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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

Design and validation of functional fluorescent protein-tagged insulin receptors that mimic endogenous insulin receptor localization. (A) Islets in pancreatic tissue sections from 24 week-old mice are labeled with mouse monoclonal antibody to the insulin receptor and the DRAQ5 DNA stain (blue). Scale bar = 50 μm. (A′) 3D reconstruction of InsR staining in a pancreatic mouse tissue section. (B) Confocal imaging of endogenous insulin receptor localization in dispersed, fixed primary human beta-cells. Insets show AMCA-insulin immunofluorescence. Scale bar = 10 μm. (C) STED super resolution microscopy identifies small ∼80 nm clusters of plasma membrane insulin receptors in fixed MIN6 cells (arrow). Scale bar = 5 μm (D) Schematic of inter-domain tagging strategy used in the present study. Orange structure = InsR; Red structure = TagRFP. (E) Representative confocal image of colocalization between internalized FITC-insulin (200 nM, 1 h) and interdomain-tagged InsRA-TagRFP in live MIN6 cells cultured at 0 mM glucose. Scale bar = 10 μm. (F, G) Expression of InsRA-TagRFP and InsRB-TagRFP sustains Erk phosphorylation in HEK 293T cells stimulated with 50 nM insulin (n = 4) at 0 mM Glucose. (H) Colocalization of identical insulin receptor isoforms tagged with different fluorescent proteins in fixed MIN6 cells (n = 10). Scale bar = 10 μm. Inset Venn diagrams, here and throughout, can be used visualize the colocalization of the color-coded proteins and represent the relative size of the puncta pools and their overlap. (I) Colocalization of fluorescently labeled insulin receptor A and B isoforms in fixed MIN6 cells (n = 10). Identical results were obtained with InsRA-TagBFP and InsRB-TagRFP (not shown). Scale bar = 10 μm. (J, K) C-terminal-tagged insulin receptors have a primarily plasma membrane localization that does not substantially colocalize with interdomain-tagged insulin receptors in fixed MIN6 cells (n = 10). Scale bar = 10 μm
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fig1: Design and validation of functional fluorescent protein-tagged insulin receptors that mimic endogenous insulin receptor localization. (A) Islets in pancreatic tissue sections from 24 week-old mice are labeled with mouse monoclonal antibody to the insulin receptor and the DRAQ5 DNA stain (blue). Scale bar = 50 μm. (A′) 3D reconstruction of InsR staining in a pancreatic mouse tissue section. (B) Confocal imaging of endogenous insulin receptor localization in dispersed, fixed primary human beta-cells. Insets show AMCA-insulin immunofluorescence. Scale bar = 10 μm. (C) STED super resolution microscopy identifies small ∼80 nm clusters of plasma membrane insulin receptors in fixed MIN6 cells (arrow). Scale bar = 5 μm (D) Schematic of inter-domain tagging strategy used in the present study. Orange structure = InsR; Red structure = TagRFP. (E) Representative confocal image of colocalization between internalized FITC-insulin (200 nM, 1 h) and interdomain-tagged InsRA-TagRFP in live MIN6 cells cultured at 0 mM glucose. Scale bar = 10 μm. (F, G) Expression of InsRA-TagRFP and InsRB-TagRFP sustains Erk phosphorylation in HEK 293T cells stimulated with 50 nM insulin (n = 4) at 0 mM Glucose. (H) Colocalization of identical insulin receptor isoforms tagged with different fluorescent proteins in fixed MIN6 cells (n = 10). Scale bar = 10 μm. Inset Venn diagrams, here and throughout, can be used visualize the colocalization of the color-coded proteins and represent the relative size of the puncta pools and their overlap. (I) Colocalization of fluorescently labeled insulin receptor A and B isoforms in fixed MIN6 cells (n = 10). Identical results were obtained with InsRA-TagBFP and InsRB-TagRFP (not shown). Scale bar = 10 μm. (J, K) C-terminal-tagged insulin receptors have a primarily plasma membrane localization that does not substantially colocalize with interdomain-tagged insulin receptors in fixed MIN6 cells (n = 10). Scale bar = 10 μm

Mentions: In most cell types, insulin receptors have been found in multiple cellular compartments, including the plasma membrane, endosomes, lysosomes, and perhaps secretory granules and the nucleus [8], [32], [33], [34]. In vivo, we observed that the majority of insulin receptors were within the beta-cell cytoplasm in mouse pancreatic sections (Figure 1A, A′). In vitro, confocal imaging of dispersed cells demonstrated that insulin receptors were located on the membranes of cytoplasmic vesicles in isolated human and mouse beta-cells (Figure 1B, Figure S1A), pointing to a possible role for receptor internalization in this cell type [35]. The size of the insulin receptor-containing structures was near the limit of conventional light microscopy. Using stimulated emission depletion (STED) super-resolution microscopy [36], we found that insulin receptors in the membrane were clustered in domains that were ∼60–80 nm and confirmed the intracellular location of a large fraction of the endogenous insulin receptors (Figure 1C). These imaging data were confirmed by sub-cellular fractionation of primary human islets, which showed that insulin receptors were in fractions enriched for markers of intracellular membranes (Figure S1B). Thus, in vivo and in vitro data indicate that endogenous insulin receptors exist primarily in an internalized state in beta-cells.


