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ABSTRACT: Receptor tyrosine kinases (RTK) act through dimerization. Previously it was thought that only bivalent ligands could be agonistic, whereas monovalent ligands should be antagonistic. This notion changed after the demonstration that monovalent ligands can be agonistic, including our report of a small molecule monovalent ligand "D3" that is a partial agonist of the NGF receptor TrkA. A bivalent "D3-linker-D3" was expected to increase agonism.
Dimeric analogs were synthesized and tested in binding, biochemical, and biological assays.
One analog, 1-ss, binds TrkA with higher affinity than D3 and induces or stabilizes receptor dimers. However, 1-ss exhibited antagonistic activity, through two mechanisms. One mechanism is that 1-ss blocks NGF binding, unlike D3 which is non-competitive. Inhibition of NGF binding may be due to the linker of 1-ss filling the inter-receptor space that NGF traverses before docking. In a second mechanism, 1-ss acts as a pure antagonist, inhibiting NGF-independent TrkA activity in cells over-expressing receptors. Inhibition is likely due to 1-ss "freezing" the TrkA dimer in the inactive state.
Dimerization of an RTK can result in antagonism, through two independent mechanisms.
we report a small molecule monovalent agonist being converted to a bivalent antagonist.
Biochimica et Biophysica Acta 09/2010; 1800(9):1018-26. · 4.66 Impact Factor
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ABSTRACT: The D5 domain of TrkC receptors is a docking site for Neurotrophin-3 (NT-3), but other domains may be relevant for function or harmonizing signals with p75(NTR) coreceptors. We report a monoclonal antibody (mAb) 2B7 targeting the juxtamembrane domain of TrkC. mAb 2B7 binds to murine and human TrkC receptors and is a functional agonist that affords activation of TrkC, AKT, and MAPK. These signals result in cell survival but not in cellular differentiation. Monomeric 2B7 Fabs also affords cell survival. Binding of 2B7 mAb and 2B7 Fabs to TrkC are blocked by NT-3 in a dose-dependent manner but not by pro-NT-3. Expression of p75(NTR) coreceptors on the cell surface block the binding and function of mAb 2B7, whereas NT-3 binding and function are enhanced. mAb 2B7 defines a previously unknown neurotrophin receptor functional hot spot; that exclusively generates survival signals; that can be activated by non-dimeric ligands; and potentially unmasks a site for p75-TrkC interactions.
Developmental Neurobiology 12/2009; 70(3):150-64. · 3.55 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 07/2007; 282(23):16754-63. · 4.77 Impact Factor
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ABSTRACT: The neurotrophins neurotrophin-3 (NT-3), brain-derived growth factor (BDNF) and nerve growth factor (NGF) bind to the p75 receptor, but each neurotrophin also binds a more selective Trk receptor (e.g. TrkA-NGF and TrkC-NT-3). The biochemical signals following engagement of either Trk or p75 with ligands are well understood, but long-term biological outcomes (trophic, proapoptotic or differentiative) remain unclear because they are cell/tissue specific. For example, Trk receptors are usually trophic but when overexpressed they can be proapoptotic in neuroblastomas and medulloblastomas. We hypothesized that coexpression of Trk and p75 receptors may lead to cross-regulation of signals and different biological outcomes; and used receptor-selective ligands to study cross-regulation by these receptors. We show that in the absence of Trk activation, expression of TrkC is permissive of p75 trophic and differentiation signals induced by p75 ligands, whereas expression of TrkA abolishes trophic and differentiation signals induced by p75 ligands. In contrast, in the presence of Trk activation, p75 ligands can regulate TrkA-mediated survival and TrkC-mediated differentiation. Therefore, a complex homeostasis of p75-selective and Trk-selective signals may determine the fate of cells expressing both receptors.
