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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 is not functional on the plasma membrane in resting PC12 cells. (A) PC12 cells were treated with a cell-surface biotinylating agent. Biotinylated cell-surface proteins were purified by affinity trap with NeutrAvidin beads. Total lysate (Lysate) and biotinylated proteins eluted from the beads (Surface) were resolved in SDS–PAGE and immunoblotted with the indicated antibodies. Lysate protein fractions represent 10% of the protein loaded in the surface fraction. (B) Cells loaded with BCECF were subjected to cytosolic acidification by briefly exposing cells to NH4Cl followed by washout with NaCl-free solution. NHE activity was induced by reintroducing the Na+-containing solution in the absence or presence of the NHE1-specific inhibitor cariporide (10 μM). No recovery from the internal acid load was observed in the presence of cariporide, indicating that NHE1 is the only functional NHE on the plasma membrane in PC12 cells. Records represent pHi measurements obtained simultaneously from 40 control and 34 cariporide-treated cells and are representative of three independent experiments under each condition.
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Figure 1: NHE5 is not functional on the plasma membrane in resting PC12 cells. (A) PC12 cells were treated with a cell-surface biotinylating agent. Biotinylated cell-surface proteins were purified by affinity trap with NeutrAvidin beads. Total lysate (Lysate) and biotinylated proteins eluted from the beads (Surface) were resolved in SDS–PAGE and immunoblotted with the indicated antibodies. Lysate protein fractions represent 10% of the protein loaded in the surface fraction. (B) Cells loaded with BCECF were subjected to cytosolic acidification by briefly exposing cells to NH4Cl followed by washout with NaCl-free solution. NHE activity was induced by reintroducing the Na+-containing solution in the absence or presence of the NHE1-specific inhibitor cariporide (10 μM). No recovery from the internal acid load was observed in the presence of cariporide, indicating that NHE1 is the only functional NHE on the plasma membrane in PC12 cells. Records represent pHi measurements obtained simultaneously from 40 control and 34 cariporide-treated cells and are representative of three independent experiments under each condition.

Mentions: We previously reported that NHE1 and NHE5 are the two major NHEs expressed in PC12 cells and generated short hairpin RNA (shRNA) PC12 cell lines that have constitutively reduced expression of these proteins (Diering et al., 2011). Using cell-surface biotinylation and Western blot, we observed that little NHE5 is present on the plasma membrane, whereas NHE1 and transferrin receptor (TfnR) are abundantly expressed on the plasma membrane of resting PC12 cells (Figure 1A). Actin was not detectable on the cell surface, indicating that membrane integrity was retained during these experiments. To test for NHE5 activity on the cell surface, we loaded PC12 cells with the pH-sensitive fluorescent dye 2′-7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and monitored the recovery of cytosolic pH (pHi) from an imposed intracellular acid load using the ammonium prepulse technique (Figure 1B). After washout of ammonium with Na+-free buffer, pHi failed to recover from the internal acid load and remained acidic until the reintroduction of external Na+, at which time it rapidly recovered to normal values. The specific NHE1 inhibitor cariporide (10 μM; Masereel et al., 2003) completely prevented the recovery of pHi observed after the reintroduction of external Na+ and, in agreement with previous work (Szabo et al., 2000), 10 μM cariporide failed to inhibit NHE5 activity heterologously expressed in NHE-deficient Chinese hamster ovary mutant AP-1 cells (Supplemental Figure S1). These findings indicate that NHE1, but not NHE5, is the major functional NHE on the plasma membrane in PC12 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 is not functional on the plasma membrane in resting PC12 cells. (A) PC12 cells were treated with a cell-surface biotinylating agent. Biotinylated cell-surface proteins were purified by affinity trap with NeutrAvidin beads. Total lysate (Lysate) and biotinylated proteins eluted from the beads (Surface) were resolved in SDS–PAGE and immunoblotted with the indicated antibodies. Lysate protein fractions represent 10% of the protein loaded in the surface fraction. (B) Cells loaded with BCECF were subjected to cytosolic acidification by briefly exposing cells to NH4Cl followed by washout with NaCl-free solution. NHE activity was induced by reintroducing the Na+-containing solution in the absence or presence of the NHE1-specific inhibitor cariporide (10 μM). No recovery from the internal acid load was observed in the presence of cariporide, indicating that NHE1 is the only functional NHE on the plasma membrane in PC12 cells. Records represent pHi measurements obtained simultaneously from 40 control and 34 cariporide-treated cells and are representative of three independent experiments under each condition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: NHE5 is not functional on the plasma membrane in resting PC12 cells. (A) PC12 cells were treated with a cell-surface biotinylating agent. Biotinylated cell-surface proteins were purified by affinity trap with NeutrAvidin beads. Total lysate (Lysate) and biotinylated proteins eluted from the beads (Surface) were resolved in SDS–PAGE and immunoblotted with the indicated antibodies. Lysate protein fractions represent 10% of the protein loaded in the surface fraction. (B) Cells loaded with BCECF were subjected to cytosolic acidification by briefly exposing cells to NH4Cl followed by washout with NaCl-free solution. NHE activity was induced by reintroducing the Na+-containing solution in the absence or presence of the NHE1-specific inhibitor cariporide (10 μM). No recovery from the internal acid load was observed in the presence of cariporide, indicating that NHE1 is the only functional NHE on the plasma membrane in PC12 cells. Records represent pHi measurements obtained simultaneously from 40 control and 34 cariporide-treated cells and are representative of three independent experiments under each condition.
Mentions: We previously reported that NHE1 and NHE5 are the two major NHEs expressed in PC12 cells and generated short hairpin RNA (shRNA) PC12 cell lines that have constitutively reduced expression of these proteins (Diering et al., 2011). Using cell-surface biotinylation and Western blot, we observed that little NHE5 is present on the plasma membrane, whereas NHE1 and transferrin receptor (TfnR) are abundantly expressed on the plasma membrane of resting PC12 cells (Figure 1A). Actin was not detectable on the cell surface, indicating that membrane integrity was retained during these experiments. To test for NHE5 activity on the cell surface, we loaded PC12 cells with the pH-sensitive fluorescent dye 2′-7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and monitored the recovery of cytosolic pH (pHi) from an imposed intracellular acid load using the ammonium prepulse technique (Figure 1B). After washout of ammonium with Na+-free buffer, pHi failed to recover from the internal acid load and remained acidic until the reintroduction of external Na+, at which time it rapidly recovered to normal values. The specific NHE1 inhibitor cariporide (10 μM; Masereel et al., 2003) completely prevented the recovery of pHi observed after the reintroduction of external Na+ and, in agreement with previous work (Szabo et al., 2000), 10 μM cariporide failed to inhibit NHE5 activity heterologously expressed in NHE-deficient Chinese hamster ovary mutant AP-1 cells (Supplemental Figure S1). These findings indicate that NHE1, but not NHE5, is the major functional NHE on the plasma membrane in PC12 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