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GGA3 mediates TrkA endocytic recycling to promote sustained Akt phosphorylation and cell survival.

Li X, Lavigne P, Lavoie C - Mol. Biol. Cell (2015)

Bottom Line: Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood.We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival.We also show that GGA3's effect on TrkA recycling is dependent on the activation of Arf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.

No MeSH data available.


Related in: MedlinePlus

GGA3 is required for TrkA recycling. (A) Schematic of internalization assay. PC12 (615) cells were biotinylated at 4°C and stimulated with NGF for 7 min at 37°C to allow for internalization. After glutathione stripping of the remaining cell-surface biotin, cells were reincubated at 37°C for another 7 or 45 min to allow for the recycling of internalized receptors. Cells were then stripped again with glutathione to remove biotin from the surface-exposed biotinylated receptors recycled back to the cell surface. The remaining biotinylated proteins were then collected with avidin and immunoblotted with TrkA antibodies. The TrkA signal lost in the second stripping procedure was considered the fraction of recycled receptors. (B) Representative Western blots of the TrkA recycling assay performed in control and GGA3-depleted PC12 (615) cells. Recy 0 refers to the internalized biotinylated receptors before rewarming; Recy 7 and 45 refer to the remaining biotinylated receptors after rewarming. (C) Quantification of the degree of TrkA recycling from three independent experiments (as described in A and B). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA following the 7-min internalization period and before rewarming. Student’s t test, *p < 0.05. (D) Confocal microscopy images comparing the distribution of endogenous GGA3 with that of cell surface–labeled TrkA (5C3 antibodies) and transferrin receptor (Alexa Fluor 594–conjugated transferrin) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate colocalization. Scale bar, 10 μm.
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Figure 3: GGA3 is required for TrkA recycling. (A) Schematic of internalization assay. PC12 (615) cells were biotinylated at 4°C and stimulated with NGF for 7 min at 37°C to allow for internalization. After glutathione stripping of the remaining cell-surface biotin, cells were reincubated at 37°C for another 7 or 45 min to allow for the recycling of internalized receptors. Cells were then stripped again with glutathione to remove biotin from the surface-exposed biotinylated receptors recycled back to the cell surface. The remaining biotinylated proteins were then collected with avidin and immunoblotted with TrkA antibodies. The TrkA signal lost in the second stripping procedure was considered the fraction of recycled receptors. (B) Representative Western blots of the TrkA recycling assay performed in control and GGA3-depleted PC12 (615) cells. Recy 0 refers to the internalized biotinylated receptors before rewarming; Recy 7 and 45 refer to the remaining biotinylated receptors after rewarming. (C) Quantification of the degree of TrkA recycling from three independent experiments (as described in A and B). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA following the 7-min internalization period and before rewarming. Student’s t test, *p < 0.05. (D) Confocal microscopy images comparing the distribution of endogenous GGA3 with that of cell surface–labeled TrkA (5C3 antibodies) and transferrin receptor (Alexa Fluor 594–conjugated transferrin) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate colocalization. Scale bar, 10 μm.

Mentions: To assess whether GGA3 participated in the postendocytic recycling of TrkA, we performed a cleavable biotinylation assay (schematized in Figure 3A) in which the biotin-labeled cell surface receptors were internalized after 7 min of NGF treatment, followed by stripping with glutathione. The cells were then returned to 37°C for 7 or 45 min to allow for recycling, and any reappearing cell surface–biotinylated receptors were stripped again (Figure 3A). The TrkA signal lost in the second stripping procedure was considered the fraction of receptors that had been recycled. In GGA3-depleted cells, only 31% of TrkA had returned to the PM by 45 min, whereas in control cells, >60% had returned (Figure 3, B and C). However, GGA3 knockdown did not significantly alter the TrkA recycling ratio at 7 min (Figure 3C), a time point representing rapid recycling (Goh and Sorkin, 2013). Thus these biotinylation data indicate that GGA3 knockdown leads to slower recycling of TrkA, suggesting that in the absence of GGA3, TrkA traverses the endosomal sorting pathway to lysosomes.


