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A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals.

Garcia-Marcos M, Ear J, Farquhar MG, Ghosh P - Mol. Biol. Cell (2011)

Bottom Line: Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown.Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a.Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 , USA. mgarciamarcos@ucsd.edu

ABSTRACT
Autophagy is the major catabolic process responsible for the removal of aggregated proteins and damaged organelles. Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown. Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a. Girdin), a nonreceptor guanine nucleotide exchange factor for Gα(i3), plays a key role in regulating autophagy and that dynamic interplay between Gα(i3), activator of G-protein signaling 3 (AGS3, its guanine nucleotide dissociation inhibitor), and GIV determines whether autophagy is promoted or inhibited. We found that AGS3 directly binds light chain 3 (LC3), recruits Gα(i3) to LC3-positive membranes upon starvation, and promotes autophagy by inhibiting the G protein. Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein. These results provide mechanistic insights into how reversible modulation of Gα(i3) activity by AGS3 and GIV maintains the delicate equilibrium between promotion and inhibition of autophagy.

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GIV’s GEF motif is required for insulin to trigger a shift in Gαi3 binding from AGS3 to GIV. (A) Gαi3 coimmunoprecipitates with AGS3 in serum-starved cells and with GIV when cells are insulin stimulated. Cos7 cells transiently transfected with FLAG-tagged Gαi3 (Gαi3-FLAG) or vector control were serum starved (-) and stimulated with 100 nM insulin (+) for 15 min before lysis. Equal aliquots of cell lysates (left panel) were incubated with anti-FLAG mAb. Immunoprecipitated complexes (right panel) were analyzed for GIV, AGS3, and FLAG (Gαi3) by immunoblotting (IB). In serum-starved cells (−), Gαi3-bound immune complexes are enriched in AGS3, whereas after insulin treatment (+) these immune complexes are depleted of AGS3 and enriched in GIV. Identical observations were made after EGF treatment (Supplemental Figure S4). (B) Changes in the abundance of Gαi–AGS3 complexes in response to insulin require an intact GEF motif in GIV. Immunoprecipitation was carried out on lysates of serum-starved and insulin-stimulated control, GIV-WT, and GIV-FA cells with anti-Gαi3 IgG, and the immune complexes were analyzed for Gαi3, AGS3, and GIV by IB. In GIV-WT cells, the amount of Gαi3-bound GIV increased and the amount of Gαi3-bound AGS3 decreased upon insulin treatment. In GIV-FA cells (GEF-deficient mutant), GIV does not coimmunoprecipitate with Gαi3, and the extent of Gαi3-bound AGS3 remains unaltered after insulin treatment.
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Figure 4: GIV’s GEF motif is required for insulin to trigger a shift in Gαi3 binding from AGS3 to GIV. (A) Gαi3 coimmunoprecipitates with AGS3 in serum-starved cells and with GIV when cells are insulin stimulated. Cos7 cells transiently transfected with FLAG-tagged Gαi3 (Gαi3-FLAG) or vector control were serum starved (-) and stimulated with 100 nM insulin (+) for 15 min before lysis. Equal aliquots of cell lysates (left panel) were incubated with anti-FLAG mAb. Immunoprecipitated complexes (right panel) were analyzed for GIV, AGS3, and FLAG (Gαi3) by immunoblotting (IB). In serum-starved cells (−), Gαi3-bound immune complexes are enriched in AGS3, whereas after insulin treatment (+) these immune complexes are depleted of AGS3 and enriched in GIV. Identical observations were made after EGF treatment (Supplemental Figure S4). (B) Changes in the abundance of Gαi–AGS3 complexes in response to insulin require an intact GEF motif in GIV. Immunoprecipitation was carried out on lysates of serum-starved and insulin-stimulated control, GIV-WT, and GIV-FA cells with anti-Gαi3 IgG, and the immune complexes were analyzed for Gαi3, AGS3, and GIV by IB. In GIV-WT cells, the amount of Gαi3-bound GIV increased and the amount of Gαi3-bound AGS3 decreased upon insulin treatment. In GIV-FA cells (GEF-deficient mutant), GIV does not coimmunoprecipitate with Gαi3, and the extent of Gαi3-bound AGS3 remains unaltered after insulin treatment.

