p75 Co-receptors Regulate Ligand-dependent and Ligand-independent Trk Receptor Activation, in Part by Altering Trk Docking Subdomains (original) (raw)
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Reciprocal modulation of TrkA and p75NTR affinity states is mediated by direct receptor interactions
European Journal of Neuroscience, 1998
Equilibrium binding of 125 I-nerve growth factor ( 125 I-NGF) to cells coexpressing the tyrosine kinase receptor A (TrkA) and common neurotrophin receptor (p75 NTR ), cells coexpressing both receptors where p75 NTR is occupied, and cells expressing only p75 NTR , revealed reciprocal modulation of receptor affinity states. Analysis of receptor affinity states in PC12 cells, PC12 cells in the presence of brain-derived neurotrophic factor (BDNF), and PC12 nnr5 cells suggested that liganded and unliganded p75 NTR induce a higher affinity state within TrkA, while TrkA induces a lower affinity state within p75 NTR . These data are consistent with receptor allosterism, and prompted a search for TrkA/p75 NTR complexes in the absence of NGF. Chemical crosslinking studies revealed high molecular weight receptor complexes that specifically bound 125 I-NGF, and were immunoprecipitated by antibodies to both receptors. The heteroreceptor complex of TrkA and p75 NTR alters conformation and/or dissociates in the presence of NGF, as indicated by the ability of low concentrations of NGF to prevent heteroreceptor crosslinking. These data suggest a new model of receptor interaction, whereby structural changes within a heteroreceptor complex are induced by ligand binding.
Journal of Biological Chemistry, 2001
Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K d ؍ 10 ؊9 M), but they produce high affinity binding sites (K d ؍ 10 ؊11 M) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.
The effect of P75 on Trk receptors in neuroblastomas
Cancer Letters, 2011
Neuroblastomas (NBs) with favorable outcome usually express TrkA, whereas unfavorable NBs frequently express TrkB and its cognate ligand BDNF. P75 (p75 LNTR , NGFR, TNFRSF16) binds NGF-related neurotrophins with low affinity and usually is co-expressed with Trk receptors in NBs. Here, we investigated the importance of p75 coexpression with Trk receptors in NBs. We transfected p75 into two Trk-null NB cell lines, SH-SY5Y and NLF that were also engineered to stably express TrkA or TrkB. Cell numbers were compared between single (Trk alone) and double (Trk+p75) transfectants, and proliferation was assessed by flow cytometry. P75 coexpression had little effect on cell growth in Trk NB cells in the absence of ligand, but it increased sensitivity and greatly enhanced the effect of cognate ligand. Exogenous NGF induced greater phosphorylation of TrkA and AKT. This was associated with increased cell number in TrkA/p75 cells compared to TrkA cells (p<0.01), which was due to increased proliferation in TrkA/p75 cells (p<0.05), followed by differentiation. Exogenous BDNF also increased cell number in TrkB/p75 compared to TrkB cells (p<0.01), due to an increase in proliferation, but without differentiation. Coexpression of p75 also increased specificity of Trk-expressing cells to ligand. NT3-induced phosphorylation of TrkA and AKT was reduced in TrkA/p75 cells. NT3-induced phosphorylation of TrkB (as well as AKT and MAPK) was also reduced with p75 coexpression. Our results suggest that p75 plays an important role in enhancing both the sensitivity of Trk receptors to low levels of ligand, as well as increasing the specificity of Trks to their cognate ligands. It also enhances ligand-induced differentiation in TrkA/p75 but not TrkB/p75 cells.
EMBO reports, 2005
Target-derived neurotrophins regulate neuronal survival and growth by interacting with cell-surface tyrosine kinase receptors. The p75 neurotrophin receptor (p75NTR) 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 liganddependent ubiquitination that is dependent on activation of the endogenous Trk activity of the receptors. Coexpression of p75NTR attenuated ubiquitination of TrkA and TrkB and delayed nerve growth factor-induced TrkA receptor internalization and receptor degradation. These results indicate that p75NTR may prolong cell-surface Trk-dependent signalling events by negatively regulating receptor ubiquitination. EMBO reports (2005) 6, 936-941.
TrkA NGF receptor plays a role in the modulation of p75NTR expression
Neuroscience Letters, 2005
The cellular response to nerve growth factor (NGF) is mediated by two structurally unrelated receptors, TrkA and p75 neurotrophin receptor (p75NTR), which have been shown to interact resulting in reciprocal modulation of function. In this study, we have examined the modulation of p75NTR protein expression by specific TrkA autophosphorylation sites in the presence or absence of NGF. We have used cell lines derived from PC12 cells that express either no endogenous TrkA (PC12nnr5) or TrkA receptors mutated via site-directed mutagenesis to abrogate individual tyrosine autophosphorylation sites on the cytoplasmic tail (Y490F, Y785F and Y490/785F). Results indicate that in the absence of TrkA in PC12nnr5 cells there is reduced constitutive p75NTR expression, which can be restored to different degrees by transfection of the Y490F TrkA or the Y490/785F TrkA, but not by transfection of the Y785F TrkA. In addition, the expression of p75NTR was upregulated in the presence of NGF in the parental and Y490F cell lines only. Together these results indicate a role for the individual tyrosine autophosphorylation sites of TrkA in regulating p75NTR expression.
