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Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

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NHE5 knockdown affects TrkA steady-state surface abundance but not NGF-induced internalization. (A–E) Control PC12 cells (PC12) and PC12 cells stably expressing shRNAs against NHE5 (shNHE5) or NHE1 (shNHE1) were incubated with a cell-surface biotinylation reagent, and total and cell-surface TrkA, NKA, or TfnR were detected by Western blot. Western blot shown in A is representative of three independent experiments. (B) No-biotin control confirmed that there was no fraction being pulled down nonspecifically. The intensity of the bands was determined by densitometry, and the data represent the mean ± SEM of relative levels of total TrkA (C), cell-surface TrkA (D), and TfnR (E) of at least three experiments. **p < 0.01 (paired Student's t test compared with control). (F, G) Cells were serum starved overnight and then treated with 50 ng/ml NGF as indicated, and the total and cell-surface abundance of TrkA, as well as of TfnR, at each time point were determined. Western blots shown are representative of three independent experiments. (G) Relative NGF-induced TrkA endocytosis was measured by quantifying the TrkA cell-surface abundance at various times after NGF treatment. The rate of TrkA endocytosis was not different between the cell lines tested. Error bars represent SEM. (H, I) After surface biotinylation, cells were incubated for 60 min at 37°C. Biotin remaining on cell-surface proteins was removed by glutathione, and then cells were subjected to the second incubation with NGF (50 ng/ml) at 37°C for 0, 15, or 30 min and treated with glutathione to remove biotin from the proteins recycled back to the plasma membrane, and relative TrkA abundance was determined. For pull down, 130 and 400 μg of total protein were used for PC12 and shNHE5 cells, respectively. The densitometry data of relative levels of total TrkA are presented with mean ± SEM. *p < 0.05 (paired Student's t test, n = 6 experiments).
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Figure 4: NHE5 knockdown affects TrkA steady-state surface abundance but not NGF-induced internalization. (A–E) Control PC12 cells (PC12) and PC12 cells stably expressing shRNAs against NHE5 (shNHE5) or NHE1 (shNHE1) were incubated with a cell-surface biotinylation reagent, and total and cell-surface TrkA, NKA, or TfnR were detected by Western blot. Western blot shown in A is representative of three independent experiments. (B) No-biotin control confirmed that there was no fraction being pulled down nonspecifically. The intensity of the bands was determined by densitometry, and the data represent the mean ± SEM of relative levels of total TrkA (C), cell-surface TrkA (D), and TfnR (E) of at least three experiments. **p < 0.01 (paired Student's t test compared with control). (F, G) Cells were serum starved overnight and then treated with 50 ng/ml NGF as indicated, and the total and cell-surface abundance of TrkA, as well as of TfnR, at each time point were determined. Western blots shown are representative of three independent experiments. (G) Relative NGF-induced TrkA endocytosis was measured by quantifying the TrkA cell-surface abundance at various times after NGF treatment. The rate of TrkA endocytosis was not different between the cell lines tested. Error bars represent SEM. (H, I) After surface biotinylation, cells were incubated for 60 min at 37°C. Biotin remaining on cell-surface proteins was removed by glutathione, and then cells were subjected to the second incubation with NGF (50 ng/ml) at 37°C for 0, 15, or 30 min and treated with glutathione to remove biotin from the proteins recycled back to the plasma membrane, and relative TrkA abundance was determined. For pull down, 130 and 400 μg of total protein were used for PC12 and shNHE5 cells, respectively. The densitometry data of relative levels of total TrkA are presented with mean ± SEM. *p < 0.05 (paired Student's t test, n = 6 experiments).

