Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4.
Several structural features of the cytoplasmic domain of CD4 including phosphorylation of Ser-408 have been shown to be important in its endocytosis (Shin, J., Doyle, C., Yang, Z., Kappes, D., and Strominger, J.L. (1990) EMBO J. 9, 425-434). A series of cytoplasmic domain truncations have now indicated that the membrane proximal region of the cytoplasmic domain from Arg-396 to Lys-417 is sufficient for phorbol ester-induced internalization; this segment is predicted to be an alpha-helix. The severe impairment of endocytosis resulting from the mutation Ser-408 to Ala-408 is largely restored by a compensating mutation Ala-404 to Ser-404; phosphorylation of Ser-404 has been directly demonstrated. Furthermore, mutation of Met-407, Ile-410, Leu-413, or Leu-414 to a hydrophilic residue eliminated CD4 endocytosis as did domain truncation at Arg-412. Ser-408 was normally phosphorylated in all of these mutants, suggesting that other residues in this region, including the four hydrophobic amino acids, are also required for CD4 endocytosis. Immunofluorescence microscopy following staining of intact and permeabilized cells showed that all endocytosis defective mutants indeed remained on the cell surface even after phorbol ester treatment, while wild type CD4 was endocytosed and degraded in lysosomes. These data indicate that endocytosis requiring residues 397-417 and binding of lymphocyte tyrosine kinase at residues 417-429 are functions of independent segments of the cytoplasmic region and lead to a hypothesis regarding some features of the endocytic process.
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
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Shin
R L
RL
Dunbrack
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Lee
J L
JL
Strominger
D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D015704:CD4 Antigens; D011505:Protein-Tyrosine Kinases