GLUT-4 NH2 TERMINUS CONTAINS A PHENYLALANINE-BASED TARGETING MOTIF THAT REGULATES INTRACELLULAR SEQUESTRATION

Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated t hat the cytoplasmic NH2 terminus of GLUT-4 contains important targetin g information that mediates intracellular sequestration of this isofor m (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies , the amino acid constituents of the GLUT-4 NH2-terminal targeting dom ain have been identified. GLUT-4 constructs containing NH2-terminal de letions or alanine substitutions within the NH2 terminus were expresse d in CHO cells using a Sindbis virus expression system. Deletion of ei ght amino acids from the GLUT-4 NH2 terminus or substituting alanine f or phenylalanine at position 5 in GLUT-4 resulted in a marked accumula tion of the transporter at the plasma membrane. Mutations at other ami no acids surrounding Phe5 also caused increased cell surface expressio n of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 wa s also localized to clathrin lattices and this colocalization was abol ished when either the first 13 amino acids were deleted or when Phe5 w as changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function indepen dently of the glucose transporter structure this domain was inserted i nto the cytoplasmic tail of the H1 subunit of the asialoglycoprotein r eceptor. H1 with the GLUT-4 NH2 terminus was predominantly localized t o an intracellular compartment similar to GLUT-4 and was sequestered m ore from the cell surface than was the wild-type H1 protein. It is con cluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic mac hinery located at the cell surface.