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Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells.

Boothe T, Lim GE, Cen H, Skovsø S, Piske M, Li SN, Nabi IR, Gilon P, Johnson JD - Mol Metab (2016)

Bottom Line: Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes.Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact.We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT

Objective: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells.

Methods: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies.

Results: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact.

Conclusions: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

No MeSH data available.


Related in: MedlinePlus

Caveolin-1 enhances insulin-stimulated Erk, but not Akt, signaling in vitro. (A, B) Overexpression of wildtype Cav1-mRFP or the phosphomimetic mutant Cav1-Y14D significantly increases basal insulin signaling to Erk. Erk signaling stimulated by 2 nM added insulin is significantly reduced in cells overexpressing the dominant negative mutant Cav1-Y14F. Akt signaling is unaffected by the overexpression of Cav-1 mutants. Experiments were performed in MIN6 cells treated for 5 min with the indicated doses of insulin (n = 10). (C) Insulin stimulated (5 min) Erk, but not Akt signaling is significantly reduced in NIH-3T3 cells previously treated for 2 h with 10 μM of Src kinase inhibitor PP2 (n = 4). *p < 0.05. (D) Erk, but not Akt signaling is significantly reduced in MIN6 cells treated with Cav1-siRNA and a physiological dose of insulin for 5 min (n = 4). *p < 0.05. (A–D) MIN6 cells and NIH-3T3 cells were incubated in 0 mM glucose during 5 min of insulin treatments. (E) Knockdown efficiency in MIN6 cells cultured at 25 mM glucose and treated with a siRNA pool against murine Cav1 (n = 6 independent samples per condition). *p < 0.05. (F) Reduced insulin receptor immunolabeling of intracellular vesicles in islets of Cav1−/− mice. *p < 0.05. (n = 9 islets). Cav1+/+ = white, Cav1−/− = black, throughout this figure. Scale bar = 25 μm. (G) Reduced Erk signaling visualized by nuclear translocation in islet cells from 12 week old Cav1−/− mice (n = 3 mice, 22 analyzed islets). Scale bar = 50 μm.
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fig6: Caveolin-1 enhances insulin-stimulated Erk, but not Akt, signaling in vitro. (A, B) Overexpression of wildtype Cav1-mRFP or the phosphomimetic mutant Cav1-Y14D significantly increases basal insulin signaling to Erk. Erk signaling stimulated by 2 nM added insulin is significantly reduced in cells overexpressing the dominant negative mutant Cav1-Y14F. Akt signaling is unaffected by the overexpression of Cav-1 mutants. Experiments were performed in MIN6 cells treated for 5 min with the indicated doses of insulin (n = 10). (C) Insulin stimulated (5 min) Erk, but not Akt signaling is significantly reduced in NIH-3T3 cells previously treated for 2 h with 10 μM of Src kinase inhibitor PP2 (n = 4). *p < 0.05. (D) Erk, but not Akt signaling is significantly reduced in MIN6 cells treated with Cav1-siRNA and a physiological dose of insulin for 5 min (n = 4). *p < 0.05. (A–D) MIN6 cells and NIH-3T3 cells were incubated in 0 mM glucose during 5 min of insulin treatments. (E) Knockdown efficiency in MIN6 cells cultured at 25 mM glucose and treated with a siRNA pool against murine Cav1 (n = 6 independent samples per condition). *p < 0.05. (F) Reduced insulin receptor immunolabeling of intracellular vesicles in islets of Cav1−/− mice. *p < 0.05. (n = 9 islets). Cav1+/+ = white, Cav1−/− = black, throughout this figure. Scale bar = 25 μm. (G) Reduced Erk signaling visualized by nuclear translocation in islet cells from 12 week old Cav1−/− mice (n = 3 mice, 22 analyzed islets). Scale bar = 50 μm.

Mentions: Next, we sought to establish the functional consequences of altered Cav1 phosphorylation in insulin signaling in beta-cells, which even under basal conditions have insulin present in their cultures [50]. Overexpressing wildtype Cav1 or the constitutively active Cav1-Y14D mutant was sufficient to significantly increase Erk phosphorylation in beta-cells in the absence of added insulin (Figure 6A). In the presence of 2 nM added insulin, the dominant negative Cav1-Y14F mutant significantly suppressed Erk activation (Figure 6A). On the other hand, neither basal nor exogenous insulin-stimulated Akt phosphorylation was affected by the expression of wildtype or mutant Cav1 (Figure 6B). Similar results were obtained in experiments where endogenous Cav1 phosphorylation was inhibited in NIH-3T3 and MIN6 cells by the Src kinase inhibitor PP2 (Figs 6C, S4). While Src has multiple cellular targets, collectively these data suggest a role for Cav1 phosphorylation in insulin receptor internalization and Erk signaling.


Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells.

