Limits...
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.

Show MeSH

Related in: MedlinePlus

NTRAP interacts with Trk receptor tyrosine kinases. (A) The amino acid sequence of NTRAP. The first underlined region represents a RING finger domain, and the next five underlined regions are five tandem C2H2 zinc finger motifs. The two proline-rich regions are shown in bold. (B) Interaction of NTRAP with the intracellular domain of TrkA, TrkB, TrkC, or Met in yeast two-hybrid assays. Laminin was used as a negative control (CTL). The interaction strength was determined by measuring the activity of the reporter β-galactosidase. (C) Specificity of NTRAP antibody. Protein extracts from PC12 cells or E18.5 rat DRGs were blotted with the affinity-purified NTRAP antibody. (D) Enhancement of interaction between NTRAP and TrkC by NT3. Lysates of HEK293 cells were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were blotted with the anti-NTRAP antibody. Cell lysates (25 μg) were also blotted with antibodies to Myc, NTRAP, and α-tubulin for examination of expression. (E) Enhancement of interaction between NTRAP and TrkA by NGF. HEK293 cells were transfected with constructs expressing Flag-tagged TrkA and NTRAP. Cell lysates were immunoprecipitated with anti-Flag antibodies, and immunoprecipitates were blotted with anti-NTRAP antibodies. Cell lysates (25 μg) were also blotted with antibodies to Flag, NTRAP, and α-tubulin. (F) Interaction of endogenous NTRAP and TrkA. PC12 cell lysates were immunoprecipitated with the mouse IgG against TrkA, and immunoprecipitates were blotted with the rabbit IgG against NTRAP or TrkA. Cell lysates were blotted with the antibody to NTRAP. NGF treatment enhanced the association of the two proteins. (G) Tissue distribution of NTRAP mRNA in adult mice, as revealed by Northern hybridization. (H) High levels of NTRAP in the nervous system and liver of an E12.5 mouse embryo, as revealed by immunohistochemistry. (I) NTRAP expression in DRG ganglia of an E12.5 mouse embryo. Note that axons of DRG neurons also contain NTRAP (arrow). (J) Localization of NTRAP in the cytoplasm of DRG neurons. Scale bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2801717&req=5

Figure 1: NTRAP interacts with Trk receptor tyrosine kinases. (A) The amino acid sequence of NTRAP. The first underlined region represents a RING finger domain, and the next five underlined regions are five tandem C2H2 zinc finger motifs. The two proline-rich regions are shown in bold. (B) Interaction of NTRAP with the intracellular domain of TrkA, TrkB, TrkC, or Met in yeast two-hybrid assays. Laminin was used as a negative control (CTL). The interaction strength was determined by measuring the activity of the reporter β-galactosidase. (C) Specificity of NTRAP antibody. Protein extracts from PC12 cells or E18.5 rat DRGs were blotted with the affinity-purified NTRAP antibody. (D) Enhancement of interaction between NTRAP and TrkC by NT3. Lysates of HEK293 cells were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were blotted with the anti-NTRAP antibody. Cell lysates (25 μg) were also blotted with antibodies to Myc, NTRAP, and α-tubulin for examination of expression. (E) Enhancement of interaction between NTRAP and TrkA by NGF. HEK293 cells were transfected with constructs expressing Flag-tagged TrkA and NTRAP. Cell lysates were immunoprecipitated with anti-Flag antibodies, and immunoprecipitates were blotted with anti-NTRAP antibodies. Cell lysates (25 μg) were also blotted with antibodies to Flag, NTRAP, and α-tubulin. (F) Interaction of endogenous NTRAP and TrkA. PC12 cell lysates were immunoprecipitated with the mouse IgG against TrkA, and immunoprecipitates were blotted with the rabbit IgG against NTRAP or TrkA. Cell lysates were blotted with the antibody to NTRAP. NGF treatment enhanced the association of the two proteins. (G) Tissue distribution of NTRAP mRNA in adult mice, as revealed by Northern hybridization. (H) High levels of NTRAP in the nervous system and liver of an E12.5 mouse embryo, as revealed by immunohistochemistry. (I) NTRAP expression in DRG ganglia of an E12.5 mouse embryo. Note that axons of DRG neurons also contain NTRAP (arrow). (J) Localization of NTRAP in the cytoplasm of DRG neurons. Scale bar, 10 μm.

Mentions: One of the novel Trk-interacting proteins is a 96-kDa protein that contains one RING finger, five tandem C2H2 zinc fingers, and two proline-rich regions (Figure 1A). This protein, which we named NTRAP, is identified as Zfp598 in the NCBI nucleotide database (Accession no. NM_183149) with no known functions and is conserved from yeast S. cerevisiae to humans. Yeast two-hybrid assays showed that NTRAP was also able to interact with TrkA, TrkB, and Met (the receptor for hepatocyte growth factor; Figure 1B) but not the receptor for epidermal growth factor (Supplemental Figure S1).


