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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 phosphorylation modulates insulin receptor domain size and internalization. (A–C) TIRF microscopy of live MIN6 cells expressing InsRA-TagRFP and Cav1 mutant proteins. Scale bar = 10 μm. (D–F) Overexpression of the Cav1-Y14F mutant leads to increased InsRA-TagRFP domain density, increased InsRA-TagRFP domain size, and a higher content of InsRA-TagRFP at the plasma membrane of fixed MIN6 cells (n = 10 cells per condition). (G) Quantification of TIRF time lapse imaging of InsRA-lum-eGFP domain lifetime at the plasma membrane of stable MIN6 cell lines expressing various Cav1 mutants. *p < 0.05, n > 4 cells per condition. (A–G) MIN6 cells were cultured in 20 mM glucose during image acquisition.
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fig5: Caveolin-1 phosphorylation modulates insulin receptor domain size and internalization. (A–C) TIRF microscopy of live MIN6 cells expressing InsRA-TagRFP and Cav1 mutant proteins. Scale bar = 10 μm. (D–F) Overexpression of the Cav1-Y14F mutant leads to increased InsRA-TagRFP domain density, increased InsRA-TagRFP domain size, and a higher content of InsRA-TagRFP at the plasma membrane of fixed MIN6 cells (n = 10 cells per condition). (G) Quantification of TIRF time lapse imaging of InsRA-lum-eGFP domain lifetime at the plasma membrane of stable MIN6 cell lines expressing various Cav1 mutants. *p < 0.05, n > 4 cells per condition. (A–G) MIN6 cells were cultured in 20 mM glucose during image acquisition.

Mentions: Colocalization studies showed a clear association between Cav1 and insulin receptors, but loss-of-function studies were required to determine whether Cav1 drives the process of insulin receptor internalization. Thus, we co-expressed mTFP-labeled Cav1 mutants, a constitutively active form harboring phosphomimetic at tyrosine 14 or a dominant negative form harboring non-phosphorylatable mutations at tyrosine 14 [24], [49], along with TagRFP-labeled insulin receptors. TIRF microscopy demonstrated that expression of the dominant negative Cav1-Y14F mutant increased InsRA-TagRFP density at the plasma membrane when compared to cells expressing wildtype Cav1 (Figure 5A–F). Subsequently, we tested the effects of modified Cav1 phosphorylation on InsR internalization with time-lapse TIRF microscopy. For this experiment, we cloned an eGFP interdomain-tagged InsR construct (InsR-lum-eGFP) to take advantage of the superior photostability of eGFP (eGFP pH-sensitivity was not a confounder in the extracellular pH neutral environment). Stable MIN6 cell lines expressing Cav1 mutants showed that the expression of the phospho-mimetic Cav1-Y14D mutant leads to a significantly shorter lifetime of InsRA domains at the plasma membrane compared to controls (Figure 5G, Supplemental Movie 4). These data are consistent with the concept that Cav1 phosphorylation promotes insulin receptor internalization 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)

Caveolin-1 phosphorylation modulates insulin receptor domain size and internalization. (A–C) TIRF microscopy of live MIN6 cells expressing InsRA-TagRFP and Cav1 mutant proteins. Scale bar = 10 μm. (D–F) Overexpression of the Cav1-Y14F mutant leads to increased InsRA-TagRFP domain density, increased InsRA-TagRFP domain size, and a higher content of InsRA-TagRFP at the plasma membrane of fixed MIN6 cells (n = 10 cells per condition). (G) Quantification of TIRF time lapse imaging of InsRA-lum-eGFP domain lifetime at the plasma membrane of stable MIN6 cell lines expressing various Cav1 mutants. *p < 0.05, n > 4 cells per condition. (A–G) MIN6 cells were cultured in 20 mM glucose during image acquisition.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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fig5: Caveolin-1 phosphorylation modulates insulin receptor domain size and internalization. (A–C) TIRF microscopy of live MIN6 cells expressing InsRA-TagRFP and Cav1 mutant proteins. Scale bar = 10 μm. (D–F) Overexpression of the Cav1-Y14F mutant leads to increased InsRA-TagRFP domain density, increased InsRA-TagRFP domain size, and a higher content of InsRA-TagRFP at the plasma membrane of fixed MIN6 cells (n = 10 cells per condition). (G) Quantification of TIRF time lapse imaging of InsRA-lum-eGFP domain lifetime at the plasma membrane of stable MIN6 cell lines expressing various Cav1 mutants. *p < 0.05, n > 4 cells per condition. (A–G) MIN6 cells were cultured in 20 mM glucose during image acquisition.
Mentions: Colocalization studies showed a clear association between Cav1 and insulin receptors, but loss-of-function studies were required to determine whether Cav1 drives the process of insulin receptor internalization. Thus, we co-expressed mTFP-labeled Cav1 mutants, a constitutively active form harboring phosphomimetic at tyrosine 14 or a dominant negative form harboring non-phosphorylatable mutations at tyrosine 14 [24], [49], along with TagRFP-labeled insulin receptors. TIRF microscopy demonstrated that expression of the dominant negative Cav1-Y14F mutant increased InsRA-TagRFP density at the plasma membrane when compared to cells expressing wildtype Cav1 (Figure 5A–F). Subsequently, we tested the effects of modified Cav1 phosphorylation on InsR internalization with time-lapse TIRF microscopy. For this experiment, we cloned an eGFP interdomain-tagged InsR construct (InsR-lum-eGFP) to take advantage of the superior photostability of eGFP (eGFP pH-sensitivity was not a confounder in the extracellular pH neutral environment). Stable MIN6 cell lines expressing Cav1 mutants showed that the expression of the phospho-mimetic Cav1-Y14D mutant leads to a significantly shorter lifetime of InsRA domains at the plasma membrane compared to controls (Figure 5G, Supplemental Movie 4). These data are consistent with the concept that Cav1 phosphorylation promotes insulin receptor internalization 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