Mentions: Next we investigated whether growth factors influence the competitive binding of GIV and AGS3 to Gαi3 in vivo. When we immunoprecipitated Gαi3 from Cos7 cells expressing Gαi3-FLAG and immunoblotted for AGS3 and GIV, we found that AGS3 interacted with Gαi3 preferentially upon starvation, and this interaction was reduced by ∼75–80% upon insulin stimulation (Figure 4A). In contrast, GIV displayed an approximately twofold increase in binding to Gαi3 compared with the starved state. Identical results were obtained when the cells were stimulated with EGF (Supplemental Figure S4), indicating that the pattern is common to several growth factors. These results demonstrate that starvation favors formation of AGS3–Gαi3 complexes, whereas growth factors favor formation of GIV–Gαi3 complexes and dissociation of AGS3–Gαi3 complexes. We conclude that AGS3–Gαi3 and GIV–Gαi3 complexes exist in equilibrium in living cells and that growth factor stimulation shifts the equilibrium toward activation of Gαi3 by GIV. We anticipated that this shift might require the presence of a functional GEF motif in GIV and found that this is indeed the case. Comparison of the endogenous Gαi3-bound complexes in starved and insulin-stimulated GIV-WT and GIV-FA cells revealed that the insulin-dependent shift from AGS3–Gαi3 to GIV–Gαi3 complexes occurred exclusively in GIV-WT cells (Figure 4B). The ability of insulin to increase GIV–Gαi3 complexes and reduce AGS3–Gαi3 complexes in GIV-WT but not in GIV-FA cells correlates with the responsiveness of these cells to insulin (Figure 1, A and B), suggesting that GIV’s ability to antagonize AGS3 (Figures 2 and 3) is required for the anti-autophagic action of insulin. We conclude that GIV’s GEF function facilitates the anti-autophagic action of insulin in part by shifting the equilibrium from AGS3–Gαi3 toward GIV–Gαi3 and triggering activation of Gαi3.


A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals.

Garcia-Marcos M, Ear J, Farquhar MG, Ghosh P - Mol. Biol. Cell (2011)

