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A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

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Rab10 interacts with Rlf. (A) Indicated proteins were ectopically expressed in Cos-1 cells. Immunoprecipitation with HA-Rlf shows specific interaction with Rab10 in immunoblots. (B) Cos-1 cells were transfected with indicated plasmids. At 24 h later, cells were lysed and immunoprecipitated with antibodies against Myc. Level of proteins in the total cell lysate and the immunoprecipitates are shown. (C) Quantification of blots in A and B. Images from three independent experiments were used to generate error bars. *p < 0.05. (D) Schematic representation of various domains in full-length and truncated Rlf. (E) Pull-down assay with GST-Rlf-RA. Indicated plasmids were expressed in Cos-1 cells, and GST or GST-Rlf-RA beads were used for pull down. Specific and increased binding to Rab10 (QL) are shown. (F) Lysates from Cos-1 cells expressing HA-Rab10 were treated with 1.0 mM GDP or 0.5 mM GTPγS. Pull-down experiments were performed with indicated GST beads. Protein levels in the total cell lysate, as well as pull down, are shown. (G) Pull-down experiment was performed as in F, except that GST-Rlf-RA beads were used instead of full-length Rlf. (H) Quantification of blots in F and G. Binding of Rim1 to GDP-bound Rab10 was set to 1, and fold changes in binding of Rlf and Rlf-RA to Rab10 in GDP/GTP-bound states were calculated. Error bars were acquired from at least three independent experiments. **p < 0.01, *p < 0.05.
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Figure 4: Rab10 interacts with Rlf. (A) Indicated proteins were ectopically expressed in Cos-1 cells. Immunoprecipitation with HA-Rlf shows specific interaction with Rab10 in immunoblots. (B) Cos-1 cells were transfected with indicated plasmids. At 24 h later, cells were lysed and immunoprecipitated with antibodies against Myc. Level of proteins in the total cell lysate and the immunoprecipitates are shown. (C) Quantification of blots in A and B. Images from three independent experiments were used to generate error bars. *p < 0.05. (D) Schematic representation of various domains in full-length and truncated Rlf. (E) Pull-down assay with GST-Rlf-RA. Indicated plasmids were expressed in Cos-1 cells, and GST or GST-Rlf-RA beads were used for pull down. Specific and increased binding to Rab10 (QL) are shown. (F) Lysates from Cos-1 cells expressing HA-Rab10 were treated with 1.0 mM GDP or 0.5 mM GTPγS. Pull-down experiments were performed with indicated GST beads. Protein levels in the total cell lysate, as well as pull down, are shown. (G) Pull-down experiment was performed as in F, except that GST-Rlf-RA beads were used instead of full-length Rlf. (H) Quantification of blots in F and G. Binding of Rim1 to GDP-bound Rab10 was set to 1, and fold changes in binding of Rlf and Rlf-RA to Rab10 in GDP/GTP-bound states were calculated. Error bars were acquired from at least three independent experiments. **p < 0.01, *p < 0.05.

Mentions: Because Rlf is required for Rab10-stimulated activation of RalA, we tested whether Rlf might be a direct effector for Rab10. Immunoprecipitation of HA-Rlf from Cos-1 cells overexpressing Myc-Rab10 (WT or QL) revealed that Rab10 specifically binds to Rlf, and further that this binding depends on the activation state of Rab10 (Figure 4A). Constitutively active Rab10 (QL) was twofold more effective in binding to Rlf than with the wild-type G protein (Figure 4C). Rlf binding to Rab10 (QL) was comparable to that observed with Rim1-RBD, a known effector of Rab10 (Figure 4, B and 4C). In addition, reciprocal immunoprecipitation of Myc-Rab10 also showed specific binding of Rlf to active Rab10 (QL) (Supplemental Figure S3).


A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.

