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First published online 14 November 2007
doi: 10.1242/jcs.007310

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Rip11 is a Rab11- and AS160-RabGAP-binding protein required for insulin-stimulated glucose uptake in adipocytes

Gavin I. Welsh^1,*, Sophie E. Leney^1,*, Bethan Lloyd-Lewis¹, Matthew Wherlock¹, Andrew J. Lindsay², Mary W. McCaffrey² and Jeremy M. Tavaré^1,

¹ Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 ITD, UK
² Molecular Cell Biology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland

View larger version (46K): [in this window] [in a new window]   Fig. 1. Insulin and constitutively active PKB promote Rip11 translocation to the plasma membrane. (A) 3T3-L1 adipocytes were electroporated with a plasmid encoding either GFP-FIP2 (a,b), GFP-RCP (c,d) or GFP-Rip11 (e-h), and in the absence (a-g) or presence (h) of plasmid directing the expression of a constitutively active PKB (MyrPKB). The cells were imaged 24 hours later after incubation in the absence (a,c,e,h) or presence (b,d,f) of 100 nM insulin for 30 minutes. In panel (g), the cells were pre-treated with 100 nM wortmannin for 30 minutes before the addition of 100 nM insulin for 30 minutes. The cells were fixed, and visualisation of GFP fluorescence performed by laser-scanning confocal microscopy. Selected representative cells are shown. In (i) the data are expressed as the intensity of GFP fluorescence in the plasma membrane as a fraction of the total cellular GFP fluorescence (note that images for treatment with 50 ng/ml PDGF for 30 minutes are not shown). The data are expressed as means ± s.e.m., with each bar representing data from a minimum of 25 cells. (B) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP (a) and mRFP-Rip11 (b) and imaged 24 hours later. The figure shows representative laser-scanning confocal micrographs of the distribution of each protein, with a merged image provided in panel c (green is GLUT4, red is Rip11) in the absence of insulin. Both panels are representative of at least three separate preparations of adipocytes.

View larger version (62K): [in this window] [in a new window]   Fig. 2. Lack of apparent colocalisation of Rip11 with markers of early and recycling endosomes, lysosomes and Golgi elements. 3T3-L1 adipocytes were electroporated with plasmids encoding GFP-Rip11 and transferrin receptor (row A), or GFP-Rip11 alone (rows B-F). In row A, the cells were incubated 24 hours later for 30 minutes with transferrin-Alexa Fluor 633 and then fixed and imaged for the localisation of recycling transferrin receptors (red) or GFP-Rip11 (green). In rows B-F, the cells were fixed 24 hours after transfection, permeabilised and stained with antibodies against EEA1 (row B), the mannose 6-phosphate receptor (CI-MPR; row C), LAMP-1 (row D), TGN38 (row E) and giantin (row F). Each row comprises a single cell illustrating the distribution of the relevant marker (red) or GFP-Rip11 (green), with a merged image shown in the right-hand column.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Overexpression of Rip11 inhibits insulin-stimulated GLUT4 translocation to, and fusion with, the plasma membrane. (A) 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP in the presence of either mRFP-Rip11 or mRFP vector, as indicated. 24 hours later, the cells were treated in the absence or presence of 100 nM insulin for 30 minutes and were then fixed and stained with an antibody against HA to detect surface-localised GLUT4. The intensity of HA antibody staining was divided by the intensity of total cellular GFP fluorescence. This provides a ratio that represents the degree of fusion of GLUT4 with the plasma membrane, corrected for the expression level of GLUT4. (B) Representative images are provided of insulin-stimulated cells from the experiment in panel A (images of additional cells can be found in supplementary material Fig. S1). The cells were analysed for the effect of Rip11 overexpression (panels b and e; where panels a and d are from cells expressing an mRFP vector control) on insulin-stimulated GLUT4 translocation, as determined by the distribution of GFP (panels a-c, where panel c is expressed as the amount of GFP fluorescence in the plasma membrane divided by total cellular GFP fluorescence) and the extent of fusion of translocated GLUT4 with the plasma membrane (panels d-f; where panel f is expressed as the intensity of HA fluorescence in the plasma membrane divided by the intensity of GFP fluorescence found at the plasma membrane) in the presence of insulin. Both panels of the figure are representative of three separate preparations of adipocytes, with the data expressed as the mean ± s.e.m.

View larger version (17K): [in this window] [in a new window]   Fig. 4. siRNA-mediated knockdown of Rip11 reduces Rip11 but not FIP2 or RCP mRNA expression and inhibits insulin-stimulated uptake of 2-deoxyglucose. 3T3-L1 adipocytes were electroporated with scrambled siRNA duplex (S) or two different siRNA duplexes directed towards distinct regions of the Rip11 mRNA sequence (A and B, respectively). (A) The level of expression of Rip11, FIP2 and RCP mRNA was measured 48 hours later using quantitative real-time PCR. The level of expression of each mRNA was determined relative to the level of expression of 2-microglobulin mRNA, and, for each mRNA, this was normalised to the level of expression in the presence of the scrambled mRNA (therefore, equal to 1 in each case). Each bar is representative of determinations arising from five independent preparations of adipocytes, with the data expressed as the mean ± s.e.m. (B) In a parallel experiment, the adipocytes were treated in the absence or presence of 100 nM insulin, as indicated, and 2-deoxyglucose uptake measured in triplicate, as described in Materials and Methods. The panel is representative of two separate experiments, with each bar indicating the mean ± s.d.

