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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 is associated with insulin receptor internalization. (A) 3D reconstruction of subcellular Cav1 staining in murine islets cells of a pancreatic section. (B) STED super-resolution imaging of mTurquoise-tagged Cav1 and endogenous insulin in MIN6 cells. Inset shows Cav1 surrounding a cluster of insulin receptors. Scale bar = 5 μm. (C) Immunolabeling and TIRF microscopy of dispersed human islet cells cultured in 5 mM glucose demonstrates colocalization of endogenous Cav1 to endogenous insulin receptors at the plasma membrane (n = 20 cells). Scale bar = 5 μm. (D) TIRF imaging reveals a high degree of colocalization of Cav1-mTFP to InsRA-TagRFP at the plasma membrane of live MIN6 cells cultured in 20 mM glucose (n = 10). Scale bar = 5 μm. (E, F) Live-cell TIRF imaging in MIN6 cells cultured in 20 mM glucose reveals the reciprocal recruitment of InsRA-TagRFP and Cav1-mTFP to membrane domains prior to the internalization of insulin receptors. Pixel size = 0.129 μm (F, F′) Intensity analysis of a single (F) InsRA-TagRFP positive membrane domain during the process of Cav1 mediated vesicle budding. Green dots correspond to the time points of the images in E. (F′) shows the averaged and normalized intensities of 15 analyzed InsRA-TagRFP positive membrane domains during Cav1 mediated vesicle budding from the plasma membrane of MIN6 cells cultured in 20 mM glucose. *p < 0.05. (G) Co-immunoprecipitation of insulin receptors from NIH-3T3 cells reveals an insulin dependent binding of Cav1 to the insulin receptor (n = 3). NIH-3T3 cells were cultured in 0 mM glucose during insulin treatments for 5 min *p < 0.05
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fig3: Caveolin-1 is associated with insulin receptor internalization. (A) 3D reconstruction of subcellular Cav1 staining in murine islets cells of a pancreatic section. (B) STED super-resolution imaging of mTurquoise-tagged Cav1 and endogenous insulin in MIN6 cells. Inset shows Cav1 surrounding a cluster of insulin receptors. Scale bar = 5 μm. (C) Immunolabeling and TIRF microscopy of dispersed human islet cells cultured in 5 mM glucose demonstrates colocalization of endogenous Cav1 to endogenous insulin receptors at the plasma membrane (n = 20 cells). Scale bar = 5 μm. (D) TIRF imaging reveals a high degree of colocalization of Cav1-mTFP to InsRA-TagRFP at the plasma membrane of live MIN6 cells cultured in 20 mM glucose (n = 10). Scale bar = 5 μm. (E, F) Live-cell TIRF imaging in MIN6 cells cultured in 20 mM glucose reveals the reciprocal recruitment of InsRA-TagRFP and Cav1-mTFP to membrane domains prior to the internalization of insulin receptors. Pixel size = 0.129 μm (F, F′) Intensity analysis of a single (F) InsRA-TagRFP positive membrane domain during the process of Cav1 mediated vesicle budding. Green dots correspond to the time points of the images in E. (F′) shows the averaged and normalized intensities of 15 analyzed InsRA-TagRFP positive membrane domains during Cav1 mediated vesicle budding from the plasma membrane of MIN6 cells cultured in 20 mM glucose. *p < 0.05. (G) Co-immunoprecipitation of insulin receptors from NIH-3T3 cells reveals an insulin dependent binding of Cav1 to the insulin receptor (n = 3). NIH-3T3 cells were cultured in 0 mM glucose during insulin treatments for 5 min *p < 0.05

Mentions: Given the observation that insulin receptors did not significantly colocalize with proteins or cargo that mark ‘classical endosomes’ in beta-cells, we examined alternative internalization and trafficking routes. We investigated caveolin-1 (Cav1) because insulin receptors colocalize with Cav1 in adipocytes, and Cav1 knockout mice exhibit impaired insulin signaling in other tissues [15], [16], [17]. Cav1 is prominently expressed in primary beta-cells, in vivo and in vitro (Figure S3A, B), and, to a lesser extent, in beta-cell lines (Figure S3C) [40], [41]. Confocal imaging illustrated a primarily cytoplasmic distribution of Cav1 in vivo within mouse pancreas sections, and in vitro in isolated mouse beta-cells (Figures. 3A, S3A, B), consistent with previous results [40], [41]. Endogenous Cav1 exhibited robust colocalization with endogenous insulin receptors in isolated mouse beta-cells (Figure S3B). STED super-resolution imaging revealed that Cav1 could be found as structures appearing to surround insulin receptor containing complexes at the plasma membrane (Figure 3B). TIRF imaging revealed colocalization of endogenous insulin receptors and Cav1 in the <200 nm near plasma membrane space of isolated human beta-cells (Figure 3C).


