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: Previous studies demonstrated that GAP proteins preferentially interact with their cognate small GTPases during the transition state of GTP hydrolysis (Scheffzek et al., 1997; Bos et al., 2007). To identify a cellular target of AS160, we loaded glutathione S-transferase (GST)–Rab proteins with guanosine diphosphate (GDP)/AlF4 to mimic this transition state, incubating the fusion proteins with lysates from COS-1 cells overexpressing FLAG-AS160. AS160 specifically interacted with GST-Rab10 bound to GDP/AIFx but not with GST-Rab10 bound to GDP or GTPγS (Figure 1A), indicating that the interaction between AS160 and Rab10 is transition state dependent. Meanwhile, AS160 did not interact, or interacted only modestly, with other GDP/AIFx-loaded small GTPases, including the Ras subfamily member RalA and the Rab subfamily members Rab4, Rab5, and Rab11 (Figure 1, B and C). These results suggest that the transition state–dependent binding between AS160 and Rab10 is specific and characteristic of a GAP-substrate GTPase interaction, consistent with the notion that AS160 possesses GAP activity toward Rab10 in vitro.
Affiliation: Life Sciences Institute, University of Michigan Medical School, Ann Arbor, MI 48109.