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Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals.

ABSTRACT: This study was initiated to find small molecule ligands that would induce a functional response when docked with neurotrophin Trk receptors. "Minimalist" mimics of beta-turns were designed for this purpose. These mimics are (i) rigid, yet easily folded into turn-like conformations, and (ii) readily accessible from amino acids bearing most of the natural side chains. Gram quantities of 16 of these turn mimics were prepared and then assembled into 152 fluorescein-labeled bivalent peptidomimetics via a solution-phase combinatorial method. Fluorescence-based screening of these molecules using cells transfected with the Trk receptors identified 10 potential ligands of TrkC, the receptor for neurotrophin-3. Analogues of these bivalent peptidomimetics with biotin replacing the fluorescein label were then prepared and tested to confirm that binding was not due to the fluorescein. Several assays were conducted to find the mode of action of these biotinylated compounds. Thus, direct binding, survival and neuritogenic, and biochemical signal transduction assays showed 8 of the original 10 hits were agonistic ligands binding to the ectodomain of TrkC. Remarkably, some peptidomimetics afford discrete signals leading to either cell survival or neuritogenic differentiation. The significance of this work is three-fold. First, we succeeded in finding small, selective, proteolytically stable ligands for the TrkC receptor; there are very few of these in the literature. Second, we show that it is possible to activate distinct and biased signaling pathways with ligands binding at the ectodomain of wild-type receptors. Third, the discovery that some peptidomimetics initiate different modes of cell signaling increases their potential as pharmacological probes and therapeutic leads.

SUBMITTER: Chen D

PROVIDER: S-EPMC2756187 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals.

Chen Dianjun D Brahimi Fouad F Angell Yu Y Li Yu-Chin YC Moscowicz Jennifer J Saragovi H Uri HU Burgess Kevin K

ACS chemical biology 20090901 9

This study was initiated to find small molecule ligands that would induce a functional response when docked with neurotrophin Trk receptors. "Minimalist" mimics of beta-turns were designed for this purpose. These mimics are (i) rigid, yet easily folded into turn-like conformations, and (ii) readily accessible from amino acids bearing most of the natural side chains. Gram quantities of 16 of these turn mimics were prepared and then assembled into 152 fluorescein-labeled bivalent peptidomimetics v ...[more]

PMID: 19735123

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Project description:UnlabelledNeurotrophin-3 (NT-3) is a secreted neurotrophic factor that binds neurotrophin receptor tyrosine kinase C (TrkC), which in turn binds to presynaptic protein tyrosine phosphatase σ (PTPσ) to govern excitatory synapse development. However, whether and how NT-3 cooperates with the TrkC-PTPσ synaptic adhesion pathway and TrkC-mediated intracellular signaling pathways in rat cultured neurons has remained unclear. Here, we report that NT-3 enhances TrkC binding affinity for PTPσ. Strikingly, NT-3 treatment bidirectionally regulates the synaptogenic activity of TrkC: at concentrations of 10-25 ng/ml, NT-3 further enhanced the increase in synapse density induced by TrkC overexpression, whereas at higher concentrations, NT-3 abrogated TrkC-induced increases in synapse density. Semiquantitative immunoblotting and optogenetics-based imaging showed that 25 ng/ml NT-3 or light stimulation at a power that produced a comparable level of NT-3 (6.25 μW) activated only extracellular signal-regulated kinase (ERK) and Akt, whereas 100 ng/ml NT-3 (light intensity, 25 μW) further triggered the activation of phospholipase C-γ1 and CREB independently of PTPσ. Notably, disruption of TrkC intracellular signaling pathways, extracellular ligand binding, or kinase activity by point mutations compromised TrkC-induced increases in synapse density. Furthermore, only sparse, but not global, TrkC knock-down in cultured rat neurons significantly decreased synapse density, suggesting that intercellular differences in TrkC expression level are critical for its synapse-promoting action. Together, our data demonstrate that NT-3 is a key factor in excitatory synapse development that may direct higher-order assembly of the TrkC/PTPσ complex and activate distinct intracellular signaling cascades in a concentration-dependent manner to promote competition-based synapse development processes.Significance statementIn this study, we present several lines of experimental evidences to support the conclusion that neurotrophin-3 (NT-3) modulates the synaptic adhesion pathway involving neurotrophin receptor tyrosine kinase C (TrkC) and presynaptic protein tyrosine phosphatase σ (PTPσ) in a bidirectional manner at excitatory synapses. NT-3 acts in concentration-independent manner to facilitate TrkC-mediated presynaptic differentiation, whereas it acts in a concentration-dependent manner to exert differential effects on TrkC-mediated organization of postsynaptic development. We further investigated TrkC extracellular ligand binding, intracellular signaling pathways, and kinase activity in NT-3-induced synapse development. Last, we found that interneuronal differences in TrkC levels regulate the synapse number. Overall, these results suggest that NT-3 functions as a positive modulator of synaptogenesis involving TrkC and PTPσ.

Dataset Information

Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals.

Publications

Bivalent peptidomimetic ligands of TrkC are biased agonists and selectively induce neuritogenesis or potentiate neurotrophin-3 trophic signals.

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