Oncogene 09/2003; 22(36):5677-85. · 6.37 Impact Factor
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Ljubica Ivanisevic
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ABSTRACT: The Trk family of tyrosine kinase receptors and the common p75NTR receptor are neurotrophin receptors. Nerve growth factor (NGF) binds TrkA, brain-derived neurotrophic factor (BDNF) binds TrkB, and neurotrophin-3 (NT-3) binds TrkC. The extracellular domain of the Trk receptor has five subdomains: a leucine-rich motif (D2), two cysteine-rich motifs (D1, D3) and immunoglobulin-like subdomains Ig-C1 (D4) and Ig-C2(D5). The Trk D4 subdomain regulates ligand-independent activation. The TrkA-D5 and TrkB-D5 subdomains regulate cognate ligand binding and Trk activation. However, the p75NTR receptor binds all neurotrophins and regulates ligand affinity and Trk signals. We showed that p75NTR affects Trk ligand - binding and activation of Trks by changing Trk subdomain utilization. When p75NTR is coexpressed, NGF can activate TrkA via the cysteine-1 subdomain (D1), and BDNF can activate TrkB via leucine-rich motif (D2) and cysteine-2 (D3) subdomains. We hypothesized conformational or allosteric regulatory mechanisms. To further study the interactions between ligands and Trks, we examined TrkA binding to NT-3 as a heterologous ligand because these interactions are biologically relevant. We found the TrkA “hot spot” functional docking sites used by NT-3. We demonstrate that TrkA-D5 has partially overlapping but distinct binding and activation “hot spots” for both, NGF and NT-3. Moreover, ligand - binding studies have identified additional NT-3 binding/allosteric site on TrkA-D4. NT-3 binding to both sites induces full agonism. Conversely, the TrkA-D5 NT-3 binding site is partially agonistic, but antagonizes NGF activity. Lasly, we address NT-3 binding and activation sites on the TrkC receptor by raising a monoclonal antibody that recognizes the juxtamembrane-linker domain of the TrkC receptor. This antibody is an artificial TrkC receptor agonist. The epitope of mAb 2B7 defines a previously unknown hot spot of TrkC. Binding to this “hot spot” induces survival but not differentiation of TrkC expressing cells. Interestingly, the functional and structural availability of this hot spot is regulated by expression of p75NTR; and the ectodomain of p75NTR masks the hot spot. This finding is important for understanding the molecular mechanisms underlying p75NTR -Trk interactions. Taken together, results presented in this thesis define multiple ligand binding and activation domains on Trk receptors for cognate and heterologous ligands as well as ligand binding and activation sites regulated by p75NTR, possibly by conformational or allosteric control. These findings have implications in understanding the molecular mechanisms of p75NTR -Trk interactiona and rational design and development of Trk receptor artificial ligands with potential therapeutic applications. La famille de récepteurs de Trk tyrosine kinase et le récepteur p75NTR sont des récepteurs de neurotrophines. Le facteur de croissance nerveuse (NGF) intéragit avec le récepteur TrkA, le facteur neurotrophique dérivé du cerveau (BDNF) intéragit avec le récepteur TrkB et la neurotrophine-3 (NT-3) intéragit avec TrkC. Le domaine extracellulaire du récepteur Trk contient cinq sous-domaines: un motif riche en leucine (D2), deux motifs riches en cysteine (D1, D3) et des sous-domaines de type immunoglobuline Ig-C1(D4) et Ig-C2(D5). Le sous-domaine Trk D4 régule l'activation indépendante de ligand. Les sous-domaines TrkA-D5 et TrkB-D5 régulent la liaison de ligands endogènes ainsi que l'activation du récepteur Trk. Le récepteur p75NTR intéragit avec toutes les neurotrophines et régule l'affinité des ligands et les signaux issues de l'activation du récepteur Trk. Par ailleurs, nous avons démontré que le p75NTR affecte la liaison du ligand au récepteur Trk en changeant l'activation de l'utilisation des sous-domaines. Lorsque le recepteur de p75NTR est coexprimé, le NGF peut activer le récepteur TrkA via le sous-domaine cysteine-1 (D1) et BDNF peut activer TrkB via le motif riche en leucine (D2) ainsi que via le sous-domaine cysteine-2 (D3). Nous avons examiné la liaison d'un ligand hétérologue, NT-3 sur le récepteur TrkA afin d'étudier plus profondément les interactions entre les ligands et le récepteur TrkA. Ces interactions sont biologiquement pertinentes. Pour faire ceci, nous avons tout d'abord identifié les « points chauds » présents sur le récepteur TrkA qui servent des sites d'amarrage fonctionnels du ligand NT-3. Nous avons démontré que le sous domaine TrkA-D5 possède deux points chauds distincts, notamment un point chaud qui sert comme le site d'amarrage et d'activation du NGF et un point chaud qui sert comme le site d'amarrage et d'activation de la NT-3. Toutefois, ces deux sites d'amarrage se chevauchent partiellement. De plus, nous avons identifié un site additionnel de liaison de NT-3 situé sur le sous domaine TrkA-D4. La liaison de NT-3 sur les deux sites (TrkA-D4 and TrkA-D5) induit un agonisme complet. Au contraire, la liaison du NT-3 sur le sous domaine TrkA-D5 seul entraîne l'agonisme partiel de l'activité de la NT-3 et un antagonisme de l'activité du NGF. Finalement, nous avons étudié la liaison de la NT-3 sur le récepteur TrkC et l'activation du récepteur TrkC. Pour faire ceci nous avons crée des anticorps monoclonaux qui reconnaissent le domaine juxtamembranaire-lieur du récepteur TrkC. Ces anticorps sont les agonistes artificiels du récepteur TrkC. Nous avons trouvé que l'épitope de mAb 2B7 possède un point chaud de TrkC, inconnu auparavant, qui lorsque activé par des ligands monomériques, induit la survie des cellules exprimant le récepteur TrkC et inhibe leur différentiation. De façon intéressante, la disponibilité de ce point chaud est régulée par l'expression du récepteur p75NTR. Cette découverte est importante afin de comprendre mieux les mécanismes moléculaires impliqués dans les interactions entre le récepteur Trk et le récepteur p75NTR. En conclusion, ce travail avait pour but de caractériser les multiples domaines de liaison de ligands (endogènes et hétérologues) et d'activation des récepteurs Trk ainsi que des sites de liaison de ligands et d'activation régulés par p75NTR, possiblement par un contrôle conformationnel ou allostérique. Les résultats obtenus par ce travail auront une implication importante dans la caractérisation des mécanismes moléculaires impliqués dans les interactions entre le récepteur Trk et le p75NTR. Par ailleurs, les résultats auront une applicabilité médicale dans le développement des ligands artificiels de Trk ayant une application thérapeutique.
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Ljubica. Ivanisevic
