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. ECEPTOR-mediated endocytosis is a process used for the efficient internalization of a number of macromolecules. Included in the list of ligands taken up by endocytosis are ligands which fulfill nutritional requirements (e.g., transferrin-mediated iron accumulation) as well as ligands responsible for transmitting signals (e.g., epidermal growth factor). The hallmark of receptor-mediated endocytosis is the internalization of these disparate receptorligand complexes through clathrin-coated pits. Only a specific subset of plasma membrane proteins are efficiently clustered in and thereby internalized through coated pits, suggesting that these proteins contain specific clustering signals. Since a variety of receptors are known to be internalized through a single-coated pit (Dickson et al., 1981; Willingham et al., 1981; Carpentier et al., 1982), it is likely that a common clustering signal exists. The sequences of a large number of receptors (>25) have been determined, but examination of the predicted amino acid sequences has not revealed a cytoplasmic sequence motif, found in all receptors, that could potentially be assigned as the clustering or internalization signal. This observation suggests that the clathrin-coated pit clustering signal is more complex than a simple linear stretch of amino acids. Furthermore, although it is compelling to consider one clathrin-coated pit internalization signal it is also possible that there are a number of "signals" that can function in this process (Verrey et al., 1990). The only known general feature for efficient internalization through coated pits is the presence of a tyrosine on the cytoplasmic domain of the receptor. The requirement for a tyrosine in maintaining efficient internalization was first shown for the human low density lipoprotein (LDL) receptor (Davis et al., 1986), and has since been extended to include the human transferrin receptor (McGraw and Maxfield, 1990; Alvarez et al., 1990; Jing et al., 1990), the polymeric immunoglobulin receptor (Breitfeld et al., 1990), and the 275-kD mannose-6-phosphate receptor (Lobel et al., 1989). The functional significance of the cytoplasmic tyrosine in clathrin-coated pit endocytosis has not been determined. It has recently been shown that amino acids near the tyrosine influence internalization of the LDL receptor (Chen et al., 1990). The sequence near the required tyrosine of the LDL receptor is asparagine-proline-valine-tyrosine. Mutagenesis of the LDL receptor, by substitutions of native residues with alanines, has shown that the asparagine and proline are required for efficient internalization whereas the 1. Abbreviations used in this paper: LDL, low density lipoprotein; S/I, surface/internal; TR, transferrin receptor. © The Rockefeller University Press, 0021-9525/91/031853/9 $2.00 The Journal of Cell Biology, Volume I 12, Number 5, March 1991 853-861 853 on A uust 0, 2017 jcb.rress.org D ow nladed fom valine is not. This amino acid sequence, NPXY, is found in the cytoplasmic domains of 13-membrane proteins and thus potentially could be a clathrin-coated pit clustering signal. However, a number of receptors that are internalized through coated pits do not contain this sequence (Chen et al., 1990). Therefore, although the presence of a cytoplasmic tyrosine appears to be a common requirement for efficient internalization, the NPXY motif cannot be the only clustering signal. The role of the single cytoplasmic tyrosine of the human transferrin receptor (TR) for efficient internalization has been studied. Efficient internalization requires an aromatic amino acid at this position, rather than there being a stringent requirement for tyrosine, because both phenylalanine and tryptophan can be substituted for tyrosine without a significant effect on internalization (McGraw and Maxfield, 1990). The TR does not contain obvious homology with the cytoplasmic domains of other receptors and specifically does not contain the NPXY sequence. To further define the requirements for efficient internalization of the human TR we have extended our in vitro mutagenesis studies of the cytoplasmic domain. In this paper we show that substitution of the phenylalanine at position 23 (see Fig. 1 a) with alanine results in a threefold reduction in the internalization constant. This effect is similar to that observed when the tyrosine at position 20 is substituted with a nonaromatic amino acid (McGraw and Maxfield, 1990). Substitution of the phenylalanine at position 13 with alanine results in a smaller effect on the internalization rate constant. These results demonstrate that aromatic amino acids, other than the tyrosine, are required for efficient internalization of the human TR. To further explore the influence of the local amino acid environment on the functioning of the tyrosine in internalization, we have replaced the native tyrosine with a cysteine and substituted a tyrosine for the native serine at position 34. Surprisingly, this double mutant is internalized as efficiently as the wild-type receptor. This result demonstrates that a second-site mutation is capable of reverting the internalization defective phenotype of the cysteine 20 TR. Unlike the requirement for an aromatic amino acid at position 20 of the TR, reversion of the internalization-defective phenotype of the cysteine 20 receptor requires a tyrosine, as a cysteine 20phenylalanine 34 double mutant is as internalization-defective as the single cysteine 20 mutation. Materials and Methods