Limits...
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-mediated TrkA recycling requires Arf6. (A) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or GGA3 mutant unable to bind Arf-GTP (GGA3-N194A). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (B) Quantification of the degree of TrkA recycling from three independent experiments (as described in A). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, *p < 0.05, **p < 0.01. (C) Analysis of TrkA recycling in PC12 (615) cells treated with control, Arf1, Arf3, and Arf6 siRNA. Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (D) Quantification of the degree of TrkA recycling from three independent experiments (as described in C). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, **p < 0.01. (E) Analysis of TrkA recycling in PC12 (615) cells treated with dimethyl sulfoxide (vehicle), MyrArf1, MyrArf6, and NonMyrArf6 (nonpermeant Arf6 peptide control). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (F) Quantification of the effects of MyrArf1, MyrArf6, and NonMyrArf6 peptides on the degree of TrkA recycling from three independent experiments (as described in A). One-way ANOVA, *p < 0.05. (G) Confocal microscopy images comparing the distribution of Arf6-GFP, endogenous GGA3, and cell surface–labeled TrkA receptors (5C3 antibodies) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate TrkA-labeled vesicles. Scale bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4666136&req=5

Figure 6: GGA3-mediated TrkA recycling requires Arf6. (A) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or GGA3 mutant unable to bind Arf-GTP (GGA3-N194A). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (B) Quantification of the degree of TrkA recycling from three independent experiments (as described in A). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, *p < 0.05, **p < 0.01. (C) Analysis of TrkA recycling in PC12 (615) cells treated with control, Arf1, Arf3, and Arf6 siRNA. Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (D) Quantification of the degree of TrkA recycling from three independent experiments (as described in C). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, **p < 0.01. (E) Analysis of TrkA recycling in PC12 (615) cells treated with dimethyl sulfoxide (vehicle), MyrArf1, MyrArf6, and NonMyrArf6 (nonpermeant Arf6 peptide control). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (F) Quantification of the effects of MyrArf1, MyrArf6, and NonMyrArf6 peptides on the degree of TrkA recycling from three independent experiments (as described in A). One-way ANOVA, *p < 0.05. (G) Confocal microscopy images comparing the distribution of Arf6-GFP, endogenous GGA3, and cell surface–labeled TrkA receptors (5C3 antibodies) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate TrkA-labeled vesicles. Scale bar, 10 μm.

Mentions: Having found that GGA3 binds directly to TrkA, we next investigated the mechanism by which GGA3 regulates the sorting of TrkA into the recycling pathway. Given that GGAs are recruited to the membrane by activated Arfs, we then examined the role of Arfs in GGA3-mediated TrkA recycling by rescue experiments using GGA3 N194A, a mutant that specifically uncouples GGA from interactions with Arf-GTP proteins (Puertollano et al., 2001b). Expression of siRNA-resistant GGA3, but not GGA3 N194A, restored TrkA recycling to the PM in GGA3- depleted PC12 (615) cells (Figure 6, A and B), suggesting that an interaction between GGA3 and Arf-GTP is required for the sorting of TrkA to the recycling pathway.


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

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

GGA3-mediated TrkA recycling requires Arf6. (A) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or GGA3 mutant unable to bind Arf-GTP (GGA3-N194A). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (B) Quantification of the degree of TrkA recycling from three independent experiments (as described in A). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, *p < 0.05, **p < 0.01. (C) Analysis of TrkA recycling in PC12 (615) cells treated with control, Arf1, Arf3, and Arf6 siRNA. Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (D) Quantification of the degree of TrkA recycling from three independent experiments (as described in C). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, **p < 0.01. (E) Analysis of TrkA recycling in PC12 (615) cells treated with dimethyl sulfoxide (vehicle), MyrArf1, MyrArf6, and NonMyrArf6 (nonpermeant Arf6 peptide control). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (F) Quantification of the effects of MyrArf1, MyrArf6, and NonMyrArf6 peptides on the degree of TrkA recycling from three independent experiments (as described in A). One-way ANOVA, *p < 0.05. (G) Confocal microscopy images comparing the distribution of Arf6-GFP, endogenous GGA3, and cell surface–labeled TrkA receptors (5C3 antibodies) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate TrkA-labeled vesicles. Scale bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4666136&req=5

Figure 6: GGA3-mediated TrkA recycling requires Arf6. (A) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or GGA3 mutant unable to bind Arf-GTP (GGA3-N194A). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (B) Quantification of the degree of TrkA recycling from three independent experiments (as described in A). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, *p < 0.05, **p < 0.01. (C) Analysis of TrkA recycling in PC12 (615) cells treated with control, Arf1, Arf3, and Arf6 siRNA. Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (D) Quantification of the degree of TrkA recycling from three independent experiments (as described in C). The amount of recycled TrkA is expressed as the percentage of the pool of biotinylated TrkA after the 7-min internalization period and before rewarming. One-way ANOVA, **p < 0.01. (E) Analysis of TrkA recycling in PC12 (615) cells treated with dimethyl sulfoxide (vehicle), MyrArf1, MyrArf6, and NonMyrArf6 (nonpermeant Arf6 peptide control). Recycling assays were performed as described in Figure 3A. int, internalized biotinylated TrkA before rewarming; rec, remaining biotinylated receptors after 45 min of rewarming (recycling). (F) Quantification of the effects of MyrArf1, MyrArf6, and NonMyrArf6 peptides on the degree of TrkA recycling from three independent experiments (as described in A). One-way ANOVA, *p < 0.05. (G) Confocal microscopy images comparing the distribution of Arf6-GFP, endogenous GGA3, and cell surface–labeled TrkA receptors (5C3 antibodies) internalized for 15 min in PC12 (615) cells. Insets, regions of higher magnification; arrowheads indicate TrkA-labeled vesicles. Scale bar, 10 μm.
Mentions: Having found that GGA3 binds directly to TrkA, we next investigated the mechanism by which GGA3 regulates the sorting of TrkA into the recycling pathway. Given that GGAs are recruited to the membrane by activated Arfs, we then examined the role of Arfs in GGA3-mediated TrkA recycling by rescue experiments using GGA3 N194A, a mutant that specifically uncouples GGA from interactions with Arf-GTP proteins (Puertollano et al., 2001b). Expression of siRNA-resistant GGA3, but not GGA3 N194A, restored TrkA recycling to the PM in GGA3- depleted PC12 (615) cells (Figure 6, A and B), suggesting that an interaction between GGA3 and Arf-GTP is required for the sorting of TrkA to the recycling pathway.

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