Limits...
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.

Show MeSH

Related in: MedlinePlus

Intracellular localization of TrkA and NHE5. (A) Double immunofluorescence microscopy of TrkA (green) and EEA1 (red) or Rab11 (red) in PC12 control cells (PC12) and PC12 cells stably expressing shRNA for NHE5 (shNHE5). Fixed cells were treated with rabbit polyclonal anti-TrkA antibody and mouse monoclonal anti-EEA1 antibody or anti-Rab11 antibody. Alexa Fluor 488–conjugated goat anti-mouse and Alexa Fluor 568–conjugated goat anti-rabbit antibodies were used to visualize the signals. (B) Double immunofluorescence microscopy of NHE5 (green) and TrkA (red) in PC12 cells. Fixed cells were treated with rabbit polyclonal anti-NHE5 antibody and mouse monoclonal anti-TrkA antibody to visualize the endogenous protein localization. Scale bars, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814139&req=5

Figure 3: Intracellular localization of TrkA and NHE5. (A) Double immunofluorescence microscopy of TrkA (green) and EEA1 (red) or Rab11 (red) in PC12 control cells (PC12) and PC12 cells stably expressing shRNA for NHE5 (shNHE5). Fixed cells were treated with rabbit polyclonal anti-TrkA antibody and mouse monoclonal anti-EEA1 antibody or anti-Rab11 antibody. Alexa Fluor 488–conjugated goat anti-mouse and Alexa Fluor 568–conjugated goat anti-rabbit antibodies were used to visualize the signals. (B) Double immunofluorescence microscopy of NHE5 (green) and TrkA (red) in PC12 cells. Fixed cells were treated with rabbit polyclonal anti-NHE5 antibody and mouse monoclonal anti-TrkA antibody to visualize the endogenous protein localization. Scale bars, 10 μm.

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)

Intracellular localization of TrkA and NHE5. (A) Double immunofluorescence microscopy of TrkA (green) and EEA1 (red) or Rab11 (red) in PC12 control cells (PC12) and PC12 cells stably expressing shRNA for NHE5 (shNHE5). Fixed cells were treated with rabbit polyclonal anti-TrkA antibody and mouse monoclonal anti-EEA1 antibody or anti-Rab11 antibody. Alexa Fluor 488–conjugated goat anti-mouse and Alexa Fluor 568–conjugated goat anti-rabbit antibodies were used to visualize the signals. (B) Double immunofluorescence microscopy of NHE5 (green) and TrkA (red) in PC12 cells. Fixed cells were treated with rabbit polyclonal anti-NHE5 antibody and mouse monoclonal anti-TrkA antibody to visualize the endogenous protein localization. Scale bars, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814139&req=5

Figure 3: Intracellular localization of TrkA and NHE5. (A) Double immunofluorescence microscopy of TrkA (green) and EEA1 (red) or Rab11 (red) in PC12 control cells (PC12) and PC12 cells stably expressing shRNA for NHE5 (shNHE5). Fixed cells were treated with rabbit polyclonal anti-TrkA antibody and mouse monoclonal anti-EEA1 antibody or anti-Rab11 antibody. Alexa Fluor 488–conjugated goat anti-mouse and Alexa Fluor 568–conjugated goat anti-rabbit antibodies were used to visualize the signals. (B) Double immunofluorescence microscopy of NHE5 (green) and TrkA (red) in PC12 cells. Fixed cells were treated with rabbit polyclonal anti-NHE5 antibody and mouse monoclonal anti-TrkA antibody to visualize the endogenous protein localization. Scale bars, 10 μm.
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