Analysis of trafficking motifs in the insulin-responsive glucose transporter isoform, GLUT4

The complex system that comprises the intracellular trafficking pathway of the insulin-responsive glucose transporter isoform, GLUT4, remains to be fully characterised. Many questions remain as to how the trafficking and targeting of this recycling protein occurs in the basal state and how this process is affected by treatment with the peptide hormone insulin. In this thesis I have attempted to address some of these issues. Much of the trafficking of GLUT4 is thought to be governed by targeting motifs that are present in the cytoplasmic amino- and carboxy-termini of the protein. These sequences have previously been shown to be involved in the internalisation of the protein from the cell surface in both the basal and post- insulin-stimulated states. In this thesis I have undertaken to determine whether these signal motifs can also function in the trafficking of GLUT4 to specific intracellular locations. My results suggest that indeed these motifs may function at more than one intracellular loci and that they may have more than one role in the complex pattern of GLUT4 trafficking. These results have led to the proposal of an alternative intracellular trafficking pathway for GLUT4 within 3T3-L1 adipocytes. I have also examined the role of the major phosphorylation site on the sequence of GLUT4, a serine residue at position 488, in the regulation of GLUT4 trafficking. Results suggest that the phosphorylation state of S488 is likely to play a role in the intracellular sorting of GLUT4, but is not involved in the insulin-stimulated recruitment of GLUT4 to the plasma membrane. Further studies examined the role of residues distal to the carboxy-terminal di-leucine motif in the cytoplasmic tail of GLUT4 in the targeting of this isoform in 3T3-L1 adipocytes. Such residues have previously been thought to be involved in the targeting of GLUT4 to specific intracellular compartments. Results suggest that the cytoplasmic carboxy-terminus of GLUT4 contains an additional targeting signal distal to the L489L490 motif that regulates sorting of GLUT4 from endosomes into a post-endocytic storage compartment. To complement the above targeting studies, I undertook the construction and characterisation of a series of GLUT2/GLUT4 recombinant chimeric glucose transporters in a further attempt to define the roles of the cytoplasmic signal sequences on the trafficking of GLUT4. Results here were in concert with much of the published literature but also concurred strongly with the proposed alternative trafficking pathway of GLUT4. My final study was undertaken during my period working at SmithKline Beecham as part of my GASE studentship. Analysis was performed into the expression levels of proteins known to be involved in the trafficking of GLUT4 in animal models of diabetes mellitus. This study identified selective changes in hindlimb skeletal muscle tissues from the Zucker diabetic jalfa model of non-insulin-dependent diabetes mellitus and also demonstrated further alterations in levels of expression after treatment with a thiazolidinedione compound. It was concluded that these selective changes were a result of the state of hyperinsuhnaemia that is prevalent in such animals