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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.


The interaction between GGA3 and TrkA is independent of TrkA activation and ubiquitination. (A) Interaction of TrkA and GGA3 in PC12 (615) cells incubated with or without 10 ng/ml NGF for 10 min. Lysates were immunoprecipitated with GGA3 antibody and immunoblotted as shown. (B) Immunoprecipitation of TrkA from HEK293T cells transfected with a TrkA wild type, TrkA kinase-dead mutant (TrkA-K547A), GGA3 wild type, or GGA3 mutant unable to bind ubiquitin (GGA3-L276A) and immunoblotted as shown. (C) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or mutant GGA3-L276A. 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 < 0.05, **p < 0.01.
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Figure 4: The interaction between GGA3 and TrkA is independent of TrkA activation and ubiquitination. (A) Interaction of TrkA and GGA3 in PC12 (615) cells incubated with or without 10 ng/ml NGF for 10 min. Lysates were immunoprecipitated with GGA3 antibody and immunoblotted as shown. (B) Immunoprecipitation of TrkA from HEK293T cells transfected with a TrkA wild type, TrkA kinase-dead mutant (TrkA-K547A), GGA3 wild type, or GGA3 mutant unable to bind ubiquitin (GGA3-L276A) and immunoblotted as shown. (C) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or mutant GGA3-L276A. 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 < 0.05, **p < 0.01.

Mentions: To establish the mechanism by which GGA3 regulates TrkA recycling, we characterized the interaction between TrkA and GGA3. Given that TrkA is ubiquitinated after NGF stimulation (Arevalo et al., 2006; Geetha and Wooten, 2008) and that GGA3 binds to ubiquitin and is involved in endosomal sorting of ubiquitinated cargo (Puertollano and Bonifacino, 2004; Kang et al., 2010), we first examined whether NGF treatment (10 min) increases the amount of endogenous GGA3 that associates with TrkA in PC12 (615) cells. The amount of TrkA that coimmunoprecipitated with GGA3 from NGF-treated cells was comparable to that from untreated cells (Figure 4A), suggesting that the interaction between TrkA and GGA3 is independent of TrkA activation and ubiquitination. This result was validated in two different manners. First, we observed interactions between GGA3 and a kinase-dead TrkA mutant (TrkA-K547A; Meakin et al., 1999) and between TrkA and the GGA3-L276A mutant, which cannot bind ubiquitin (Puertollano and Bonifacino, 2004; Figure 4B). Second, after reintroduction of GGA3-L276A into GGA3-depleted cells with an siRNA-resistant form of GGA3-L276A, the level of TrkA recycling was similar to that in cells rescued with siRNA-resistant wild-type GGA3 or in control cells (Figure 4, C and D), confirming that the ability of GGA3 to bind ubiquitin is not required for endosomal sorting of TrkA.


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

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

The interaction between GGA3 and TrkA is independent of TrkA activation and ubiquitination. (A) Interaction of TrkA and GGA3 in PC12 (615) cells incubated with or without 10 ng/ml NGF for 10 min. Lysates were immunoprecipitated with GGA3 antibody and immunoblotted as shown. (B) Immunoprecipitation of TrkA from HEK293T cells transfected with a TrkA wild type, TrkA kinase-dead mutant (TrkA-K547A), GGA3 wild type, or GGA3 mutant unable to bind ubiquitin (GGA3-L276A) and immunoblotted as shown. (C) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or mutant GGA3-L276A. 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 < 0.05, **p < 0.01.
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Figure 4: The interaction between GGA3 and TrkA is independent of TrkA activation and ubiquitination. (A) Interaction of TrkA and GGA3 in PC12 (615) cells incubated with or without 10 ng/ml NGF for 10 min. Lysates were immunoprecipitated with GGA3 antibody and immunoblotted as shown. (B) Immunoprecipitation of TrkA from HEK293T cells transfected with a TrkA wild type, TrkA kinase-dead mutant (TrkA-K547A), GGA3 wild type, or GGA3 mutant unable to bind ubiquitin (GGA3-L276A) and immunoblotted as shown. (C) Analysis of TrkA recycling in GGA3-depleted PC12 (615) cells rescued with control cDNA (vector) or siRNA-resistant GGA3 wild type or mutant GGA3-L276A. 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 < 0.05, **p < 0.01.
Mentions: To establish the mechanism by which GGA3 regulates TrkA recycling, we characterized the interaction between TrkA and GGA3. Given that TrkA is ubiquitinated after NGF stimulation (Arevalo et al., 2006; Geetha and Wooten, 2008) and that GGA3 binds to ubiquitin and is involved in endosomal sorting of ubiquitinated cargo (Puertollano and Bonifacino, 2004; Kang et al., 2010), we first examined whether NGF treatment (10 min) increases the amount of endogenous GGA3 that associates with TrkA in PC12 (615) cells. The amount of TrkA that coimmunoprecipitated with GGA3 from NGF-treated cells was comparable to that from untreated cells (Figure 4A), suggesting that the interaction between TrkA and GGA3 is independent of TrkA activation and ubiquitination. This result was validated in two different manners. First, we observed interactions between GGA3 and a kinase-dead TrkA mutant (TrkA-K547A; Meakin et al., 1999) and between TrkA and the GGA3-L276A mutant, which cannot bind ubiquitin (Puertollano and Bonifacino, 2004; Figure 4B). Second, after reintroduction of GGA3-L276A into GGA3-depleted cells with an siRNA-resistant form of GGA3-L276A, the level of TrkA recycling was similar to that in cells rescued with siRNA-resistant wild-type GGA3 or in control cells (Figure 4, C and D), confirming that the ability of GGA3 to bind ubiquitin is not required for endosomal sorting of TrkA.

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.