Karunanithi S, Xiong T, Uhm M, Leto D, Sun J, Chen XW, Saltiel AR - Mol. Biol. Cell (2014)

Rab10 interacts with Rlf. (A) Indicated proteins were ectopically expressed in Cos-1 cells. Immunoprecipitation with HA-Rlf shows specific interaction with Rab10 in immunoblots. (B) Cos-1 cells were transfected with indicated plasmids. At 24 h later, cells were lysed and immunoprecipitated with antibodies against Myc. Level of proteins in the total cell lysate and the immunoprecipitates are shown. (C) Quantification of blots in A and B. Images from three independent experiments were used to generate error bars. *p < 0.05. (D) Schematic representation of various domains in full-length and truncated Rlf. (E) Pull-down assay with GST-Rlf-RA. Indicated plasmids were expressed in Cos-1 cells, and GST or GST-Rlf-RA beads were used for pull down. Specific and increased binding to Rab10 (QL) are shown. (F) Lysates from Cos-1 cells expressing HA-Rab10 were treated with 1.0 mM GDP or 0.5 mM GTPγS. Pull-down experiments were performed with indicated GST beads. Protein levels in the total cell lysate, as well as pull down, are shown. (G) Pull-down experiment was performed as in F, except that GST-Rlf-RA beads were used instead of full-length Rlf. (H) Quantification of blots in F and G. Binding of Rim1 to GDP-bound Rab10 was set to 1, and fold changes in binding of Rlf and Rlf-RA to Rab10 in GDP/GTP-bound states were calculated. Error bars were acquired from at least three independent experiments. **p < 0.01, *p < 0.05.
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Figure 4: Rab10 interacts with Rlf. (A) Indicated proteins were ectopically expressed in Cos-1 cells. Immunoprecipitation with HA-Rlf shows specific interaction with Rab10 in immunoblots. (B) Cos-1 cells were transfected with indicated plasmids. At 24 h later, cells were lysed and immunoprecipitated with antibodies against Myc. Level of proteins in the total cell lysate and the immunoprecipitates are shown. (C) Quantification of blots in A and B. Images from three independent experiments were used to generate error bars. *p < 0.05. (D) Schematic representation of various domains in full-length and truncated Rlf. (E) Pull-down assay with GST-Rlf-RA. Indicated plasmids were expressed in Cos-1 cells, and GST or GST-Rlf-RA beads were used for pull down. Specific and increased binding to Rab10 (QL) are shown. (F) Lysates from Cos-1 cells expressing HA-Rab10 were treated with 1.0 mM GDP or 0.5 mM GTPγS. Pull-down experiments were performed with indicated GST beads. Protein levels in the total cell lysate, as well as pull down, are shown. (G) Pull-down experiment was performed as in F, except that GST-Rlf-RA beads were used instead of full-length Rlf. (H) Quantification of blots in F and G. Binding of Rim1 to GDP-bound Rab10 was set to 1, and fold changes in binding of Rlf and Rlf-RA to Rab10 in GDP/GTP-bound states were calculated. Error bars were acquired from at least three independent experiments. **p < 0.01, *p < 0.05.
Mentions: Because Rlf is required for Rab10-stimulated activation of RalA, we tested whether Rlf might be a direct effector for Rab10. Immunoprecipitation of HA-Rlf from Cos-1 cells overexpressing Myc-Rab10 (WT or QL) revealed that Rab10 specifically binds to Rlf, and further that this binding depends on the activation state of Rab10 (Figure 4A). Constitutively active Rab10 (QL) was twofold more effective in binding to Rlf than with the wild-type G protein (Figure 4C). Rlf binding to Rab10 (QL) was comparable to that observed with Rim1-RBD, a known effector of Rab10 (Figure 4, B and 4C). In addition, reciprocal immunoprecipitation of Myc-Rab10 also showed specific binding of Rlf to active Rab10 (QL) (Supplemental Figure S3).

Bottom Line: Once activated, Rab10 can increase the GTP binding of RalA by recruiting the Ral guanyl nucleotide exchange factor, Rlf/Rgl2.Overexpression of membrane-tethered Rlf compensates for the loss of Rab10 in Glut4 translocation, suggesting that Rab10 recruits Rlf to membrane compartments for RalA activation and that RalA is downstream of Rab10.Together these studies identify a new G protein cascade in the regulation of insulin-stimulated Glut4 trafficking and glucose uptake.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.

Show MeSH
Related in: MedlinePlus