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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

Insulin receptors do not associate with markers of clathrin-dependent endocytosis. (A, A′) Pulse-chase colocalization analysis of Alexafluor488-labeled EGF relative to InsRA-TagRFP indicates separate entry pathways but eventual fusion into a shared mature pool of vesicles in MIN6 cells cultured in 0 mM glucose. A significant increase in the degree of colocalization was observed at a 45 min chase time compared to 0 min chase time (n = 4 images per time point). Scale bar = 5 μm. (B–D) Colocalization analysis of InsRA-TagRFP with TagBFP-tagged Rab5a (early endosomes), TagBFP-Rab7 (late endosomes), and TagBFP-Rab11a (recycling endosomes) in fixed MIN6 cells (n = 10). Similar results observed with InsRB-TagRFP. Scale bar = 5 μm. (E) Colocalization analysis of immunolabeled endogenous insulin receptors with immunolabeled endogenous clathrin in MIN6 cells (n = 10). Scale bar = 10 μm. (F–H) Colocalization analysis of tagged insulin receptors with immunolabeled endogenous Rab5a, Rab7, and Rab4a (alternate marker for recycling endosomes) in MIN6 cells (n = 10). Scale bar = 10 μm. (B–H) Prior fixation, MIN6 cells were cultured in 25 mM glucose.
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fig2: Insulin receptors do not associate with markers of clathrin-dependent endocytosis. (A, A′) Pulse-chase colocalization analysis of Alexafluor488-labeled EGF relative to InsRA-TagRFP indicates separate entry pathways but eventual fusion into a shared mature pool of vesicles in MIN6 cells cultured in 0 mM glucose. A significant increase in the degree of colocalization was observed at a 45 min chase time compared to 0 min chase time (n = 4 images per time point). Scale bar = 5 μm. (B–D) Colocalization analysis of InsRA-TagRFP with TagBFP-tagged Rab5a (early endosomes), TagBFP-Rab7 (late endosomes), and TagBFP-Rab11a (recycling endosomes) in fixed MIN6 cells (n = 10). Similar results observed with InsRB-TagRFP. Scale bar = 5 μm. (E) Colocalization analysis of immunolabeled endogenous insulin receptors with immunolabeled endogenous clathrin in MIN6 cells (n = 10). Scale bar = 10 μm. (F–H) Colocalization analysis of tagged insulin receptors with immunolabeled endogenous Rab5a, Rab7, and Rab4a (alternate marker for recycling endosomes) in MIN6 cells (n = 10). Scale bar = 10 μm. (B–H) Prior fixation, MIN6 cells were cultured in 25 mM glucose.

Mentions: We examined the trafficking route of these functional insulin receptors by comparing their localization to endosomal pathway markers. First, we examined whether insulin receptors colocalized with epidermal growth factor (EGF), an established endosomal cargo of the clathrin pathway, in a pulse chase experiment. Fluorescent Alexa-488 labeled EGF showed very limited colocalization (<5%) to InsRA-TagRFP at short-chase times, indicating that the proximal routes of endocytosis were likely distinct (Figure 2A). The significantly increased colocalization at longer chase times indicated that the vesicles of the two different internalization routes eventually merged into more mature compartments such as lysosomes (Figure 2A′,A″). Next, we assessed the colocalization of InsRA-TagRFP with Rab5a, a marker associated with clathrin-dependent endocytosis [39], as well as Rab7-labeled late endosomes and Rab11a-labeled recycling endosomes and found virtually no colocalization (Figure 2B–D). Similarly, endogenous insulin receptors showed minimal colocalization with endogenous clathrin in primary mouse beta-cells (Figure 2E), and tagged insulin receptors did not appreciably colocalize with endogenous Rab5a, Rab7, or Rab4A (Figure 2F–H). Clathrin loss-of-function experiments resulted in gross changes in beta-cell plasma membrane morphology (not shown), so we were unable to rule out some small role for clathrin in insulin receptor trafficking. Notwithstanding, these data demonstrate that insulin receptors spend little, if any, time in endocytic vesicles labeled with these Rab-GTPases, or clathrin.


