The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Abstract

Abstract. Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221. To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalaninebased internalization motif, rather than retention of the transporter within intracellular structures.

Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein

“…This data suggests that the major internalisation motif is present at the N-terminus of GLUT4, and that its interaction with the internalisation machinery is sterically hindered by the presence of a large bulky GFP molecule. Studies with a chimeric transferrin receptor possessing a cytoplasmic tail derived from the N-terminus of GLUT4 suggest that this domain of GLUT4 is involved in efficient internalisation and not subcellular localisation of GLUT4 in CHO cells [33]. Our data now suggest that the N-terminal domain of full-length GLUT4 possesses a critical internalisation motif.…”

Distinct signals in the GLUT4 glucose transporter for internalization and for targeting to an insulin-responsive compartment.

“…A phenylalanine residue within this region has been suggested to function in the internalization of transporters from the cell surface (16,29). The chimeric transporter 4HB1, which contains the NH2-terminal 183 amino acids of GLUT4, also showed some intracellular localization in NIH3T3 and 3T3-L1 fibroblasts (43; and Fig.…”

Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4.

“…Mutating the phenylalanine to alanine causes a marked increase in cell surface levels of GLUT-4 and a decreased association of GLUT-4 with cell surface clathrincoated pits . The GLUT-4 NH2-terminus confers intracellular targeting when substituted for the cytoplasmic tail of either the H1 subunit of the asialoglycoprotein receptor or the transferrin receptor (Garippa et al, 1994) and in both cases mutating the phenylalanine at position 5 to alanine causes accumulation of the protein at the cell surface. Kinetic analysis of the transferrin receptor/GLUT-4 hybrids in CHO cells reveals that the GLUT-4 NH, terminus regulates efficient internalization but not intracellular retention (Garippa et al, 1994).…”