GIV’s GEF motif is required for insulin to trigger a shift in Gαi3 binding from AGS3 to GIV. (A) Gαi3 coimmunoprecipitates with AGS3 in serum-starved cells and with GIV when cells are insulin stimulated. Cos7 cells transiently transfected with FLAG-tagged Gαi3 (Gαi3-FLAG) or vector control were serum starved (-) and stimulated with 100 nM insulin (+) for 15 min before lysis. Equal aliquots of cell lysates (left panel) were incubated with anti-FLAG mAb. Immunoprecipitated complexes (right panel) were analyzed for GIV, AGS3, and FLAG (Gαi3) by immunoblotting (IB). In serum-starved cells (−), Gαi3-bound immune complexes are enriched in AGS3, whereas after insulin treatment (+) these immune complexes are depleted of AGS3 and enriched in GIV. Identical observations were made after EGF treatment (Supplemental Figure S4). (B) Changes in the abundance of Gαi–AGS3 complexes in response to insulin require an intact GEF motif in GIV. Immunoprecipitation was carried out on lysates of serum-starved and insulin-stimulated control, GIV-WT, and GIV-FA cells with anti-Gαi3 IgG, and the immune complexes were analyzed for Gαi3, AGS3, and GIV by IB. In GIV-WT cells, the amount of Gαi3-bound GIV increased and the amount of Gαi3-bound AGS3 decreased upon insulin treatment. In GIV-FA cells (GEF-deficient mutant), GIV does not coimmunoprecipitate with Gαi3, and the extent of Gαi3-bound AGS3 remains unaltered after insulin treatment.
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Figure 4: GIV’s GEF motif is required for insulin to trigger a shift in Gαi3 binding from AGS3 to GIV. (A) Gαi3 coimmunoprecipitates with AGS3 in serum-starved cells and with GIV when cells are insulin stimulated. Cos7 cells transiently transfected with FLAG-tagged Gαi3 (Gαi3-FLAG) or vector control were serum starved (-) and stimulated with 100 nM insulin (+) for 15 min before lysis. Equal aliquots of cell lysates (left panel) were incubated with anti-FLAG mAb. Immunoprecipitated complexes (right panel) were analyzed for GIV, AGS3, and FLAG (Gαi3) by immunoblotting (IB). In serum-starved cells (−), Gαi3-bound immune complexes are enriched in AGS3, whereas after insulin treatment (+) these immune complexes are depleted of AGS3 and enriched in GIV. Identical observations were made after EGF treatment (Supplemental Figure S4). (B) Changes in the abundance of Gαi–AGS3 complexes in response to insulin require an intact GEF motif in GIV. Immunoprecipitation was carried out on lysates of serum-starved and insulin-stimulated control, GIV-WT, and GIV-FA cells with anti-Gαi3 IgG, and the immune complexes were analyzed for Gαi3, AGS3, and GIV by IB. In GIV-WT cells, the amount of Gαi3-bound GIV increased and the amount of Gαi3-bound AGS3 decreased upon insulin treatment. In GIV-FA cells (GEF-deficient mutant), GIV does not coimmunoprecipitate with Gαi3, and the extent of Gαi3-bound AGS3 remains unaltered after insulin treatment.
Mentions: Next we investigated whether growth factors influence the competitive binding of GIV and AGS3 to Gαi3 in vivo. When we immunoprecipitated Gαi3 from Cos7 cells expressing Gαi3-FLAG and immunoblotted for AGS3 and GIV, we found that AGS3 interacted with Gαi3 preferentially upon starvation, and this interaction was reduced by ∼75–80% upon insulin stimulation (Figure 4A). In contrast, GIV displayed an approximately twofold increase in binding to Gαi3 compared with the starved state. Identical results were obtained when the cells were stimulated with EGF (Supplemental Figure S4), indicating that the pattern is common to several growth factors. These results demonstrate that starvation favors formation of AGS3–Gαi3 complexes, whereas growth factors favor formation of GIV–Gαi3 complexes and dissociation of AGS3–Gαi3 complexes. We conclude that AGS3–Gαi3 and GIV–Gαi3 complexes exist in equilibrium in living cells and that growth factor stimulation shifts the equilibrium toward activation of Gαi3 by GIV. We anticipated that this shift might require the presence of a functional GEF motif in GIV and found that this is indeed the case. Comparison of the endogenous Gαi3-bound complexes in starved and insulin-stimulated GIV-WT and GIV-FA cells revealed that the insulin-dependent shift from AGS3–Gαi3 to GIV–Gαi3 complexes occurred exclusively in GIV-WT cells (Figure 4B). The ability of insulin to increase GIV–Gαi3 complexes and reduce AGS3–Gαi3 complexes in GIV-WT but not in GIV-FA cells correlates with the responsiveness of these cells to insulin (Figure 1, A and B), suggesting that GIV’s ability to antagonize AGS3 (Figures 2 and 3) is required for the anti-autophagic action of insulin. We conclude that GIV’s GEF function facilitates the anti-autophagic action of insulin in part by shifting the equilibrium from AGS3–Gαi3 toward GIV–Gαi3 and triggering activation of Gαi3.

Bottom Line: Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown.Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a.Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 , USA. mgarciamarcos@ucsd.edu

ABSTRACT
Autophagy is the major catabolic process responsible for the removal of aggregated proteins and damaged organelles. Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown. Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a. Girdin), a nonreceptor guanine nucleotide exchange factor for Gα(i3), plays a key role in regulating autophagy and that dynamic interplay between Gα(i3), activator of G-protein signaling 3 (AGS3, its guanine nucleotide dissociation inhibitor), and GIV determines whether autophagy is promoted or inhibited. We found that AGS3 directly binds light chain 3 (LC3), recruits Gα(i3) to LC3-positive membranes upon starvation, and promotes autophagy by inhibiting the G protein. Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein. These results provide mechanistic insights into how reversible modulation of Gα(i3) activity by AGS3 and GIV maintains the delicate equilibrium between promotion and inhibition of autophagy.

Show MeSH
Related in: MedlinePlus