Date of Award
Open Access Thesis
Medical Doctor (MD)
The glucose transporter 4 (GLUT4) is the major insulin-responsive glucose transporter in adipose and muscle tissues. Although the early steps in the insulin signaling pathway governing translocation of GLUT4 to the plasma membrane are well understood, the final steps in this pathway are not. TUG is a protein which has been shown to affect trafficking of GLUT4 both in the basal state and in response to insulin. One protein-protein interaction between TUG and the large cytosolic loop of GLUT4 has previously been identified. Based on reports of the requirement of the GLUT4 N-terminal domain for its proper targeting to the plasma membrane, we postulated that an interaction might also exist between TUG and the N-terminal domain of GLUT4, and we tested this hypothesis using two sets of pull-down experiments. In the first set, using the N-terminal domain of GLUT4 fused with glutathione S-transferase (GST), we were able to pull TUG down from the lysates of TUG-transfected HEK 293 cells. TUG was also pulled down by the GLUT4 cytosolic loop and, to a much lesser extent, its C-terminal domain. However, there was no specific interaction between these fusion proteins and the lysates of cells transfected with a truncated form of TUG lacking its own N-terminal domain. In the second set of experiments, using a biotinylated synthetic GLUT4 N-terminal peptide, we pulled down a protein detected by an anti-TUG antibody and running at ~64 kDa, a slightly higher molecular weight than wild-type TUG. We believe that this band represents modified full-length TUG. This interaction was not seen using synthetic GLUT4 N-terminal peptide mutated at 4 amino acids previously identified as necessary for proper GLUT4 retention and insulin-responsive trafficking. We conclude that TUG interacts not only with the large cytosolic loop of GLUT4, but also with the N-terminal domain of GLUT4, and that this latter interaction can be disrupted by mutations in GLUT4 that cause defective trafficking, suggesting that this interaction is critical for GLUT4 intracellular retention and insulin-responsive GLUT4 trafficking.
Mansourian, Stefan V., "Interactions between the GLUT4 Glucose Transporter and Its Regulator, TUG" (2008). Yale Medicine Thesis Digital Library. 352.