Mentions: NGF-induced differentiation in PC12 cells involves NGF signaling through the high-affinity receptor tyrosine kinase TrkA and the low-affinity p75 receptor (Huang and Reichardt, 2001; Vaudry et al., 2002; Chao, 2003). TrkA traffics between the cell surface and the recycling endosomal compartment, and this recycling controls the steady-state surface abundance and activity of TrkA (Arimura et al., 2009; Ascano et al., 2009; Vaegter et al., 2011). Consistent with this, we found that TrkA exhibits some association with the recycling endosomal marker Rab11 at the perinuclear region and, to a lesser extent, the early endosomal marker EEA1 (Figure 3A, PC12). Reduced NHE5 expression did not disrupt the endosomal association of TrkA (Figure 3A, shNHE5). Because NHE5 is predominantly associated with endosomal compartments in hippocampal neurons in primary culture (Diering et al., 2011), we addressed whether NHE5 and TrkA associate in endosomes of PC12 cells. NHE5 and TrkA exhibited close association mostly in intracellular compartments (Figure 3B). To test the potential involvement of NHE5 in TrkA trafficking behavior, we next examined the total and cell-surface abundance of TrkA by surface biotinylation and Western blot. There was no difference in the total abundance of TrkA between control cells and PC12 cells stably expressing shNHE5 or shNHE1 (Figure 4, A and C), suggesting that reduced expression of NHE5 or NHE1 has little effect on TrkA degradation or synthesis. In contrast, the cell-surface abundance of TrkA was significantly reduced in cells expressing shRNA for NHE5 (shNHE5; by approximately half) compared with control PC12 cells or PC12 cells expressing shRNA for NHE1 (shNHE1; Figure 4, A and D). When cell lysates were not incubated with the biotinylating agent, TrkA was not detected (Figure 4B). Similar levels of cell-surface populations of Na+/K+ ATPase pump α-subunit and TfnR across the cell lines were observed (Figure 4, A and E), suggesting the specific effects of NHE5 on TrkA trafficking. After NGF treatment, there was a time-dependent reduction in cell-surface TrkA abundance in control PC12 cells (Figure 4, F and G), as reported previously (Grimes et al., 1996; Jullien et al., 2002; Kuruvilla et al., 2004; Chen et al., 2005). To test the potential involvement of NHE5 in NGF-induced TrkA internalization, shRNA-expressing cells were treated with NGF for the indicated times, and TrkA retained on the cell surface was detected by biotinylation (see Materials and Methods). Although the initial cell-surface abundance of TrkA is significantly lower in shNHE5 cells than in shNHE1 or control cells (time 0, Figure 4F), densitometric analysis showed that the rates of NGF-induced TrkA endocytosis were comparable among the three cell lines (Figure 4G). These results suggest that knockdown of NHE5 but not NHE1 reduces steady-state TrkA abundance on the cell surface but not overall TrkA protein levels or rates of endocytosis. To test whether TrkA trafficking from recycling endosomes to the plasma membrane is affected by NHE5 knockdown, we next determined recycling rates of biotinylated TrkA from endosomes to the plasma membrane. As shown in Figure 4, H and I, a slight but significant delay in recycling rates was detected in shNHE5 cells.


Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling.

Diering GH, Numata Y, Fan S, Church J, Numata M - Mol. Biol. Cell (2013)

