Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype.
Site-specific mutagenesis has been used to define the sequences required for efficient internalization of the human transferrin receptor. It has previously been shown that the sole cytoplasmic tyrosine, at position 20, is required for efficient internalization. When two other cytoplasmic aromatic residues, the phenylalanines at positions 13 and 23, are substituted with alanines internalization is also reduced. The phenylalanine 23 mutation decreases the internalization rate constant approximately threefold, and mutation of phenylalanine 13 decreases it by approximately twofold. The mutation at position 23 has as serious an effect on internalization as substitution with a nonaromatic amino acid for the single tyrosine. These results demonstrate the importance of several aromatic amino acids in maintaining efficient internalization of the transferrin receptor. Substitution of a tyrosine at a second site, for a serine at position 34, within the cytoplasmic domain of a transferrin receptor with a nonaromatic amino acid at position 20, results in a complete reversion of the internalization-defective phenotype. This reversion is completely dependent upon a tyrosine, as phenylalanine substituted at position 34 does not revert the internalization-defective phenotype. This result demonstrates that a tyrosine placed outside of its native context can still function in the internalization of the transferrin receptor, suggesting a flexibility in surrounding sequences required for efficient internalization.
McGraw
T E
TE
Department of Pathology, Columbia University College of Physicians and Surgeons, New York 10032.
Pytowski
B
B
Arzt
J
J
Ferrone
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010649:Phenylalanine / Q000737:chemistry*; D011990:Receptors, Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D011990:Receptors, Transferrin; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D007501:Iron