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)

Insulin receptors do not associate with markers of clathrin-dependent endocytosis. (A, A′) Pulse-chase colocalization analysis of Alexafluor488-labeled EGF relative to InsRA-TagRFP indicates separate entry pathways but eventual fusion into a shared mature pool of vesicles in MIN6 cells cultured in 0 mM glucose. A significant increase in the degree of colocalization was observed at a 45 min chase time compared to 0 min chase time (n = 4 images per time point). Scale bar = 5 μm. (B–D) Colocalization analysis of InsRA-TagRFP with TagBFP-tagged Rab5a (early endosomes), TagBFP-Rab7 (late endosomes), and TagBFP-Rab11a (recycling endosomes) in fixed MIN6 cells (n = 10). Similar results observed with InsRB-TagRFP. Scale bar = 5 μm. (E) Colocalization analysis of immunolabeled endogenous insulin receptors with immunolabeled endogenous clathrin in MIN6 cells (n = 10). Scale bar = 10 μm. (F–H) Colocalization analysis of tagged insulin receptors with immunolabeled endogenous Rab5a, Rab7, and Rab4a (alternate marker for recycling endosomes) in MIN6 cells (n = 10). Scale bar = 10 μm. (B–H) Prior fixation, MIN6 cells were cultured in 25 mM glucose.
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fig2: Insulin receptors do not associate with markers of clathrin-dependent endocytosis. (A, A′) Pulse-chase colocalization analysis of Alexafluor488-labeled EGF relative to InsRA-TagRFP indicates separate entry pathways but eventual fusion into a shared mature pool of vesicles in MIN6 cells cultured in 0 mM glucose. A significant increase in the degree of colocalization was observed at a 45 min chase time compared to 0 min chase time (n = 4 images per time point). Scale bar = 5 μm. (B–D) Colocalization analysis of InsRA-TagRFP with TagBFP-tagged Rab5a (early endosomes), TagBFP-Rab7 (late endosomes), and TagBFP-Rab11a (recycling endosomes) in fixed MIN6 cells (n = 10). Similar results observed with InsRB-TagRFP. Scale bar = 5 μm. (E) Colocalization analysis of immunolabeled endogenous insulin receptors with immunolabeled endogenous clathrin in MIN6 cells (n = 10). Scale bar = 10 μm. (F–H) Colocalization analysis of tagged insulin receptors with immunolabeled endogenous Rab5a, Rab7, and Rab4a (alternate marker for recycling endosomes) in MIN6 cells (n = 10). Scale bar = 10 μm. (B–H) Prior fixation, MIN6 cells were cultured in 25 mM glucose.
Mentions: We examined the trafficking route of these functional insulin receptors by comparing their localization to endosomal pathway markers. First, we examined whether insulin receptors colocalized with epidermal growth factor (EGF), an established endosomal cargo of the clathrin pathway, in a pulse chase experiment. Fluorescent Alexa-488 labeled EGF showed very limited colocalization (<5%) to InsRA-TagRFP at short-chase times, indicating that the proximal routes of endocytosis were likely distinct (Figure 2A). The significantly increased colocalization at longer chase times indicated that the vesicles of the two different internalization routes eventually merged into more mature compartments such as lysosomes (Figure 2A′,A″). Next, we assessed the colocalization of InsRA-TagRFP with Rab5a, a marker associated with clathrin-dependent endocytosis [39], as well as Rab7-labeled late endosomes and Rab11a-labeled recycling endosomes and found virtually no colocalization (Figure 2B–D). Similarly, endogenous insulin receptors showed minimal colocalization with endogenous clathrin in primary mouse beta-cells (Figure 2E), and tagged insulin receptors did not appreciably colocalize with endogenous Rab5a, Rab7, or Rab4A (Figure 2F–H). Clathrin loss-of-function experiments resulted in gross changes in beta-cell plasma membrane morphology (not shown), so we were unable to rule out some small role for clathrin in insulin receptor trafficking. Notwithstanding, these data demonstrate that insulin receptors spend little, if any, time in endocytic vesicles labeled with these Rab-GTPases, or clathrin.

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