GGA3 mediates TrkA endocytic recycling to promote sustained Akt phosphorylation and cell survival.

Li X, Lavigne P, Lavoie C - Mol. Biol. Cell (2015)

GGA3 is required for TrkA recycling. (A) Schematic of internalization assay. PC12 (615) cells were biotinylated at 4°C and stimulated with NGF for 7 min at 37°C to allow for internalization. After glutathione stripping of the remaining cell-surface biotin, cells were reincubated at 37°C for another 7 or 45 min to allow for the recycling of internalized receptors. Cells were then stripped again with glutathione to remove biotin from the surface-exposed biotinylated receptors recycled back to the cell surface. The remaining biotinylated proteins were then collected with avidin and immunoblotted with TrkA antibodies. The TrkA signal lost in the second stripping procedure was considered the fraction of recycled receptors. (B) Representative Western blots of the TrkA recycling assay performed in control and GGA3-depleted PC12 (615) cells. Recy 0 refers to the internalized biotinylated receptors before rewarming; Recy 7 and 45 refer to the remaining biotinylated receptors after rewarming. (C) Quantification of the degree of TrkA recycling from three independent experiments (as described in A and B). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA following the 7-min internalization period and before rewarming. Student’s t test, *p < 0.05. (D) Confocal microscopy images comparing the distribution of endogenous GGA3 with that of cell surface–labeled TrkA (5C3 antibodies) and transferrin receptor (Alexa Fluor 594–conjugated transferrin) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate colocalization. Scale bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: GGA3 is required for TrkA recycling. (A) Schematic of internalization assay. PC12 (615) cells were biotinylated at 4°C and stimulated with NGF for 7 min at 37°C to allow for internalization. After glutathione stripping of the remaining cell-surface biotin, cells were reincubated at 37°C for another 7 or 45 min to allow for the recycling of internalized receptors. Cells were then stripped again with glutathione to remove biotin from the surface-exposed biotinylated receptors recycled back to the cell surface. The remaining biotinylated proteins were then collected with avidin and immunoblotted with TrkA antibodies. The TrkA signal lost in the second stripping procedure was considered the fraction of recycled receptors. (B) Representative Western blots of the TrkA recycling assay performed in control and GGA3-depleted PC12 (615) cells. Recy 0 refers to the internalized biotinylated receptors before rewarming; Recy 7 and 45 refer to the remaining biotinylated receptors after rewarming. (C) Quantification of the degree of TrkA recycling from three independent experiments (as described in A and B). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA following the 7-min internalization period and before rewarming. Student’s t test, *p < 0.05. (D) Confocal microscopy images comparing the distribution of endogenous GGA3 with that of cell surface–labeled TrkA (5C3 antibodies) and transferrin receptor (Alexa Fluor 594–conjugated transferrin) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate colocalization. Scale bar, 10 μm.
Mentions: To assess whether GGA3 participated in the postendocytic recycling of TrkA, we performed a cleavable biotinylation assay (schematized in Figure 3A) in which the biotin-labeled cell surface receptors were internalized after 7 min of NGF treatment, followed by stripping with glutathione. The cells were then returned to 37°C for 7 or 45 min to allow for recycling, and any reappearing cell surface–biotinylated receptors were stripped again (Figure 3A). The TrkA signal lost in the second stripping procedure was considered the fraction of receptors that had been recycled. In GGA3-depleted cells, only 31% of TrkA had returned to the PM by 45 min, whereas in control cells, >60% had returned (Figure 3, B and C). However, GGA3 knockdown did not significantly alter the TrkA recycling ratio at 7 min (Figure 3C), a time point representing rapid recycling (Goh and Sorkin, 2013). Thus these biotinylation data indicate that GGA3 knockdown leads to slower recycling of TrkA, suggesting that in the absence of GGA3, TrkA traverses the endosomal sorting pathway to lysosomes.

Bottom Line: Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood.We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival.We also show that GGA3's effect on TrkA recycling is dependent on the activation of Arf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.

No MeSH data available.


Related in: MedlinePlus