THE AMINO-TERMINUS OF GLUT4 FUNCTIONS AS AN INTERNALIZATION MOTIF BUTNOT AN INTRACELLULAR RETENTION SIGNAL WHEN SUBSTITUTED FOR THE TRANSFERRIN RECEPTOR CYTOPLASMIC DOMAIN

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that d etermines its steady state distribution between the surface and the in terior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Purl, and D. E. James. 1993 . J. Cell Biol. 121:1221). To directly measure the effect that the GLU T4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence w as substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chines e hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surfa ce with a rate similar to the transferrin receptor, indicating that th e GLUT4 sequence was not promoting intracellular retention of the chim era. The GLUT4-transferrin receptor chimera was internalized at half t he rate of the transferrin receptor. Substitution of an alanine for ph enylalanine at position 5 slowed internalization of the chimera by two fold, to a level characteristic of bulk membrane internalization. Howe ver, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutio ns significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major fun ction of the GLUT4 amino-terminal domain is to promote the effective i nternalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of th e transporter within intracellular structures.