An internalization motif is created in the cytoplasmic domain of the transferrin receptor by substitution of a tyrosine at the first position of a predicted tight turn.

Receptors are internalized from the plasma membrane at approximately 10 times the rate of bulk membrane. The predominant model for the motif that promotes rapid internalization proposes a requirement for a tyrosine located in the first position of a tight turn. In this report we show that an internalization motif can be created de novo by substituting a tyrosine for the first or last residues of a tetrapeptide GDNS (residues 31-34) that is predicted to form a tight turn within the cytoplasmic domain of the human transferrin receptor. These substitutions restore wild-type levels of internalization to transferrin receptors that are poorly internalized due to missense mutations in the native internalization motif. The introduction of a tyrosine at the first or last position of the GDNS tetrapeptide in a transferrin receptor containing an unmodified wild-type internalization motif significantly increases the internalization rate above that of the wild-type receptor. Our results indicate that a functional novel internalization motif can be created by placing specific aromatic amino acids within the overall structure of an existing beta-turn in a cytoplasmic domain of a receptor.

The amino terminus of GLUT4 functions as an internalization motif but not 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 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 phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.