Boothe T, Lim GE, Cen H, Skovsø S, Piske M, Li SN, Nabi IR, Gilon P, Johnson JD - Mol Metab (2016)

Caveolin-1 enhances insulin-stimulated Erk, but not Akt, signaling in vitro. (A, B) Overexpression of wildtype Cav1-mRFP or the phosphomimetic mutant Cav1-Y14D significantly increases basal insulin signaling to Erk. Erk signaling stimulated by 2 nM added insulin is significantly reduced in cells overexpressing the dominant negative mutant Cav1-Y14F. Akt signaling is unaffected by the overexpression of Cav-1 mutants. Experiments were performed in MIN6 cells treated for 5 min with the indicated doses of insulin (n = 10). (C) Insulin stimulated (5 min) Erk, but not Akt signaling is significantly reduced in NIH-3T3 cells previously treated for 2 h with 10 μM of Src kinase inhibitor PP2 (n = 4). *p < 0.05. (D) Erk, but not Akt signaling is significantly reduced in MIN6 cells treated with Cav1-siRNA and a physiological dose of insulin for 5 min (n = 4). *p < 0.05. (A–D) MIN6 cells and NIH-3T3 cells were incubated in 0 mM glucose during 5 min of insulin treatments. (E) Knockdown efficiency in MIN6 cells cultured at 25 mM glucose and treated with a siRNA pool against murine Cav1 (n = 6 independent samples per condition). *p < 0.05. (F) Reduced insulin receptor immunolabeling of intracellular vesicles in islets of Cav1−/− mice. *p < 0.05. (n = 9 islets). Cav1+/+ = white, Cav1−/− = black, throughout this figure. Scale bar = 25 μm. (G) Reduced Erk signaling visualized by nuclear translocation in islet cells from 12 week old Cav1−/− mice (n = 3 mice, 22 analyzed islets). Scale bar = 50 μm.
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fig6: Caveolin-1 enhances insulin-stimulated Erk, but not Akt, signaling in vitro. (A, B) Overexpression of wildtype Cav1-mRFP or the phosphomimetic mutant Cav1-Y14D significantly increases basal insulin signaling to Erk. Erk signaling stimulated by 2 nM added insulin is significantly reduced in cells overexpressing the dominant negative mutant Cav1-Y14F. Akt signaling is unaffected by the overexpression of Cav-1 mutants. Experiments were performed in MIN6 cells treated for 5 min with the indicated doses of insulin (n = 10). (C) Insulin stimulated (5 min) Erk, but not Akt signaling is significantly reduced in NIH-3T3 cells previously treated for 2 h with 10 μM of Src kinase inhibitor PP2 (n = 4). *p < 0.05. (D) Erk, but not Akt signaling is significantly reduced in MIN6 cells treated with Cav1-siRNA and a physiological dose of insulin for 5 min (n = 4). *p < 0.05. (A–D) MIN6 cells and NIH-3T3 cells were incubated in 0 mM glucose during 5 min of insulin treatments. (E) Knockdown efficiency in MIN6 cells cultured at 25 mM glucose and treated with a siRNA pool against murine Cav1 (n = 6 independent samples per condition). *p < 0.05. (F) Reduced insulin receptor immunolabeling of intracellular vesicles in islets of Cav1−/− mice. *p < 0.05. (n = 9 islets). Cav1+/+ = white, Cav1−/− = black, throughout this figure. Scale bar = 25 μm. (G) Reduced Erk signaling visualized by nuclear translocation in islet cells from 12 week old Cav1−/− mice (n = 3 mice, 22 analyzed islets). Scale bar = 50 μm.
Mentions: Next, we sought to establish the functional consequences of altered Cav1 phosphorylation in insulin signaling in beta-cells, which even under basal conditions have insulin present in their cultures [50]. Overexpressing wildtype Cav1 or the constitutively active Cav1-Y14D mutant was sufficient to significantly increase Erk phosphorylation in beta-cells in the absence of added insulin (Figure 6A). In the presence of 2 nM added insulin, the dominant negative Cav1-Y14F mutant significantly suppressed Erk activation (Figure 6A). On the other hand, neither basal nor exogenous insulin-stimulated Akt phosphorylation was affected by the expression of wildtype or mutant Cav1 (Figure 6B). Similar results were obtained in experiments where endogenous Cav1 phosphorylation was inhibited in NIH-3T3 and MIN6 cells by the Src kinase inhibitor PP2 (Figs 6C, S4). While Src has multiple cellular targets, collectively these data suggest a role for Cav1 phosphorylation in insulin receptor internalization and Erk signaling.

Bottom Line: Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes.Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact.We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.

ABSTRACT

Objective: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells.

Methods: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies.

Results: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact.

Conclusions: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.

No MeSH data available.


Related in: MedlinePlus