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 is associated with insulin receptor internalization. (A) 3D reconstruction of subcellular Cav1 staining in murine islets cells of a pancreatic section. (B) STED super-resolution imaging of mTurquoise-tagged Cav1 and endogenous insulin in MIN6 cells. Inset shows Cav1 surrounding a cluster of insulin receptors. Scale bar = 5 μm. (C) Immunolabeling and TIRF microscopy of dispersed human islet cells cultured in 5 mM glucose demonstrates colocalization of endogenous Cav1 to endogenous insulin receptors at the plasma membrane (n = 20 cells). Scale bar = 5 μm. (D) TIRF imaging reveals a high degree of colocalization of Cav1-mTFP to InsRA-TagRFP at the plasma membrane of live MIN6 cells cultured in 20 mM glucose (n = 10). Scale bar = 5 μm. (E, F) Live-cell TIRF imaging in MIN6 cells cultured in 20 mM glucose reveals the reciprocal recruitment of InsRA-TagRFP and Cav1-mTFP to membrane domains prior to the internalization of insulin receptors. Pixel size = 0.129 μm (F, F′) Intensity analysis of a single (F) InsRA-TagRFP positive membrane domain during the process of Cav1 mediated vesicle budding. Green dots correspond to the time points of the images in E. (F′) shows the averaged and normalized intensities of 15 analyzed InsRA-TagRFP positive membrane domains during Cav1 mediated vesicle budding from the plasma membrane of MIN6 cells cultured in 20 mM glucose. *p < 0.05. (G) Co-immunoprecipitation of insulin receptors from NIH-3T3 cells reveals an insulin dependent binding of Cav1 to the insulin receptor (n = 3). NIH-3T3 cells were cultured in 0 mM glucose during insulin treatments for 5 min *p < 0.05
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fig3: Caveolin-1 is associated with insulin receptor internalization. (A) 3D reconstruction of subcellular Cav1 staining in murine islets cells of a pancreatic section. (B) STED super-resolution imaging of mTurquoise-tagged Cav1 and endogenous insulin in MIN6 cells. Inset shows Cav1 surrounding a cluster of insulin receptors. Scale bar = 5 μm. (C) Immunolabeling and TIRF microscopy of dispersed human islet cells cultured in 5 mM glucose demonstrates colocalization of endogenous Cav1 to endogenous insulin receptors at the plasma membrane (n = 20 cells). Scale bar = 5 μm. (D) TIRF imaging reveals a high degree of colocalization of Cav1-mTFP to InsRA-TagRFP at the plasma membrane of live MIN6 cells cultured in 20 mM glucose (n = 10). Scale bar = 5 μm. (E, F) Live-cell TIRF imaging in MIN6 cells cultured in 20 mM glucose reveals the reciprocal recruitment of InsRA-TagRFP and Cav1-mTFP to membrane domains prior to the internalization of insulin receptors. Pixel size = 0.129 μm (F, F′) Intensity analysis of a single (F) InsRA-TagRFP positive membrane domain during the process of Cav1 mediated vesicle budding. Green dots correspond to the time points of the images in E. (F′) shows the averaged and normalized intensities of 15 analyzed InsRA-TagRFP positive membrane domains during Cav1 mediated vesicle budding from the plasma membrane of MIN6 cells cultured in 20 mM glucose. *p < 0.05. (G) Co-immunoprecipitation of insulin receptors from NIH-3T3 cells reveals an insulin dependent binding of Cav1 to the insulin receptor (n = 3). NIH-3T3 cells were cultured in 0 mM glucose during insulin treatments for 5 min *p < 0.05
Mentions: Given the observation that insulin receptors did not significantly colocalize with proteins or cargo that mark ‘classical endosomes’ in beta-cells, we examined alternative internalization and trafficking routes. We investigated caveolin-1 (Cav1) because insulin receptors colocalize with Cav1 in adipocytes, and Cav1 knockout mice exhibit impaired insulin signaling in other tissues [15], [16], [17]. Cav1 is prominently expressed in primary beta-cells, in vivo and in vitro (Figure S3A, B), and, to a lesser extent, in beta-cell lines (Figure S3C) [40], [41]. Confocal imaging illustrated a primarily cytoplasmic distribution of Cav1 in vivo within mouse pancreas sections, and in vitro in isolated mouse beta-cells (Figures. 3A, S3A, B), consistent with previous results [40], [41]. Endogenous Cav1 exhibited robust colocalization with endogenous insulin receptors in isolated mouse beta-cells (Figure S3B). STED super-resolution imaging revealed that Cav1 could be found as structures appearing to surround insulin receptor containing complexes at the plasma membrane (Figure 3B). TIRF imaging revealed colocalization of endogenous insulin receptors and Cav1 in the <200 nm near plasma membrane space of isolated human beta-cells (Figure 3C).

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