Characterization of Trafficking Regulator of GLUT4-1 (TRARG1)

Abstract

Insulin resistance is a precursor of most metabolic diseases including Type 2 diabetes, and is largely recapitulated by impaired insulin-stimulated glucose transport mediated by the glucose transporter, GLUT4. However, understanding precisely how insulin orchestrates regulation of GLUT4 traffic at the various transport steps, has been a longstanding puzzle. GLUT4 storage vesicles (GSVs) are specialized intracellular storage compartment of GLUT4 and translocate to the plasma membrane (PM) in response to insulin stimulation. In pursuit of novel proteins regulating GLUT4 traffic, our laboratory performed proteomic studies of GSVs purified from adipocytes and uncovered TRARG1 as a novel positive regulator of insulin-stimulated GLUT4 trafficking. To elucidate the mechanism(s) by which TRARG1 functions, I began with demonstrating that TRARG1 contains a single transmembrane domain (TMD) and one re-entrant loop, with a cytosolic N-terminus, contrary to the consensus predicted topology. Mass spectrometry (MS) analysis revealed a range of post-translational modifications (PTMs) on TRARG1, including phosphorylation. Examination of MS analysis of insulin-regulated protein phosphorylation in 3T3-L1 adipocytes and biochemical validation suggested that TRARG1 dephosphorylation by insulin is PI3K/Akt-dependent. Furthermore, TRARG1 is a novel substrate of the protein kinase GSK3. TRARG1 protein-protein interaction analysis revealed that insulin-mediated TRARG1 dephosphorylation regulates its binding to a range of proteins including Bcl9l. These findings have elucidated novel functions of TRARG1 in that it is predicted to function at multiple steps along the GLUT4 trafficking pathway as well as other functions thought to interact with GLUT4 trafficking. These latter include the Wnt-β-catenin signalling pathway which may regulate insulin sensitivity. My studies have revealed many novel functions of TRARG1 and highlighted this to be a key mediator of GLUT4 trafficking in fat cells.