Characteristics of the Tyrosine Recognition Signal for Internalization of Transmernbrane Surface Glycoproteins

A tyrosine residue in the cytoplasmic domain of a class of cell surface receptors is necessary, but not sufficient, for internalization through coated pits. To identify the amino acid context enabling a tyrosine to serve as a signal for endocytosis, we mutated the short cytoplasmic domain of a mutant influenza virus hemagglutinin that is competent for internalization, HA-Y543, and determined the effect of each change on internalization. From these results and a comparison of sequences of other proteins recognized by coated pits, a "tyrosine internalization signal" was proposed. Site-directed mutagenesis was employed to insert complete, or incomplete "tyrosine internalization signals" into the cytoplasmic domain of a protein normally not endocytosed, human glycophorin A. Only the complete signal caused internalization of mutant glycophorins by coated pits. The signal is formed by a short amino acid sequence, with polar or basic residues preferred at certain positions on either side of the tyrosine. Amino acids, which in proteins of known structure are frequently found in turns, are clustered near the tyrosine on the side of the signal nearest the transmembrane domain. T HE plasma membranes of animal cells contain small specialized domains, the clathrin-coated pits, designed for the rapid internalization of cell surface receptors. Coated pits appear to sort transmembrane surface proteins into at least three classes: those that localize with high affinity to coated pits and are internalized rapidly (Goldstein et al., 1985), those that can enter coated pits slowly and are internalized at approximately the same rate as the bulk of membrane (Roth et al., 1986), and proteins that are excluded from coated pits (Bretscher et al., 1980; Roth et al., 1986). The information necessary to specify rapid internalization through coated pits resides in the cytoplasmic domain of at least some transmembrane proteins. Deletion of the cytoplasmic domain of all receptors studied to date results in proteins that travel to the cell surface but are internalized very slowly (Lehrman et al., 1985; Prywes et al., 1986; Mostov et al., 1986; Rothenberg et al., 1987; Miettinen et al., 1989; Lobel et al., 1989). Domain switching experiments have shown that when the cytoplasmic domains of proteins with high affinity for coated pits are transferred to a protein normally excluded from coated pits, the resulting chimeric proteins are efficiently internalized. Although the cytoplasmic epitope recognized by coated pits has not been identified, several lines of evidence suggest that a relatively short stretch of amino acids containing a tyrosine is necessary and sufficient to specify internalization. Internalization of the receptors for LDL or for mannose-6-phosphate (M-6-P) is not affected by deletion of the distal half of the cytoplasmic domain (Davis et al., 1987; Lobel et al., 1989). Mutation of a single tyrosine within the stretch of cytoplasmic amino acids required for internalization of the receptors for LDL, M-6-P, or transferrin (Tf), results in receptors unable to be efficiently internalized (Davis et al., 1987; Lobel et al., 1989; Jing et al., 1990; McGraw and Maxfield, 1990). Introduction of a tyrosine residue at position 543 of the short cytoplasmic domain of the influenza virus hemagglutinin (HA)' converts HA from a protein that is ordinarily excluded from coated pits into one that enters coated pits and is rapidly internalized (Lazarovits and Roth, 1988). Significantly, introduction of tyrosines at positions 540, 545, and 546 of the HA cytoplasmic domain does not result in endocytosed proteins (Lazarovits and Roth, 1988; and this work). In addition, the LDL receptor cytoplasmic domain contains three tyrosines, but only one (at position 807) participates in signaling internalization of this protein (Davis et al., 1987). Thus, in addition to the presence of a tyrosine, other features of the amino acid sequence of the cytoplasmic domain are necessary to allow a protein to enter the endocytic pathway. To identify the amino acid context required for a tyrosine to function as an endocytosis signal, the amino acid sequence surrounding tyrosine 543 in the HA cytoplasmic domain was systematically mutated and the resulting proteins were analyzed for their ability to be internalized. From the results of these experiments, and from comparison of amino acid sequences of the region surrounding tyrosines in the cytoplasmic domains of proteins known to be internalized through coated pits, we have identified a "tyrosine'internali 1. Abbreviation used in this paper: HA, hemagglutinin. © The Rockefeller University Press, 0021-9525/90/10/1393/15 $2.00 The Journal of Cell Biology, Volume 111, October 199