Insulin-dependent Interactions of Proteins with GLUT4 Revealed through Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)*

The insulin-regulated glucose transporter (GLUT4) translocates to the plasma membrane in response
to insulin in order to facilitate the postprandial uptake of glucose into fat and muscle cells. While early
insulin receptor signaling steps leading to this translocation are well defined, the integration of signaling
and regulation of GLUT4 traffic remains elusive. Several lines of evidence suggest an important role
for the actin cytoskeleton and for protein−protein interactions in regulating GLUT4 localization by insulin.
Here, we applied stable isotope labeling by amino acids in cell culture (SILAC) to identify proteins that
interact with GLUT4 in an insulin-regulated manner. Myc-tagged GLUT4 (GLUT4myc) stably expressed
in L6 myotubes was immunoprecipitated via the myc epitope from total membranes isolated from
basal and insulin-stimulated cells grown in medium containing normal isotopic abundance leucine or
deuterated leucine, respectively. Proteins coprecipitating with GLUT4myc were analyzed by liquid
chromatography/ tandem mass spectrometry. Of 603 proteins quantified, 36 displayed an insulin-dependent change of their interaction with GLUT4myc of more than 1.5-fold in either direction. Several
cytoskeleton-related proteins were elevated in immunoprecipates from insulin-treated cells, whereas
components of the ubiquitin-proteasome degradation system were generally reduced. Proteins
participating in vesicle traffic also displayed insulin-regulated association. Of cytoskeleton-related
proteins, α-actinin-4 recovery in GLUT4 immunoprecipitates rose in response to insulin 2.1 ± 0.5-fold
by SILAC and 2.9 ± 0.8-fold by immunoblotting. Insulin caused GLUT4 and α-actinin-4 co-localization
as revealed by confocal immunofluorescence microscopy. We conclude that insulin elicits changes in
interactions between diverse proteins and GLUT4, and that cytoskeletal proteins, notably α-actinin-4,
associate with the transporter, potentially to facilitate its routing to the plasma membrane.
Keywords: alpha-actinin 4 • L6 muscle cells • protein−protein interactions • mass spectrometry • quantitative
proteomics • insulin • GLUT4 binding proteins