NHE5 knockdown affects TrkA steady-state surface abundance but not NGF-induced internalization. (A–E) Control PC12 cells (PC12) and PC12 cells stably expressing shRNAs against NHE5 (shNHE5) or NHE1 (shNHE1) were incubated with a cell-surface biotinylation reagent, and total and cell-surface TrkA, NKA, or TfnR were detected by Western blot. Western blot shown in A is representative of three independent experiments. (B) No-biotin control confirmed that there was no fraction being pulled down nonspecifically. The intensity of the bands was determined by densitometry, and the data represent the mean ± SEM of relative levels of total TrkA (C), cell-surface TrkA (D), and TfnR (E) of at least three experiments. **p < 0.01 (paired Student's t test compared with control). (F, G) Cells were serum starved overnight and then treated with 50 ng/ml NGF as indicated, and the total and cell-surface abundance of TrkA, as well as of TfnR, at each time point were determined. Western blots shown are representative of three independent experiments. (G) Relative NGF-induced TrkA endocytosis was measured by quantifying the TrkA cell-surface abundance at various times after NGF treatment. The rate of TrkA endocytosis was not different between the cell lines tested. Error bars represent SEM. (H, I) After surface biotinylation, cells were incubated for 60 min at 37°C. Biotin remaining on cell-surface proteins was removed by glutathione, and then cells were subjected to the second incubation with NGF (50 ng/ml) at 37°C for 0, 15, or 30 min and treated with glutathione to remove biotin from the proteins recycled back to the plasma membrane, and relative TrkA abundance was determined. For pull down, 130 and 400 μg of total protein were used for PC12 and shNHE5 cells, respectively. The densitometry data of relative levels of total TrkA are presented with mean ± SEM. *p < 0.05 (paired Student's t test, n = 6 experiments).
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Figure 4: NHE5 knockdown affects TrkA steady-state surface abundance but not NGF-induced internalization. (A–E) Control PC12 cells (PC12) and PC12 cells stably expressing shRNAs against NHE5 (shNHE5) or NHE1 (shNHE1) were incubated with a cell-surface biotinylation reagent, and total and cell-surface TrkA, NKA, or TfnR were detected by Western blot. Western blot shown in A is representative of three independent experiments. (B) No-biotin control confirmed that there was no fraction being pulled down nonspecifically. The intensity of the bands was determined by densitometry, and the data represent the mean ± SEM of relative levels of total TrkA (C), cell-surface TrkA (D), and TfnR (E) of at least three experiments. **p < 0.01 (paired Student's t test compared with control). (F, G) Cells were serum starved overnight and then treated with 50 ng/ml NGF as indicated, and the total and cell-surface abundance of TrkA, as well as of TfnR, at each time point were determined. Western blots shown are representative of three independent experiments. (G) Relative NGF-induced TrkA endocytosis was measured by quantifying the TrkA cell-surface abundance at various times after NGF treatment. The rate of TrkA endocytosis was not different between the cell lines tested. Error bars represent SEM. (H, I) After surface biotinylation, cells were incubated for 60 min at 37°C. Biotin remaining on cell-surface proteins was removed by glutathione, and then cells were subjected to the second incubation with NGF (50 ng/ml) at 37°C for 0, 15, or 30 min and treated with glutathione to remove biotin from the proteins recycled back to the plasma membrane, and relative TrkA abundance was determined. For pull down, 130 and 400 μg of total protein were used for PC12 and shNHE5 cells, respectively. The densitometry data of relative levels of total TrkA are presented with mean ± SEM. *p < 0.05 (paired Student's t test, n = 6 experiments).
Mentions: NGF-induced differentiation in PC12 cells involves NGF signaling through the high-affinity receptor tyrosine kinase TrkA and the low-affinity p75 receptor (Huang and Reichardt, 2001; Vaudry et al., 2002; Chao, 2003). TrkA traffics between the cell surface and the recycling endosomal compartment, and this recycling controls the steady-state surface abundance and activity of TrkA (Arimura et al., 2009; Ascano et al., 2009; Vaegter et al., 2011). Consistent with this, we found that TrkA exhibits some association with the recycling endosomal marker Rab11 at the perinuclear region and, to a lesser extent, the early endosomal marker EEA1 (Figure 3A, PC12). Reduced NHE5 expression did not disrupt the endosomal association of TrkA (Figure 3A, shNHE5). Because NHE5 is predominantly associated with endosomal compartments in hippocampal neurons in primary culture (Diering et al., 2011), we addressed whether NHE5 and TrkA associate in endosomes of PC12 cells. NHE5 and TrkA exhibited close association mostly in intracellular compartments (Figure 3B). To test the potential involvement of NHE5 in TrkA trafficking behavior, we next examined the total and cell-surface abundance of TrkA by surface biotinylation and Western blot. There was no difference in the total abundance of TrkA between control cells and PC12 cells stably expressing shNHE5 or shNHE1 (Figure 4, A and C), suggesting that reduced expression of NHE5 or NHE1 has little effect on TrkA degradation or synthesis. In contrast, the cell-surface abundance of TrkA was significantly reduced in cells expressing shRNA for NHE5 (shNHE5; by approximately half) compared with control PC12 cells or PC12 cells expressing shRNA for NHE1 (shNHE1; Figure 4, A and D). When cell lysates were not incubated with the biotinylating agent, TrkA was not detected (Figure 4B). Similar levels of cell-surface populations of Na+/K+ ATPase pump α-subunit and TfnR across the cell lines were observed (Figure 4, A and E), suggesting the specific effects of NHE5 on TrkA trafficking. After NGF treatment, there was a time-dependent reduction in cell-surface TrkA abundance in control PC12 cells (Figure 4, F and G), as reported previously (Grimes et al., 1996; Jullien et al., 2002; Kuruvilla et al., 2004; Chen et al., 2005). To test the potential involvement of NHE5 in NGF-induced TrkA internalization, shRNA-expressing cells were treated with NGF for the indicated times, and TrkA retained on the cell surface was detected by biotinylation (see Materials and Methods). Although the initial cell-surface abundance of TrkA is significantly lower in shNHE5 cells than in shNHE1 or control cells (time 0, Figure 4F), densitometric analysis showed that the rates of NGF-induced TrkA endocytosis were comparable among the three cell lines (Figure 4G). These results suggest that knockdown of NHE5 but not NHE1 reduces steady-state TrkA abundance on the cell surface but not overall TrkA protein levels or rates of endocytosis. To test whether TrkA trafficking from recycling endosomes to the plasma membrane is affected by NHE5 knockdown, we next determined recycling rates of biotinylated TrkA from endosomes to the plasma membrane. As shown in Figure 4, H and I, a slight but significant delay in recycling rates was detected in shNHE5 cells.

Bottom Line: NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin.NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment.These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ABSTRACT
To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na(+)/H(+) exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase-Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation.

Show MeSH
Related in: MedlinePlus