine kinase receptor and not through p75NTR receptors.     

Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors.

The discoidin domain receptor (DDR1) is characterized by a discoidin I motif in the extracellular domain, an unusually long cytoplasmic juxtamembrane (JM) region, and a kinase domain that is 45% identical to that of the NGF receptor, TrkA. DDR1 also has a major splice form, which has a 37 amino acid insert in the JM region with a consensus Shc PTB site that is lacking in the shorter receptor. One class of ligands for the DDR receptors has recently been identified as being derived from the collagen family, but neither native PC12 cells, which express modest amounts of DDR1, nor transfected PC12 cells, which express much larger amounts of DDR1, respond to this ligand. A chimeric receptor, containing the extracellular domain of hPDGFRbeta fused to the transmembrane and intracellular regions of DDR1, also fails to mediate neuronal-like differentiation in stably transfected PC12 cells and is only weakly autophosphorylated. However, chimeric receptors, which are composed of combinations of intracellular regions from DDR1 and TrkA (with the extracellular domain of hPDGFRbeta), in some cases provided ligand (PDGF) -inducible receptor responses. Those with the TrkA kinase domain and the DDR1 JM regions were able to produce differentiation to varying degrees, whereas the opposite combination did not. Analysis of the signaling responses of the two chimeras with DDR1 JM sequences (with and without the insert) indicated that the shorter sequence bound and activated FRS2 whereas the insert-containing form activated Shc instead. Both activated PLCgamma through the carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue numbering). Mutation of this site (Y-->F) eliminated PLCgamma activation (indicating there are no other cryptic binding sites for PLCgamma in the DDR1 sequences) and markedly reduced the differentiative activity of the receptor. This is in contrast to TrkA (or PDGFRbeta/TrkA chimeras), where ablation of this pathway has no notable effect on PC12 cell morphogenic responses. Thus, the activation of FRS2 and Shc (leading to MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA alone, and the PLCgamma contribution becomes essential for full response. Nonetheless, both DDR1 JM regions contain potentially usable signaling sites, albeit they apparently are not activated directly in DDR1 (or DDR1 chimeras) in a ligand-dependent fashion. These findings suggest that the DDR1 receptors do have signaling capacity but may require additional components or altered conditions to fully activate their kinase domains and/or sustain the activation of the JM sites.     

Characterization of epidermal growth factor receptor function in lysophosphatidic acid signaling in PC12 cells

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.     

Sprouty4 is an endogenous negative modulator of TrkA signaling and neuronal differentiation induced by NGF

The Sprouty (Spry) family of proteins represents endogenous regulators of downstream signaling pathways induced by receptor tyrosine kinases (RTKs). Using real time PCR, we detect a significant increase in the expression of Spry4 mRNA in response to NGF, indicating that Spry4 could modulate intracellular signaling pathways and biological processes induced by NGF and its receptor TrkA. In this work, we demonstrate that overexpression of wild-type Spry4 causes a significant reduction in MAPK and Rac1 activation and neurite outgrowth induced by NGF. At molecular level, our findings indicate that ectopic expression of a mutated form of Spry4 (Y53A), in which a conserved tyrosine residue was replaced, fail to block both TrkA-mediated Erk/MAPK activation and neurite outgrowth induced by NGF, suggesting that an intact tyrosine 53 site is required for the inhibitory effect of Spry4 on NGF signaling. Downregulation of Spry4 using small interference RNA knockdown experiments potentiates PC12 cell differentiation and MAPK activation in response to NGF. Together, these findings establish a new physiological mechanism through which Spry4 regulates neurite outgrowth reducing not only the MAPK pathway but also restricting Rac1 activation in response to NGF.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.     

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.     

Nerve Growth Factor Receptor TrkA,a New Receptor in Insulin Signaling Pathway in PC12 Cells

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.     

Pincher,a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes

A central tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.     

Individual and combined effects of TrkA and p75NTR nerve growth factor receptors. A role for the high affinity receptor site

A long-standing question in neurotrophin signal transduction is whether heteromeric TrkA-p75NTR complexes possess signaling capabilities that are significantly different from homo-oligomeric TrkA or p75NTR alone. To address this issue, various combinations of transfected PC12 cells expressing a platelet-derived growth factor receptor-TrkA chimera and the p75NTR-selective nerve growth factor mutant (Delta9/13 NGF) were utilized to selectively stimulate TrkA or p75NTR signaling, respectively. The contribution of individual and combined receptor effects was analyzed in terms of downstream signaling and certain end points. The results suggest two unique functions for the high affinity heteromeric NGF receptor site: (a) integration of both the MAPK and Akt pathways in the production of NGF-induced neurite outgrowth, and (b) rapid and sustained activation of the Akt pathway, with consequent long term cellular survival. Whereas activation of TrkA signaling is sufficient for eliciting neurite outgrowth in PC12 cells, signaling through p75NTR plays a modulatory role, especially in the increased formation of fine, synaptic "bouton-like" structures, in which both TrkA and p75NTR appear to co-localize. In addition, a new interaction in the TrkA/p75NTR heteromeric receptor signal transduction network was revealed, namely that NGF-induced activation of the MAPK pathway appears to inhibit the parallel NGF-induced Akt pathway.     

Protein tyrosine phosphatase receptor type Z dephosphorylates TrkA receptors and attenuates NGF-dependent neurite outgrowth of PC12 cells

Protein tyrosine phosphatase receptor type Z (Ptprz/Ptpzeta / RPTPbeta) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Co-expression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Co-expression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signalling of TrkA receptors.     

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.     

Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography

Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF or membrane depolarization on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). Both NGF re-stimulation and high potassium-induced depolarization enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. However, the transition of uptake differed completely. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking, and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.