Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is dependent upon both the amino terminus and the large cytoplasmic loop

We have recently reported that following initial biosynthesis, the GLUT4 protein exits the Golgi apparatus and directly enters the insulin-responsive compartment(s) without transiting the plasma membrane (Watson, R. T., Khan, A. H., Furukawa, M., Hou, J. C., Li, L., Kanzaki, M., Okada, S., Kandror, K. V., and Pessin, J. E. (2004) EMBO J. 23, 2059-2070). To investigate the structural motifs involved in these initial sorting events, we have generated a variety of loss-of-function and gain-of-function GLUT4/ GLUT1 chimera proteins. Substitution of the GLUT4 carboxyl-terminal domain with GLUT1 had no significant effect on the acquisition of insulin responsiveness. In contrast, substitution of either the GLUT4 amino-terminal domain or the large cytoplasmic loop between transmembrane domains 6 and 7 resulted in the rapid default of GLUT4 to the plasma membrane with blunted insulin response. Consistent with these findings, substitution of the amino-terminal, cytoplasmic loop, or carboxyl-terminal domains individually into GLUT1 backbone did not recapitulate normal GLUT4 trafficking. Similarly, dual substitutions of the GLUT1 amino and carboxyl termini with GLUT4 domains or the combination of the cytoplasmic loop plus the carboxyl terminus failed to display normal GLUT4 trafficking. However, the dual replacement of the amino terminus plus the cytoplasmic loop of GLUT4 in the GLUT1 backbone resulted in a complete restoration of normal GLUT4 trafficking. Alanine-scanning mutagenesis of the GLUT4 amino terminus demonstrated that Phe⁵ and Ile⁸ within the FQQI motif and, to a lesser extent, Asp¹²/Gly¹³ were necessary for the appropriate initial trafficking following biosynthesis. In addition, amino acids 229-271 in the large intracellular loop between transmembrane domains 6 and 7 functionally cooperated with the amino-terminal domain. These data demonstrate that initial trafficking of GLUT4 from the Golgi to the insulin-responsive GLUT4 compartment requires the functional interaction of two distinct domains.