MUC1 Membrane Trafficking Is Modulated by Multiple Interactions

Abstract

MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [35S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr20 and Tyr60 were both required for efficient endocytosis. Mutation of Tyr20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y20HPM is recognized as a YXXϕ motif by the μ2 subunit. The tyrosine-phosphorylated Y60TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr60 blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1. MUC1 is a mucin-like transmembrane protein found on the apical surface of many epithelia. Because aberrant intracellular localization of MUC1 in tumor cells correlates with an aggressive tumor and a poor prognosis for the patient, experiments were designed to characterize the features that modulate MUC1 membrane trafficking. By following [35S]Met/Cys-labeled MUC1 in glycosylation-defective Chinese hamster ovary cells, we found previously that truncation of O-glycans on MUC1 inhibited its surface expression and stimulated its internalization by clathrin-mediated endocytosis. To identify signals for MUC1 internalization that are independent of its glycosylation state, the ectodomain of MUC1 was replaced with that of Tac, and chimera endocytosis was measured by the same protocol. Endocytosis of the chimera was significantly faster than for MUC1, indicating that features of the highly extended ectodomain inhibit MUC1 internalization. Analysis of truncation mutants and tyrosine mutants showed that Tyr20 and Tyr60 were both required for efficient endocytosis. Mutation of Tyr20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y20HPM is recognized as a YXXϕ motif by the μ2 subunit. The tyrosine-phosphorylated Y60TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr60 blocked coimmunoprecipitation of the chimera with Grb2. This is the first indication that Grb2 plays a significant role in the endocytosis of MUC1. MUC1 membrane trafficking is modulated by multiple interactions. Vol. 279 (2004) 53071-53077Journal of Biological ChemistryVol. 280Issue 31PreviewPage 53071, in the Introduction: The second sentence of the second paragraph should read as follows: “Schroeder et al. (12) found a tumor-specific complex between MUC1 and β-catenin in the cytoplasm and membrane of infiltrating ductal breast carcinoma and lymph node metastases; aberrant cytoplasmic and nuclear levels of activated β-catenin in breast tumors also correlates with a poor prognosis for the patient (13).” Full-Text PDF Open Access MUC1 is a mucin-like type 1 transmembrane protein normally expressed on the apical surface of epithelial cells (for review, see Refs. 1Hanisch F-G. Muller S. Glycobiology. 2000; 10: 439-449Crossref PubMed Scopus (237) Google Scholar and 2Gendler S.J. J. Mammary Gland. Biol. Neoplasia. 2001; 6: 339-353Crossref PubMed Scopus (469) Google Scholar). It is synthesized as a single propeptide and cleaved while in the endoplasmic reticulum to yield the large amino-terminal subunit containing O-glycosylated near-perfect tandem repeats, and the smaller carboxyl-terminal subunit containing the membrane anchor and cytoplasmic tail (3Ligtenberg M.J.L. Kruijshaar L. 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Grb2 was from of cells or with the Tyr20 or Tyr60 the with an hamster cytoplasmic tail was with as a to expression The of chimera in the with that in the is expressed as a The and are of that of the Tyr60 and levels of chimera of from that MUC1 endocytosis was it was with than O-glycans in a glycosylation-defective Y. G. Mol. Biol. Cell. 2000; PubMed Scopus Google Scholar). Because on MUC1 clathrin-mediated endocytosis was by a in or of the extended mucin-like ectodomain with a chimera was with the ectodomain of Tac, a surface type 1 transmembrane that used previously in to carboxyl-terminal cytoplasmic signals chimera in van E. L. J.S. J. 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Biol. Cell. 2003; PubMed Scopus Google the of endocytosis for mutants and is to Because endocytosis was inhibited to or to were is with binding of The MUC1 tail tyrosine of the in the MUC1 tail and are with the of binding to the μ2 subunit of Because the tyrosine the YXXϕ motif is for μ2 binding, and were the of the chimera tail and expressed in or endocytosis are the between and but a tyrosine-phosphorylated motif in previously to a binding site for the SH2 of the protein Grb2 S. Kufe D. Cancer Res. 1995; Google Scholar). chimera with the mutation was also expressed in in the mutation endocytosis by with the of a endocytosis motif in the between and mutation of or significant on chimera endocytosis. However, the mutation inhibited chimera endocytosis by with a role for Grb2 binding in MUC1 internalization. Because the the mutation blocked both were the in the mutation MUC1 endocytosis by with To Y20HPM a binding site for AP-2, the protein complex was with subunit and for both the chimera and the μ2 subunit of and levels of the μ2 subunit in all subunit as levels of the chimera in the were with the the of chimera expressed in the of chimera associated with in the was by with the chimera and with the is by the Y20HPM It is that the mutation chimera by with the chimera with the of endocytosis for in This indicates that with the chimera in the of Grb2 binding, but Grb2 binding the of MUC1 with by an To Grb2 was binding to the MUC1 cytoplasmic tail was expressed in cells, Grb2 was from cells or and for the of chimera expressed in it is that the mutation of the of with Grb2. 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In we that of the highly extended ectodomain of MUC1 with that of stimulated that the ectodomain with clathrin-mediated endocytosis of MUC1. also identified of the complex cytoplasmic tail of MUC1 that are required for its efficient endocytosis. is the between MUC1 and the Grb2 binding to MUC1 plays a role in of MUC1 The role for Grb2 is in the of to MUC1, and experiments and for and for for with the of the for and the and for