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)

Design and validation of functional fluorescent protein-tagged insulin receptors that mimic endogenous insulin receptor localization. (A) Islets in pancreatic tissue sections from 24 week-old mice are labeled with mouse monoclonal antibody to the insulin receptor and the DRAQ5 DNA stain (blue). Scale bar = 50 μm. (A′) 3D reconstruction of InsR staining in a pancreatic mouse tissue section. (B) Confocal imaging of endogenous insulin receptor localization in dispersed, fixed primary human beta-cells. Insets show AMCA-insulin immunofluorescence. Scale bar = 10 μm. (C) STED super resolution microscopy identifies small ∼80 nm clusters of plasma membrane insulin receptors in fixed MIN6 cells (arrow). Scale bar = 5 μm (D) Schematic of inter-domain tagging strategy used in the present study. Orange structure = InsR; Red structure = TagRFP. (E) Representative confocal image of colocalization between internalized FITC-insulin (200 nM, 1 h) and interdomain-tagged InsRA-TagRFP in live MIN6 cells cultured at 0 mM glucose. Scale bar = 10 μm. (F, G) Expression of InsRA-TagRFP and InsRB-TagRFP sustains Erk phosphorylation in HEK 293T cells stimulated with 50 nM insulin (n = 4) at 0 mM Glucose. (H) Colocalization of identical insulin receptor isoforms tagged with different fluorescent proteins in fixed MIN6 cells (n = 10). Scale bar = 10 μm. Inset Venn diagrams, here and throughout, can be used visualize the colocalization of the color-coded proteins and represent the relative size of the puncta pools and their overlap. (I) Colocalization of fluorescently labeled insulin receptor A and B isoforms in fixed MIN6 cells (n = 10). Identical results were obtained with InsRA-TagBFP and InsRB-TagRFP (not shown). Scale bar = 10 μm. (J, K) C-terminal-tagged insulin receptors have a primarily plasma membrane localization that does not substantially colocalize with interdomain-tagged insulin receptors in fixed MIN6 cells (n = 10). Scale bar = 10 μm
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fig1: Design and validation of functional fluorescent protein-tagged insulin receptors that mimic endogenous insulin receptor localization. (A) Islets in pancreatic tissue sections from 24 week-old mice are labeled with mouse monoclonal antibody to the insulin receptor and the DRAQ5 DNA stain (blue). Scale bar = 50 μm. (A′) 3D reconstruction of InsR staining in a pancreatic mouse tissue section. (B) Confocal imaging of endogenous insulin receptor localization in dispersed, fixed primary human beta-cells. Insets show AMCA-insulin immunofluorescence. Scale bar = 10 μm. (C) STED super resolution microscopy identifies small ∼80 nm clusters of plasma membrane insulin receptors in fixed MIN6 cells (arrow). Scale bar = 5 μm (D) Schematic of inter-domain tagging strategy used in the present study. Orange structure = InsR; Red structure = TagRFP. (E) Representative confocal image of colocalization between internalized FITC-insulin (200 nM, 1 h) and interdomain-tagged InsRA-TagRFP in live MIN6 cells cultured at 0 mM glucose. Scale bar = 10 μm. (F, G) Expression of InsRA-TagRFP and InsRB-TagRFP sustains Erk phosphorylation in HEK 293T cells stimulated with 50 nM insulin (n = 4) at 0 mM Glucose. (H) Colocalization of identical insulin receptor isoforms tagged with different fluorescent proteins in fixed MIN6 cells (n = 10). Scale bar = 10 μm. Inset Venn diagrams, here and throughout, can be used visualize the colocalization of the color-coded proteins and represent the relative size of the puncta pools and their overlap. (I) Colocalization of fluorescently labeled insulin receptor A and B isoforms in fixed MIN6 cells (n = 10). Identical results were obtained with InsRA-TagBFP and InsRB-TagRFP (not shown). Scale bar = 10 μm. (J, K) C-terminal-tagged insulin receptors have a primarily plasma membrane localization that does not substantially colocalize with interdomain-tagged insulin receptors in fixed MIN6 cells (n = 10). Scale bar = 10 μm
Mentions: In most cell types, insulin receptors have been found in multiple cellular compartments, including the plasma membrane, endosomes, lysosomes, and perhaps secretory granules and the nucleus [8], [32], [33], [34]. In vivo, we observed that the majority of insulin receptors were within the beta-cell cytoplasm in mouse pancreatic sections (Figure 1A, A′). In vitro, confocal imaging of dispersed cells demonstrated that insulin receptors were located on the membranes of cytoplasmic vesicles in isolated human and mouse beta-cells (Figure 1B, Figure S1A), pointing to a possible role for receptor internalization in this cell type [35]. The size of the insulin receptor-containing structures was near the limit of conventional light microscopy. Using stimulated emission depletion (STED) super-resolution microscopy [36], we found that insulin receptors in the membrane were clustered in domains that were ∼60–80 nm and confirmed the intracellular location of a large fraction of the endogenous insulin receptors (Figure 1C). These imaging data were confirmed by sub-cellular fractionation of primary human islets, which showed that insulin receptors were in fractions enriched for markers of intracellular membranes (Figure S1B). Thus, in vivo and in vitro data indicate that endogenous insulin receptors exist primarily in an internalized state in beta-cells.

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