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Retrograde neurotrophic signaling requires a protein interacting with receptor tyrosine kinases via C2H2 zinc fingers.

Fu X, Zang K, Zhou Z, Reichardt LF, Xu B - Mol. Biol. Cell (2009)

Bottom Line: Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor-associated protein), plays a crucial role in this signaling process.In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival.We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Georgetown University, Washington, DC 20057, USA.

ABSTRACT
Neurotrophins at axonal terminals signal to cell bodies to regulate neuronal development via signaling endosomes containing activated Trk receptor tyrosine kinases and mitogen-activated protein kinases (MAPKs). Requirements for the formation of signaling endosomes remain, however, poorly characterized. Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor-associated protein), plays a crucial role in this signaling process. NTRAP interacts with the Trk intracellular domain through its C(2)H(2) zinc fingers in a kinase-dependent manner. It is associated with vesicles, some of which contain markers for signaling endosomes. Inhibition of NTRAP function suppresses neurotrophin-induced neurite outgrowth in PC12 cells by altering TrkA endocytic traffic, inhibiting the formation of endosomes containing persistently active MAPKs. In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival. We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.

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NGF-induced localization of NTRAP in late endosomes. (A) Colocalization of NTRAP with the early endosome marker, EEA1. The graph shows percentage of NTRAP immunoreactivity that was colocalized with EEA1. Scale bar, 10 μm. (B) Colocalization of NTRAP with the late endosome marker, Rab7. PC12 cells were starved for serum overnight and then incubated with/without NGF (100 ng/ml) for 20 min. NGF treatment increased colocalization of NTRAP and Rab7. Scale bar, 10 μm.
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Figure 6: NGF-induced localization of NTRAP in late endosomes. (A) Colocalization of NTRAP with the early endosome marker, EEA1. The graph shows percentage of NTRAP immunoreactivity that was colocalized with EEA1. Scale bar, 10 μm. (B) Colocalization of NTRAP with the late endosome marker, Rab7. PC12 cells were starved for serum overnight and then incubated with/without NGF (100 ng/ml) for 20 min. NGF treatment increased colocalization of NTRAP and Rab7. Scale bar, 10 μm.

Mentions: After the addition of NGF, activated TrkA bound to NGF is internalized into vesicles, which are converted to early endosomes and then late endosomes (Howe and Mobley, 2005). Deficits in either TrkA internalization or TrkA trafficking along the endosome differentiation pathway could diminish NGF-induced persistent ERK1/2 activation in PC12 cells expressing ZF-HA. We found that the expression of ZF-HA in PC12 cells or NTRAP shRNA in DRG neurons did not affect internalization of the TrkA receptor (Supplemental Figure S2). To investigate the role of NTRAP in endocytic trafficking of Trk receptors, we first examined the distribution of NTRAP in early and late endosomes. Confocal microscopy revealed that NTRAP was localized in vesicles in PC12 cells (Figure 6A). Some NTRAP-positive vesicles contained EEA1, a marker for early endosomes (Figure 6A), but very little Rab7, a late endosome component (Figure 6B). A 20-min NGF treatment caused a trend of reduction in colocalization of NTRAP with EEA1 (Figure 6A) and significantly increased the presence of NTRAP in late endosomes (Figure 6B), suggesting that NTRAP moved along with activated TrkA from early to late endosomes, perhaps through association.


Retrograde neurotrophic signaling requires a protein interacting with receptor tyrosine kinases via C2H2 zinc fingers.

Fu X, Zang K, Zhou Z, Reichardt LF, Xu B - Mol. Biol. Cell (2009)

NGF-induced localization of NTRAP in late endosomes. (A) Colocalization of NTRAP with the early endosome marker, EEA1. The graph shows percentage of NTRAP immunoreactivity that was colocalized with EEA1. Scale bar, 10 μm. (B) Colocalization of NTRAP with the late endosome marker, Rab7. PC12 cells were starved for serum overnight and then incubated with/without NGF (100 ng/ml) for 20 min. NGF treatment increased colocalization of NTRAP and Rab7. Scale bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: NGF-induced localization of NTRAP in late endosomes. (A) Colocalization of NTRAP with the early endosome marker, EEA1. The graph shows percentage of NTRAP immunoreactivity that was colocalized with EEA1. Scale bar, 10 μm. (B) Colocalization of NTRAP with the late endosome marker, Rab7. PC12 cells were starved for serum overnight and then incubated with/without NGF (100 ng/ml) for 20 min. NGF treatment increased colocalization of NTRAP and Rab7. Scale bar, 10 μm.
Mentions: After the addition of NGF, activated TrkA bound to NGF is internalized into vesicles, which are converted to early endosomes and then late endosomes (Howe and Mobley, 2005). Deficits in either TrkA internalization or TrkA trafficking along the endosome differentiation pathway could diminish NGF-induced persistent ERK1/2 activation in PC12 cells expressing ZF-HA. We found that the expression of ZF-HA in PC12 cells or NTRAP shRNA in DRG neurons did not affect internalization of the TrkA receptor (Supplemental Figure S2). To investigate the role of NTRAP in endocytic trafficking of Trk receptors, we first examined the distribution of NTRAP in early and late endosomes. Confocal microscopy revealed that NTRAP was localized in vesicles in PC12 cells (Figure 6A). Some NTRAP-positive vesicles contained EEA1, a marker for early endosomes (Figure 6A), but very little Rab7, a late endosome component (Figure 6B). A 20-min NGF treatment caused a trend of reduction in colocalization of NTRAP with EEA1 (Figure 6A) and significantly increased the presence of NTRAP in late endosomes (Figure 6B), suggesting that NTRAP moved along with activated TrkA from early to late endosomes, perhaps through association.

Bottom Line: Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor-associated protein), plays a crucial role in this signaling process.In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival.We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Georgetown University, Washington, DC 20057, USA.

ABSTRACT
Neurotrophins at axonal terminals signal to cell bodies to regulate neuronal development via signaling endosomes containing activated Trk receptor tyrosine kinases and mitogen-activated protein kinases (MAPKs). Requirements for the formation of signaling endosomes remain, however, poorly characterized. Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor-associated protein), plays a crucial role in this signaling process. NTRAP interacts with the Trk intracellular domain through its C(2)H(2) zinc fingers in a kinase-dependent manner. It is associated with vesicles, some of which contain markers for signaling endosomes. Inhibition of NTRAP function suppresses neurotrophin-induced neurite outgrowth in PC12 cells by altering TrkA endocytic traffic, inhibiting the formation of endosomes containing persistently active MAPKs. In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival. We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.

Show MeSH
Related in: MedlinePlus