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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

InsR-TagRFP is transported to lysosomes in Cav-1 and Flot-1 positive vesicles. (A) Confocal imaging of Cav1-mRFP and InsRA-TagBFP in MIN6 cells (n = 10). Scale bars = 10 μm. (B) G-STED microscopy in MIN6 cells illustrates the presence of Flot1 on vesicles containing endogenous insulin receptors. Scale bars = 5 μm. (B′) Insets show flotillin-1 positive vesicles harboring insulin receptors. (C–G) Colocalization between InsRA-TagBFP and Flot1-mRFP, Flot1-mRFP and CAV1-eGFP, Flot1-mRFP and LAMP2-eGFP, InsRA-TagRFP and LAMP2-eGFP, CAV1-mRFP and LAMP2-eGFP in MIN6 cells demonstrates a Cav1-Flot1-LAMP2 endocytic trafficking route to lysosomes (n = 10). Scale bars = 10 μm; F: Scale bar = 5 μm. (H, I) Immunolabeling of endogenous insulin receptors, endogenous Flot1, and endogenous LAMP2 (lysosomes) in primary 12 week-old mouse islet cells (n = 10). Scale bars = 10 μm. (J) Summary of object-based colocalization of tagged proteins compared with InsRA-TagRFP in MIN6 cells. Note that InsRA controls represent the maximum colocalization in this system (n = 10 cells per condition). (K) Summary of colocalization within vesicular pools in the Cav1/Flot1/Lamp2 pathway of MIN6 cells (n = 10 cells per condition). (A–I) MIN6 cells were cultured in 25 mM glucose and primary cells were cultured in 11 mM glucose prior fixation.
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fig4: InsR-TagRFP is transported to lysosomes in Cav-1 and Flot-1 positive vesicles. (A) Confocal imaging of Cav1-mRFP and InsRA-TagBFP in MIN6 cells (n = 10). Scale bars = 10 μm. (B) G-STED microscopy in MIN6 cells illustrates the presence of Flot1 on vesicles containing endogenous insulin receptors. Scale bars = 5 μm. (B′) Insets show flotillin-1 positive vesicles harboring insulin receptors. (C–G) Colocalization between InsRA-TagBFP and Flot1-mRFP, Flot1-mRFP and CAV1-eGFP, Flot1-mRFP and LAMP2-eGFP, InsRA-TagRFP and LAMP2-eGFP, CAV1-mRFP and LAMP2-eGFP in MIN6 cells demonstrates a Cav1-Flot1-LAMP2 endocytic trafficking route to lysosomes (n = 10). Scale bars = 10 μm; F: Scale bar = 5 μm. (H, I) Immunolabeling of endogenous insulin receptors, endogenous Flot1, and endogenous LAMP2 (lysosomes) in primary 12 week-old mouse islet cells (n = 10). Scale bars = 10 μm. (J) Summary of object-based colocalization of tagged proteins compared with InsRA-TagRFP in MIN6 cells. Note that InsRA controls represent the maximum colocalization in this system (n = 10 cells per condition). (K) Summary of colocalization within vesicular pools in the Cav1/Flot1/Lamp2 pathway of MIN6 cells (n = 10 cells per condition). (A–I) MIN6 cells were cultured in 25 mM glucose and primary cells were cultured in 11 mM glucose prior fixation.

Mentions: Studies in other cell types indicate that insulin receptors travel to lysosomes [8], and we have published data illustrating the colocalization of endogenous InsR with Lysotracker in human beta-cells [23], but the intracellular route of beta-cell insulin receptors has not been reported. Confocal imaging demonstrated that tagged CAV1 colocalized with tagged InsRA on intracellular vesicular structures, but to a lesser extent than the colocalization between these proteins at the plasma membrane (Figure 3, Figure 4A). Flotillin-I (Flot1) has been described in clathrin-independent endocytosis [43], associates with caveolin-containing structures [43], [44], and has been implicated in insulin signaling in other cell types [45], [46]. Endogenous Flot1 was expressed in primary mouse beta-cells (Figure 4H). Using super-resolution imaging, we found that endogenous Flot1 was localized on intracellular vesicles containing insulin receptors (Figure 4B, B′). Similarly, InsRA-TagBFP showed robust colocalization with Flot1-mRFP positive vesicles by confocal imaging (Figure 4C). Flot1-mRFP also colocalized with both Cav1-eGFP and LAMP2-eGFP (Figure 4D, E), suggesting a distribution of internalized insulin receptors to early and late endolysosomal compartments.


