α-Actinin-4 Is Selectively Required for Insulin-induced GLUT4 Translocation

Abstract

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64 -75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of ␣-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton. Insulin-regulated glucose transporter 4 (GLUT4)4 is a member of the SLC2A facilitative glucose transporter family (2) and is responsible for glucose entry into muscle and fat tissues (3-5). GLUT4 continuously cycles to/from the cell membrane through a series of endosomal compartments. In response to insulin there is a rapid increase in the steady-state level of GLUT4 at the cell surface, at the expense of intracellular pools (6 -9). This process is defective in insulin resistance and type 2 diabetes (10 -12). Stimuli other than insulin such as muscle contraction, depolarization, or hypoxia also increase surface GLUT4 (13-16). Whereas insulin largely increases the exocytic arm of GLUT4 cycling (17), hypoxia or membrane depolarization preferentially reduce GLUT4 endocytosis in muscle cells (16,18,19). Moreover, although insulin-dependent GLUT4 translocation requires dynamic remodeling of filamentous actin (20 -23), the gain in surface GLUT4 elicited by plateletderived growth factor (PDGF), depolarization, or mitochondrial uncouplers is independent of actin dynamics (24 -26).Intensive research has recently focused on identifying the individual mechanisms participating in GLUT4 traffic and the specific events regulated by insulin (27)(28)(29)(30)(31). Hypothesizing that GLUT4 traffic may be regulated by interaction with partner proteins, it is of fundamental and clinical interest to identify such proteins. Accordingly, we recently applied the novel SILAC (stable isotope labeling by amino acids in cell culture) approach (32) to search for proteins that associate with GLUT4 in an insulin-regulated manner (1). The study took advantage of the stable expression in L6 muscle cells of GLUT4 encoding an myc tag that faces the extrace...

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GLUT4 Vesicle Recruitment and Fusion Are Differentially Regulated by Rac, AS160, and Rab8A in Muscle Cells

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New insights to the MLL recombinome of acute leukemias

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