Biochemical Journal, 2005
The topology and trafficking of receptors play a key role in their signalling capability. Indeed, receptor function is related to the microenvironment inside the cell, where specific signalling molecules are compartmentalized. The response to NGF (nerve growth factor) is strongly dependent on the trafficking of its receptor, TrkA. However, information is still scarce about the role of the cellular localization of the TrkA co-receptor, p75 NTR (where NTR is neurotrophin receptor), following stimulation by NGF. It has been shown that these two receptors play a key role in epithelial tissue and in epithelial-derived tumours, where the microenvironment at the plasma membrane is defined by the presence of tight junctions. Indeed, in thyroid carcinomas, rearrangements of TrkA are frequently found, which produce TrkA mutants that are localized exclusively in the cytoplasm. We used a thyroid cellular model in which it was possible to dissect the trafficking of the two NGF receptors upon neurotrophin stimulation. In FRT (Fischer rat thyroid) cells, endogenous TrkA is localized exclusively on the basolateral surface, while transfected p75 NTR is selectively distributed on the apical membrane. This cellular system enabled us to selectively stimulate either p75 NTR or TrkA and to analyse the role of receptor trafficking in their signalling capability. We found that, after binding to NGF, p75 NTR was co-immunoprecipitated with TrkA and was transcytosed at the basolateral membrane. We showed that the TrkA-p75 NTR interaction is necessary for this relocation of p75 NTR to the basolateral side. Interestingly, TrkAspecific stimulation by basolateral NGF loading also induced the TrkA-p75 NTR interaction and subsequent p75 NTR transcytosis at the basolateral surface. Moreover, specific stimulation of p75 NTR by NGF activated TrkA and the MAPK (mitogen-activated protein kinase) pathway. Our data indicate that TrkA regulates the subcellular localization of p75 NTR upon stimulation with neurotrophins, thus affecting the topology of the signal transduction molecules, driving the activation of a specific signal transduction pathway.
The Extracellular Domain of p75NTR Is Necessary to Inhibit Neurotrophin-3 Signaling through TrkA
Journal of Biological Chemistry, 2001
The TrkA receptor is activated primarily by nerve growth factor (NGF), but it can also be activated by high concentrations of neurotrophin 3 (NT-3). The pan-neurotrophin receptor p75 NTR strongly inhibits activation of TrkA by NT-3 but not by NGF. To examine the role of p75 NTR in regulating the specificity of TrkA signaling, we expressed both receptors in Xenopus oocytes. Application of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of 45 Ca 2؉ by a phospholipase C-␥-dependent pathway. Coexpression of p75 NTR with TrkA inhibited 45 Ca 2؉ efflux in response to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA increased with increasing expression of p75 NTR. Coexpression of a truncated p75 NTR receptor lacking all but the first 9 amino acids of the cytoplasmic domain inhibited NT-3 stimulation of 45 Ca 2؉ efflux, whereas coexpression of an epidermal growth factor receptor/p75 NTR chimera (extracellular domain of epidermal growth factor receptor with transmembrane and cytoplasmic domains of p75 NTR) did not inhibit NT-3 signaling through TrkA. These studies demonstrated that the extracellular domain of p75 NTR was necessary to inhibit NT-3 signaling through TrkA. Remarkably, p75 NTR binding to NT-3 was not required to prevent signaling through TrkA, since occupying p75 NTR with brain-derived neurotrophic factor or anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate 45 Ca 2؉ efflux. These data suggested a physical association between TrkA and p75 NTR. Documenting this physical interaction, we showed that p75 NTR and TrkA could be coimmunoprecipitated from Xenopus oocytes. Our results suggest that the interaction of these two receptors on the cell surface mediated the inhibition of NT-3-activated signaling through TrkA.
Tyrosine 785 is a major determinant of Trk--substrate interaction
The EMBO …, 1993
4Corresponding author Communicated by A.Ullrich Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co-overexpression in human 293 fibroblasts using ET-R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF-R) extraceliular ligand binding domain and the Trk transmembrane and intracellular signal-generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C7y (PLC-y), ras GTPaseactivating protein (GAP) and the non-catalytic subunit of phosphatidylinositol-3'-kinase, p85, in a liganddependent manner. Deletion of 15 C-terminal amino acids, including tyrosine 785 (Y-785) abrogated receptor and substrate phosphorylation activities. Mutation of Y-785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET-YF phosphorylation of PLCy was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine-phosphorylated synthetic C-terminal peptide competitively inhibited Trk cytoplasmic domain association with PLCy. Thus, the short C-terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y-785 is a major and selective interaction site for PLC-y.
The Neurotrophin Receptor, Gp75, Forms a Complex With the Receptor Tyrosine Kinase TrkA
Journal of Cell …, 1996
The high-affinity NGF receptor is thought to be a complex of two receptors, gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association has been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with an anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression system which allows high level expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. This association is specific, since gp75 does not copatch with other tyrosine kinase recep-tors, including TrkB, platelet-derived growth factor receptor-~, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains shows partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.