Figure 8.
Formation of the human insulin-responsive GLUT4 pathway involves membrane traffic from the ERGIC and supports a model for two routes to GLUT4 sequestration. (A and B) Insulin-stimulated GLUT4 translocation in HeLa-GLUT4 cells as quantified by FACS analysis of surface:total GLUT4. Cells were pretreated with siRNA targeting CHC22, CHC17, p115, GM130, sortilin, IRAP, sortilin plus IRAP, or nontargeting control siRNA (siCon) as in Fig. 7 A and then incubated with (+) or without (−) insulin (Ins). For the experiments in A, data are expressed as mean ± SEM, n = 9, 10,000 cells acquired per experiment. For the experiments in B, data are expressed as mean ± SEM, n = 7, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus untreated. (C) Proposed model for the roles of CHC22 in the human GLUT4 pathway. Newly synthesized GLUT4 traffics from the ER to the ERGIC. At the ERGIC, a complex forms between IRAP and p115 that promotes binding of CHC22 clathrin and sequesters GLUT4 through its IRAP interaction (Shi et al., 2008; box A). Formation of the CHC22 clathrin coat at the ERGIC then facilitates sorting of GLUT4 to the intracellular region, where GLUT4 storage vesicles (GSV) and insulin-responsive GLUT4 vesicles (IRV) are formed. After insulin-mediated GLUT4 translocation and GLUT4 reuptake (by CHC17 clathrin), a complex forms (box B) between endosomal GLUT4, sortilin, and the clathrin adaptor GGA2, which promotes CHC22 recruitment. Endosomal GLUT4 sorting also involves clathrin adaptor AP1 (Blot and McGraw, 2008; Gillingham et al., 1999), which interacts with CHC22, further participating in its recruitment to endosomes (Vassilopoulos et al., 2009). Formation of the CHC22 coat on sorting endosomes facilitates GLUT4 traffic to the TGN via the retrograde pathway, enabling replenishment of the intracellular GSV/IRV pool. The GSC comprises the entire tubulo-vesicular complex involved in sorting and sequestration of GLUT4, with CHC22 mediating two pathways to the human GSC.

Formation of the human insulin-responsive GLUT4 pathway involves membrane traffic from the ERGIC and supports a model for two routes to GLUT4 sequestration. (A and B) Insulin-stimulated GLUT4 translocation in HeLa-GLUT4 cells as quantified by FACS analysis of surface:total GLUT4. Cells were pretreated with siRNA targeting CHC22, CHC17, p115, GM130, sortilin, IRAP, sortilin plus IRAP, or nontargeting control siRNA (siCon) as in Fig. 7 A and then incubated with (+) or without (−) insulin (Ins). For the experiments in A, data are expressed as mean ± SEM, n = 9, 10,000 cells acquired per experiment. For the experiments in B, data are expressed as mean ± SEM, n = 7, 10,000 cells acquired per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus untreated. (C) Proposed model for the roles of CHC22 in the human GLUT4 pathway. Newly synthesized GLUT4 traffics from the ER to the ERGIC. At the ERGIC, a complex forms between IRAP and p115 that promotes binding of CHC22 clathrin and sequesters GLUT4 through its IRAP interaction (Shi et al., 2008; box A). Formation of the CHC22 clathrin coat at the ERGIC then facilitates sorting of GLUT4 to the intracellular region, where GLUT4 storage vesicles (GSV) and insulin-responsive GLUT4 vesicles (IRV) are formed. After insulin-mediated GLUT4 translocation and GLUT4 reuptake (by CHC17 clathrin), a complex forms (box B) between endosomal GLUT4, sortilin, and the clathrin adaptor GGA2, which promotes CHC22 recruitment. Endosomal GLUT4 sorting also involves clathrin adaptor AP1 (Blot and McGraw, 2008; Gillingham et al., 1999), which interacts with CHC22, further participating in its recruitment to endosomes (Vassilopoulos et al., 2009). Formation of the CHC22 coat on sorting endosomes facilitates GLUT4 traffic to the TGN via the retrograde pathway, enabling replenishment of the intracellular GSV/IRV pool. The GSC comprises the entire tubulo-vesicular complex involved in sorting and sequestration of GLUT4, with CHC22 mediating two pathways to the human GSC.

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