YTRF IS THE CONSERVED INTERNALIZATION SIGNAL OF THE TRANSFERRIN RECEPTOR, AND A 2ND YTRF SIGNAL AT POSITION-31-34 ENHANCES ENDOCYTOSIS

By functional analysis of mutant human transferrin receptors (TR) expr essed in chicken embryo fibroblasts, we previously identified a tetrap eptide sequence, Y20TRF23, within the 61-residue cytoplasmic tail as t he signal for high-efficiency endocytosis (Collawn, J. F., Stangel, M. , Kuhn, L. A., Esekogwu, V., Jing, S., Trowbridge, I. S., and Tainer, J. A. (1990) Cell 63, 1061-1072). It has been inferred from other stud ies, however, that the TR internalization signal was localized to a mu ch larger region, residues 7 through 26 (Girones, N., Alvarez, E., Set h, A., Lin, I-M., Latour, D. A., and Davis, R. J. (1991) J. Biol. Chem . 266,19006-19012). Additionally, Tyr20 was reported to not be conserv ed in the Chinese hamster cytoplasmic tail sequence (Alvarez, E., Giro nes, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35). In the studi es reported here, we examined the effect of insertion of an extra copy of a YTRF sequence at three different locations within the human TR c ytoplasmic domain and show that the insertion of another YTRF signal a t position 31-34 in the wild-type TR, but not the other two locations, increases the rate of endocytosis 2-fold. Furthermore, introduction o f YTRF at position 31-34 in an internalization-defective mutant recept or restores endocytosis to wild-type levels, indicating that YTRF sign als at either positions 20-23 or 31-34 are necessary and sufficient to promote TR internalization and function in an independent and additiv e manner. We also report the complete primary structure of the Chinese hamster TR deduced from its cDNA sequence and show that the Tyr20 as well as the complete YTRF motif is conserved.