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)

NTRAP interacts with Trk receptor tyrosine kinases. (A) The amino acid sequence of NTRAP. The first underlined region represents a RING finger domain, and the next five underlined regions are five tandem C2H2 zinc finger motifs. The two proline-rich regions are shown in bold. (B) Interaction of NTRAP with the intracellular domain of TrkA, TrkB, TrkC, or Met in yeast two-hybrid assays. Laminin was used as a negative control (CTL). The interaction strength was determined by measuring the activity of the reporter β-galactosidase. (C) Specificity of NTRAP antibody. Protein extracts from PC12 cells or E18.5 rat DRGs were blotted with the affinity-purified NTRAP antibody. (D) Enhancement of interaction between NTRAP and TrkC by NT3. Lysates of HEK293 cells were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were blotted with the anti-NTRAP antibody. Cell lysates (25 μg) were also blotted with antibodies to Myc, NTRAP, and α-tubulin for examination of expression. (E) Enhancement of interaction between NTRAP and TrkA by NGF. HEK293 cells were transfected with constructs expressing Flag-tagged TrkA and NTRAP. Cell lysates were immunoprecipitated with anti-Flag antibodies, and immunoprecipitates were blotted with anti-NTRAP antibodies. Cell lysates (25 μg) were also blotted with antibodies to Flag, NTRAP, and α-tubulin. (F) Interaction of endogenous NTRAP and TrkA. PC12 cell lysates were immunoprecipitated with the mouse IgG against TrkA, and immunoprecipitates were blotted with the rabbit IgG against NTRAP or TrkA. Cell lysates were blotted with the antibody to NTRAP. NGF treatment enhanced the association of the two proteins. (G) Tissue distribution of NTRAP mRNA in adult mice, as revealed by Northern hybridization. (H) High levels of NTRAP in the nervous system and liver of an E12.5 mouse embryo, as revealed by immunohistochemistry. (I) NTRAP expression in DRG ganglia of an E12.5 mouse embryo. Note that axons of DRG neurons also contain NTRAP (arrow). (J) Localization of NTRAP in the cytoplasm of DRG neurons. Scale bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801717&req=5

Figure 1: NTRAP interacts with Trk receptor tyrosine kinases. (A) The amino acid sequence of NTRAP. The first underlined region represents a RING finger domain, and the next five underlined regions are five tandem C2H2 zinc finger motifs. The two proline-rich regions are shown in bold. (B) Interaction of NTRAP with the intracellular domain of TrkA, TrkB, TrkC, or Met in yeast two-hybrid assays. Laminin was used as a negative control (CTL). The interaction strength was determined by measuring the activity of the reporter β-galactosidase. (C) Specificity of NTRAP antibody. Protein extracts from PC12 cells or E18.5 rat DRGs were blotted with the affinity-purified NTRAP antibody. (D) Enhancement of interaction between NTRAP and TrkC by NT3. Lysates of HEK293 cells were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were blotted with the anti-NTRAP antibody. Cell lysates (25 μg) were also blotted with antibodies to Myc, NTRAP, and α-tubulin for examination of expression. (E) Enhancement of interaction between NTRAP and TrkA by NGF. HEK293 cells were transfected with constructs expressing Flag-tagged TrkA and NTRAP. Cell lysates were immunoprecipitated with anti-Flag antibodies, and immunoprecipitates were blotted with anti-NTRAP antibodies. Cell lysates (25 μg) were also blotted with antibodies to Flag, NTRAP, and α-tubulin. (F) Interaction of endogenous NTRAP and TrkA. PC12 cell lysates were immunoprecipitated with the mouse IgG against TrkA, and immunoprecipitates were blotted with the rabbit IgG against NTRAP or TrkA. Cell lysates were blotted with the antibody to NTRAP. NGF treatment enhanced the association of the two proteins. (G) Tissue distribution of NTRAP mRNA in adult mice, as revealed by Northern hybridization. (H) High levels of NTRAP in the nervous system and liver of an E12.5 mouse embryo, as revealed by immunohistochemistry. (I) NTRAP expression in DRG ganglia of an E12.5 mouse embryo. Note that axons of DRG neurons also contain NTRAP (arrow). (J) Localization of NTRAP in the cytoplasm of DRG neurons. Scale bar, 10 μm.
Mentions: One of the novel Trk-interacting proteins is a 96-kDa protein that contains one RING finger, five tandem C2H2 zinc fingers, and two proline-rich regions (Figure 1A). This protein, which we named NTRAP, is identified as Zfp598 in the NCBI nucleotide database (Accession no. NM_183149) with no known functions and is conserved from yeast S. cerevisiae to humans. Yeast two-hybrid assays showed that NTRAP was also able to interact with TrkA, TrkB, and Met (the receptor for hepatocyte growth factor; Figure 1B) but not the receptor for epidermal growth factor (Supplemental Figure S1).

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