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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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Bafilomycin treatment reduces TrkA surface levels. (A) Serum-starved PC12 cells were incubated with bafilomycin A1 (250 nM) for the times indicated, and cell-surface TrkA or TfnR was detected by surface biotinylation assay. (B) TrkA or TfnR cell-surface abundance was determined by densitometry, and relative intensities were calculated. N = 3; error bars represent SEM; **p < 0.01 (Student's t test) for difference from untreated cells (time 0).
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Figure 5: Bafilomycin treatment reduces TrkA surface levels. (A) Serum-starved PC12 cells were incubated with bafilomycin A1 (250 nM) for the times indicated, and cell-surface TrkA or TfnR was detected by surface biotinylation assay. (B) TrkA or TfnR cell-surface abundance was determined by densitometry, and relative intensities were calculated. N = 3; error bars represent SEM; **p < 0.01 (Student's t test) for difference from untreated cells (time 0).

Mentions: Our data are consistent with the possibility that NHE5 may be a novel regulator of recycling endosomal pH to influence the trafficking of TrkA between endosomes and the plasma membrane. To further test this hypothesis, we examined the effect of bafilomycin on TrkA cell-surface abundance. Treatment of control PC12 cells with bafilomycin (250 nM) caused a significant decrease in TrkA surface abundance after 5–6 h of bafilomycin treatment but not in total TrkA protein expression (Figure 5, A and B). The cell-surface abundance of TfnR, another recycling receptor, was not noticeably affected by bafilomycin. Bafilomycin treatment, which is known to lead to organellar alkalinization, resulted in the accumulation of TrkA inside the cell similar to that observed in cells with stable knockdown of NHE5. Taken together, these results suggest that steady-state trafficking of TrkA through recycling endosomes is sensitive to endosomal pH.


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)

Bafilomycin treatment reduces TrkA surface levels. (A) Serum-starved PC12 cells were incubated with bafilomycin A1 (250 nM) for the times indicated, and cell-surface TrkA or TfnR was detected by surface biotinylation assay. (B) TrkA or TfnR cell-surface abundance was determined by densitometry, and relative intensities were calculated. N = 3; error bars represent SEM; **p < 0.01 (Student's t test) for difference from untreated cells (time 0).
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Related In: Results  -  Collection

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Figure 5: Bafilomycin treatment reduces TrkA surface levels. (A) Serum-starved PC12 cells were incubated with bafilomycin A1 (250 nM) for the times indicated, and cell-surface TrkA or TfnR was detected by surface biotinylation assay. (B) TrkA or TfnR cell-surface abundance was determined by densitometry, and relative intensities were calculated. N = 3; error bars represent SEM; **p < 0.01 (Student's t test) for difference from untreated cells (time 0).
Mentions: Our data are consistent with the possibility that NHE5 may be a novel regulator of recycling endosomal pH to influence the trafficking of TrkA between endosomes and the plasma membrane. To further test this hypothesis, we examined the effect of bafilomycin on TrkA cell-surface abundance. Treatment of control PC12 cells with bafilomycin (250 nM) caused a significant decrease in TrkA surface abundance after 5–6 h of bafilomycin treatment but not in total TrkA protein expression (Figure 5, A and B). The cell-surface abundance of TfnR, another recycling receptor, was not noticeably affected by bafilomycin. Bafilomycin treatment, which is known to lead to organellar alkalinization, resulted in the accumulation of TrkA inside the cell similar to that observed in cells with stable knockdown of NHE5. Taken together, these results suggest that steady-state trafficking of TrkA through recycling endosomes is sensitive to endosomal pH.

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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