A Rab10:RalA G protein cascade regulates insulin-stimulated glucose uptake in adipocytes.
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.
Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.Show MeSH
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Mentions: Recent evidence on the sequential action of G proteins in vesicular trafficking (Mizuno-Yamasaki et al., 2012) suggests that G protein cascades could be a general mechanism in the regulation of vesicle recycling. Of interest, upon insulin stimulation in fat cells, Akt1/2 phosphorylates and inactivates both AS160 (Kane et al., 2002) and the RalGAP complex RGC1/2 (Chen et al., 2011b), leading to the activation of Rab10 and RalA. However, the relative roles of these G proteins and their relationship to each other remain unknown. Whereas the overexpression of constitutively active RalA (G23V/GV) or Rab10 (Q68L/QL) (Figure 2C) independently increased basal glucose uptake in adipocytes (Figure 2A), no additive effects were observed with co-overexpression of the two active G proteins in the same cells. Expression of active RalA modestly increased insulin-stimulated glucose uptake, without further effect from coexpression of Rab10 (QL) (Figure 2B). These data suggest the possibility that RalA and Rab10 might reside in a linear pathway, and we thus determined whether either of the two G proteins might influence the activity of the other. Although overexpression of constitutively active RalA did not alter cellular Rab10 activity, measured as described (Supplemental Figure S2), Rab10 overexpression produced an increase in RalA activity under insulin-stimulated conditions (Figure 2, D and E) as measured by GST-Sec5-RBD pull-down assay. Activation of RalA was dependent on the activation status of Rab10. Overexpression of the constitutively active Rab10 (QL) allele caused hyperactivation of RalA, whereas a GDP-locked version of Rab10 (TN) decreased RalA activity (Figure 2, F and G). These data suggest that Rab10 functions upstream of RalA, and further that its activation can stimulate RalA activity.
Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.