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)

InsR-TagRFP is transported to lysosomes in Cav-1 and Flot-1 positive vesicles. (A) Confocal imaging of Cav1-mRFP and InsRA-TagBFP in MIN6 cells (n = 10). Scale bars = 10 μm. (B) G-STED microscopy in MIN6 cells illustrates the presence of Flot1 on vesicles containing endogenous insulin receptors. Scale bars = 5 μm. (B′) Insets show flotillin-1 positive vesicles harboring insulin receptors. (C–G) Colocalization between InsRA-TagBFP and Flot1-mRFP, Flot1-mRFP and CAV1-eGFP, Flot1-mRFP and LAMP2-eGFP, InsRA-TagRFP and LAMP2-eGFP, CAV1-mRFP and LAMP2-eGFP in MIN6 cells demonstrates a Cav1-Flot1-LAMP2 endocytic trafficking route to lysosomes (n = 10). Scale bars = 10 μm; F: Scale bar = 5 μm. (H, I) Immunolabeling of endogenous insulin receptors, endogenous Flot1, and endogenous LAMP2 (lysosomes) in primary 12 week-old mouse islet cells (n = 10). Scale bars = 10 μm. (J) Summary of object-based colocalization of tagged proteins compared with InsRA-TagRFP in MIN6 cells. Note that InsRA controls represent the maximum colocalization in this system (n = 10 cells per condition). (K) Summary of colocalization within vesicular pools in the Cav1/Flot1/Lamp2 pathway of MIN6 cells (n = 10 cells per condition). (A–I) MIN6 cells were cultured in 25 mM glucose and primary cells were cultured in 11 mM glucose prior fixation.
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fig4: InsR-TagRFP is transported to lysosomes in Cav-1 and Flot-1 positive vesicles. (A) Confocal imaging of Cav1-mRFP and InsRA-TagBFP in MIN6 cells (n = 10). Scale bars = 10 μm. (B) G-STED microscopy in MIN6 cells illustrates the presence of Flot1 on vesicles containing endogenous insulin receptors. Scale bars = 5 μm. (B′) Insets show flotillin-1 positive vesicles harboring insulin receptors. (C–G) Colocalization between InsRA-TagBFP and Flot1-mRFP, Flot1-mRFP and CAV1-eGFP, Flot1-mRFP and LAMP2-eGFP, InsRA-TagRFP and LAMP2-eGFP, CAV1-mRFP and LAMP2-eGFP in MIN6 cells demonstrates a Cav1-Flot1-LAMP2 endocytic trafficking route to lysosomes (n = 10). Scale bars = 10 μm; F: Scale bar = 5 μm. (H, I) Immunolabeling of endogenous insulin receptors, endogenous Flot1, and endogenous LAMP2 (lysosomes) in primary 12 week-old mouse islet cells (n = 10). Scale bars = 10 μm. (J) Summary of object-based colocalization of tagged proteins compared with InsRA-TagRFP in MIN6 cells. Note that InsRA controls represent the maximum colocalization in this system (n = 10 cells per condition). (K) Summary of colocalization within vesicular pools in the Cav1/Flot1/Lamp2 pathway of MIN6 cells (n = 10 cells per condition). (A–I) MIN6 cells were cultured in 25 mM glucose and primary cells were cultured in 11 mM glucose prior fixation.
Mentions: Studies in other cell types indicate that insulin receptors travel to lysosomes [8], and we have published data illustrating the colocalization of endogenous InsR with Lysotracker in human beta-cells [23], but the intracellular route of beta-cell insulin receptors has not been reported. Confocal imaging demonstrated that tagged CAV1 colocalized with tagged InsRA on intracellular vesicular structures, but to a lesser extent than the colocalization between these proteins at the plasma membrane (Figure 3, Figure 4A). Flotillin-I (Flot1) has been described in clathrin-independent endocytosis [43], associates with caveolin-containing structures [43], [44], and has been implicated in insulin signaling in other cell types [45], [46]. Endogenous Flot1 was expressed in primary mouse beta-cells (Figure 4H). Using super-resolution imaging, we found that endogenous Flot1 was localized on intracellular vesicles containing insulin receptors (Figure 4B, B′). Similarly, InsRA-TagBFP showed robust colocalization with Flot1-mRFP positive vesicles by confocal imaging (Figure 4C). Flot1-mRFP also colocalized with both Cav1-eGFP and LAMP2-eGFP (Figure 4D, E), suggesting a distribution of internalized insulin receptors to early and late endolysosomal compartments.

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