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: As we showed, active Rab10 increases RalA activity through Rlf (Figure 3, B– D). Because Rab10 can increase RalA activity via recruitment of Rlf (Figures 3 and 4), we attempted to rescue the effect of Rab10 knockdown by expression of an activated version of Rlf. We used a tagged version of Rlf (Rlf-CAAX) through addition of a CAAX motif to its C-terminus, thus tethering the protein to membranous compartments (Vigil et al., 2010). We confirmed that Rlf-CAAX partitioned more in the membrane fraction than in the cytosolic fraction, unlike the wild-type protein (Supplemental Figure S4A). Overexpression of Rlf-CAAX also produced a larger increase in RalA activity compared with the wild-type Rlf (Supplemental Figure S4B; Wolthuis et al., 1997). Whereas Rab10 knockdown caused a 25–30% loss in surface Glut4 levels (Figure 6, A and B), overexpression of Rlf-CAAX in these cells (Figure 6C) brought the surface Glut4 levels closer to that seen in control insulin-stimulated cells (Figure 6B). These data indicate that Rab10 affects Glut4 translocation at least partly by recruiting Rlf to activate RalA.
Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.