View larger version (16K): [in this window] [in a new window]   Fig. 5. A combination of Rip11 siRNA duplexes blocks insulin-stimulated uptake of 2-deoxyglucose but not transferrin binding by 3T3-L1 adipocytes. 3T3-L1 adipocytes were electroporated with control siRNA duplex or a combination of Rip11A and Rip11B siRNA duplexes (Rip11A/B) at two different concentrations directed to deplete Rip11. The cells were left for 48 hours before measuring: (A): uptake of 2-deoxyglucose in the absence or presence of 100 nM insulin. (B) The level of expression of Rip11 mRNA, which was measured using quantitative real-time PCR with the data shown relative to the level of expression of mRNA encoding cyclophilin A, which was itself unchanged by the Rip11 siRNA. (C) 125I-labelled transferrin binding in the absence or presence of 100 nM insulin. Each panel is representative of three separate preparations of adipocytes, with the data expressed as the mean ± s.e.m.

View larger version (45K): [in this window] [in a new window]   Fig. 6. The I630E mutation in Rip11 blocks Rab11a and Rab11b binding. (A) CHO.T cells were transfected with plasmids encoding GFP-tagged Rab11a or GFP-vector, along with plasmids encoding either Xpress-tagged wild-type Rip11 (Rip11-WT) or Rip11-I630E, as indicated. The cells were incubated in the absence or presence of 100 nM insulin for 10 minutes, as shown, before lysis. Cell lysates were blotted with antibodies against GFP (lysate: IB Rab11a) or antibodies against Xpress (lysate: IB Rip11) to assess the level of expression of these proteins. Alternatively, Rab11a was immunoprecipitated using antibody against GFP, and the complexes were blotted with antibody against Xpress (IP Rab11a: IB Rip11) or against GFP (IP Rab11a: IB Rab11a) to detect the presence of Xpress-tagged Rip11 and GFP-tagged Rab11a in the precipitates, respectively. (B) A similar experiment to panel A was performed, except that the Rab11a expression plasmid was replaced with an equivalent plasmid expressing Rab11b.

View larger version (35K): [in this window] [in a new window]   Fig. 7. AS160 interacts with Rip11. (A) CHO.T cells were transfected with plasmids encoding FLAG-tagged wild-type AS160 (AS160-WT), along with either Xpress-tagged wild-type Rip11 (Rip11-WT) or Rip11-I630E, or the Xpress vector, as indicated. The cells were incubated in the absence or presence of 100 nM insulin for 10 minutes, as shown, before lysis. Cell lysates were blotted with antibodies against Xpress (lysate: IB Rip11) or against FLAG (lysate: IB AS160) to assess the level of expression of these proteins. Alternatively, Rip11 complexes were immunoprecipitated using the antibody against the Xpress tag, and these complexes were blotted with antibodies against Xpress (IP Rip11: IB Rip11) or FLAG (IP Rip11: IB AS160) to detect the presence of Xpress-tagged Rip11 and FLAG-tagged AS160 in the precipitates, respectively. (B) The extent of the interaction of the wild-type (WT) and I630E mutant Rip11 with AS160 was quantitated by densitometry and is plotted as the mean ± s.e.m. for four separate preparations of adipocytes.

View larger version (30K): [in this window] [in a new window]   Fig. 8. AS160 becomes phosphorylated in the Rip11 complex in response to insulin. CHO.T cells were cotransfected with plasmids encoding FLAG-tagged wild-type AS160 (AS160-WT) together with either Xpress-tagged wild-type Rip11 (Rip11-WT) or Rip11-I630E, as shown. The cells were incubated in the absence or presence of 100 nM insulin for 10 minutes, as indicated, before lysis and immunoprecipitation of Rip11 complexes with antibodies against Xpress. These complexes were blotted with the antibody against FLAG (top panel) or the antibody against PAS, which recognises the PKB phosphorylation consensus sequence(s) present on AS160 (lower panel).

View larger version (18K): [in this window] [in a new window]   Fig. 9. A Rip11[I630E] mutant retains the ability to inhibit the insulin-stimulated appearance of GLUT4 at the cell surface. 3T3-L1 adipocytes were electroporated with plasmids encoding HA-GLUT4-GFP in the presence of either mRFP vector, mRFP-Rip11 or mRFP-Rip11[I630E], as indicated. 24 hours later, the cells were treated in the absence or presence of 100 nM insulin for 30 minutes and were then fixed and stained with an antibody against HA to detect surface-localised GLUT4. The intensity of HA antibody staining was divided by the intensity of total cellular GFP fluorescence, giving an estimate of the surface appearance of the HA-tagged GLUT4. The data shown are representative of two independent experiments.

View larger version (42K): [in this window] [in a new window]   Fig. 10. Model for the role of the Rip11-AS160 complex in insulin-stimulated GLUT4 translocation and fusion with the plasma membrane. See text for further details. IRAP, insulin-responsive aminopeptidase; PA, phosphatidic acid; PM, plasma membrane; RBD, Rab-binding domain.