Trk is a calmodulin-binding protein: implications for receptor processing

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for neurotrophin activation of Trk receptors and cellular signaling

Neurotrophin-induced Trk tyrosine kinase receptor activation and neuronal cell survival responses have been reported to be under the control of a membrane associated sialidase. Here, we identify an unprecedented membrane sialidase mechanism initiated by nerve growth factor (NGF) binding to TrkA to potentiate GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live primary neurons and TrkA- and TrkB-expressing cell lines. Central to this process is that Neu1/MMP-9 complex is bound to TrkA on the cell surface of naïve primary neurons and TrkA-expressing cells. Tamiflu completely blocks this sialidase activity in live TrkA-PC12 cells treated with NGF with an IC₅₀ of 3.876 μM with subsequent inhibition of Trk activation in primary neurons and neurite outgrowth in TrkA-PC12 cells. Our findings uncover a Neu1 and MMP-9 cross-talk on the cell surface that is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and cellular signaling.     

p75 neurotrophin receptor reduces ligand-induced Trk receptor ubiquitination and delays Trk receptor internalization and degradation

Target-derived neurotrophins regulate neuronal survival and growth by interacting with cell-surface tyrosine kinase receptors. The p75 neurotrophin receptor (p75 NTR) is coexpressed with Trk receptors in long-range projection neurons, in which it facilitates neurotrophin binding to Trk and enhances Trk activity. Here, we show that TrkA and TrkB receptors undergo robust ligand-dependent ubiquitination that is dependent on activation of the endogenous Trk activity of the receptors. Coexpression of p75 NTR attenuated ubiquitination of TrkA and TrkB and delayed nerve growth factor-induced TrkA receptor internalization and receptor degradation. These results indicate that p75 NTR may prolong cell-surface Trk-dependent signalling events by negatively regulating receptor ubiquitination.     

PTPsigma binds and dephosphorylates neurotrophin receptors and can suppress NGF-dependent neurite outgrowth from sensory neurons

Neurotrophin receptors of the Trk family play a vital role in the survival of developing neurons and the process of axonogenesis. The Trk family are receptor protein tyrosine kinases (RTKs) and their signalling in response to neurotrophins is critically dependent upon their ability to transphosphorylate and act as signalling centres for multiple adaptor proteins and distinct, downstream pathways. Such phosphotyrosine signalling also depends upon the appropriate counter-regulation by phosphatases. A large family of receptor-like protein tyrosine phosphatases (RPTPs) are also expressed in developing neurons and in this study we have examined the ability of the phosphatase PTPsigma to interact with and regulate Trk proteins in transfected HEK 293T cells. PTPsigma can bind differentially to Trk proteins, binding stably in complexes with TrkA and TrkC, but not TrkB. The transmembrane domains of PTPsigma and TrkA appear to be sufficient for the direct or indirect interaction between these two receptors. Furthermore, PTPsigma is shown to dephosphorylate all three Trk receptors and suppress their phosphorylation in the presence of neurotrophins. In addition, overexpression of PTPsigma in primary sensory neurons in culture inhibits neurite outgrowth without affecting the short-term survival of these neurons. PTPsigma can thus show differential complex formation with different Trk family members and in neurons can selectively target the neurite-forming signalling pathway driven by TrkA.     

Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent gamma-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including beta-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for gamma-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that gamma-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid beta-peptide 40 (Abeta40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that gamma-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that gamma-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction.     

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.     

Cell survival through Trk neurotrophin receptors is differentially regulated by ubiquitination

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.     

GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.     

The non-peptidyl fungal metabolite L-783,281 activates TRK neurotrophin receptors

Neurotrophin binding to the extracellular surface of the Trk family of tyrosine kinase receptors leads to the activation of multiple signalling cascades, culminating in neuroregenerative effects, including neuronal survival and neurite outgrowth. Since neurotrophins themselves are not ideal drug candidates due to their poor pharmacokinetic behaviour and bioavailability, small molecule neurotrophin mimetics may be beneficial in treating a number of neurodegenerative disorders. The present study demonstrates that L-783,281, a non-peptidyl fungal metabolite, is capable of stimulating TrkA, B and C phosphorylation to various extents in CHO cells stably expressing human Trk receptors. L-783,281 also stimulated Trk phosphorylation in a number of rat and human primary neuronal cultures, whereas the highly similar compound, L-767,827, was without effect. Mechanistic studies utilizing transiently transfected PDGF/TrkA and TrkA/PDGF chimeras, demonstrated that L-783,281 is likely to interact with the intracellular domain of the TrkA receptor. Further investigations suggested that L-783,281 was nevertheless able to instigate receptor dimerization by binding in a non-covalent manner. Although the cytotoxicity of the compound was shown to preclude its effects in neuronal survival and neurite outgrowth assays, it is a prototype for a small molecule neurotrophin mimetic that activates Trk by interacting at a site different from the neurotrophin-binding site.     

Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells)

Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.     

Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells)

Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.     

TrkA receptor "hot spots" for binding of NT-3 as a heterologous ligand

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.     

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.     

Crystal structures of the neurotrophin-binding domain of TrkA, TrkB and TrkC.

The Trk receptors and their neurotrophin ligands control development and maintenance of the nervous system. The crystal structures of the ligand binding domain of TrkA, TrkB, and TrkC were solved and refined to high resolution. The domains adopt an immunoglobulin-like fold, but crystallized in all three instances as dimers with the N-terminal strand of each molecule replaced by the same strand of a symmetry-related mate. Models of the correctly folded domains could be constructed by changing the position of a single residue, and the resulting model of the binding domain of TrkA is essentially identical with the bound structure as observed in a complex with nerve growth factor. An analysis of the existing mutagenesis data for TrkA and TrkC in light of these structures reveals the structural reasons for the specificity among the Trk receptors, and explains the underpinnings of the multi-functional ligands that have been reported. The overall structure of all three domains belongs to the I-set of immunoglobulin-like domains, but shows several unusual features, such as an exposed disulfide bridge linking two neighboring strands in the same beta-sheet. For all three domains, the residues that deviate from the standard fingerprint pattern common to the I-set family fall in the region of the ligand binding site observed in the complex. Therefore, identification of these deviations in the sequences of other immunoglobulin-like domain-containing receptors may help to identify their ligand binding site even in the absence of structural or mutagenesis data.     

p75 Co-receptors regulate ligand-dependent and ligand-independent Trk receptor activation, in part by altering Trk docking subdomains.

Neurotrophins signal via Trk tyrosine kinase receptors and a common receptor called p75. Nerve growth factor is the cognate ligand for TrkA, brain-derived neurotrophic factor for TrkB, and neurotrophin-3 (NT-3) for TrkC. NT-3 also binds TrkA and TrkB as a heterologous ligand. All neurotrophins bind p75, which regulates ligand affinity and Trk signals. Trk extracellular domain has five subdomains: a leucine-rich motif, two cysteine-rich clusters, and immunoglobulin-like subdomains IgG-C1 and IgG-C2. The IgG-C1 subdomain is surface exposed in the tertiary structure and regulates ligand-independent activation. The IgG-C2 subdomain is less exposed but regulates cognate ligand binding and Trk activation. NT-3 as a heterologous ligand of TrkA and TrkB optimally requires the IgG-C2 but also binds other subdomains of these receptors. When p75 is co-expressed, major changes are observed; NGF-TrkA activation can occur also via the cysteine 1 subdomain, and brain-derived neurotrophic factor-TrkB activation requires the TrkB leucine-rich motif and cysteine 2 subdomains. We propose a two-site model of Trk binding and activation, regulated conformationally by the IgG-C1 subdomain. Moreover, p75 affects Trk subdomain utilization in ligand-dependent activation, possibly by conformational or allosteric control.     

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.     

Effects of signal peptide and adenylate on the oligomerization and membrane binding of soluble SecA

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.     

Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation

The uncleaved, pro-form of nerve growth factor (proNGF) functions as a pro-apoptotic ligand for the p75 neurotrophin receptor (p75NTR). However, some reports have indicated that proneurotrophins bind and activate Trk receptors. In this study, we have examined proneurotrophin receptor binding and activation properties in an attempt to reconcile these findings. We show that proNGF readily binds p75NTR expressed in HEK293T cells but does not interact with TrkA expressed under similar circumstances. Importantly, proNGF activates TrkA tyrosine phosphorylation, induces Erk and Akt activation, and causes PC12 cell differentiation. We show that inhibiting endocytosis or furin activity reduced TrkA activation induced by proNGF but not that induced by mature NGF and that proNGF123, a mutant form of NGF lacking dibasic cleavage sites in the prodomain, does not induce TrkA phosphorylation in PC12 cells. Therefore, endocytosis and cleavage appear to be prerequisites for proNGF-induced TrkA activity. We also found that proBDNF induces activation of TrkB in cerebellar granule neurons and that proBDNF cleavage by furin and metalloproteases facilitates this effect. Taken together, these data indicate that under physiological conditions, proneurotrophins do not directly bind or activate Trk receptors. However, endocytosis and cleavage of proneurotrophins produce processed forms of neurotrophins that are capable of inducing Trk activation.     

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.