Title: 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. Buijs F. van Meijer M. Litvinov S.V. Hilkens J. J. Biol. Chem. 1992; 267: 6171-6177Abstract Full Text PDF PubMed Google Scholar). The resulting subunits remain tightly associated; the heterodimer is SDS-labile but is resistant to boiling, urea, sulfhydryl reduction, peroxide, high salt, or low pH (4Julian J. 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Oncogene. 2003; 22: 1324-1332Crossref PubMed Scopus (154) Google Scholar) found a tumor-specific complex between MUC1 and β-catenin in the cytoplasm and nucleus in metastatic lesions of breast cancer patients; aberrant cytoplasmic and nuclear levels of activated β-catenin in breast tumors also correlates with a poor prognosis for the patient (13Lin S-Y. Xiz W. Wang J.C. Kwong K.Y. Spohn B. Wen Y. Pestell R.G. Hung M.-C Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4262-4266Crossref PubMed Scopus (689) Google Scholar). β-Catenin binding to the cytoplasmic tail of MUC1 at the SXXXXXSSLS59 motif is differentially modulated by phosphorylation at several adjacent sites by Src-family kinases, the epidermal growth factor receptor (EGFR), 1The abbreviations used are: EGFR, epidermal growth factor receptor; CHO, Chinese hamster ovary; AP-2, adapter protein complex 2; MESNA, 2-mercaptoethanesulfonic acid sodium salt; HRP, horseradish peroxidase; WT, wild-type; EGF, epidermal growth factor; SH, Src homology. protein kinase Cδ, and glycogen synthase kinase 3β (14Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar, 15Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Li Y. Ren J. Yu W. Li Q. Kuwahara H. Yin L. Carraway K.L.I. Kufe D. J. Biol. Chem. 2001; 276: 35239-35242Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Ren J. Li Y. Kufe D. J. Biol. Chem. 2002; 277: 17616-17622Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 18Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 37-43Crossref Scopus (70) Google Scholar). Although trafficking of the MUC1 cytoplasmic tail and β-catenin (or γ-catenin) to the nucleus is clearly stimulated by either ligand binding to members of the EGF receptor family or stimulation of Src-family kinases, the mechanism for this trafficking is not clear (18Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 37-43Crossref Scopus (70) Google Scholar, 19Li Y. Yu W. Ren J. Chen W. Huang L. Kharbanda S. Loda M. Kufe D. Mol. Cancer Res. 2003; 1: 765-775PubMed Google Scholar, 20Wen Y. Caffrey T.C. Wheelock M.J. Johnson K.R. Hollingsworth M.A. J. Biol. Chem. 2003; 278: 38029-38039Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The large subunit of MUC1 is not found with β-catenin in the nucleus, but immunohistochemical staining with subunit-specific antibodies indicates that both MUC1 subunits are present in the cytoplasm of breast carcinomas (18Li Y. Chen W. Ren J. Yu W. Li Q. Yoshida K. Kufe D. Cancer Biol. Ther. 2003; 2: 37-43Crossref Scopus (70) Google Scholar, 20Wen Y. Caffrey T.C. Wheelock M.J. Johnson K.R. Hollingsworth M.A. J. Biol. Chem. 2003; 278: 38029-38039Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21Croce M.V. Isla-Larrain M.T. Rua C.E. Rabassa M.E. Gendler S.J. Segal-Eiras A. J. Histochem. Cytochem. 2003; 51: 781-788Crossref PubMed Scopus (48) Google Scholar). Thus, it is quite likely that trafficking of the MUC1/β-catenin complex to the nucleus involves endocytosis of MUC1 from the cell surface as a first step. MUC1 glycosylation is also altered in breast tumor cells (22Hull S.R. Bright A. Carraway K.L. Abe M. Hayes D.F. Kufe D.W. Cancer Commun. 1989; 1: 261-267PubMed Google Scholar, 23Brockhausen I. Yang J-M. Burchell J. Whitehouse C. Taylor-Papadimitriou J Eur. J. Biochem. 1995; 233: 607-617Crossref PubMed Scopus (311) Google Scholar, 24Whitehouse C. Burchell J. Gschmeissner S. Brockhausen I. Lloyd K.O. Taylor-Papadimitriou J. J. Cell Biol. 1997; 137: 1229-1241Crossref PubMed Scopus (94) Google Scholar, 25Dalziel M. Whitehouse C. McFarlane I. Brockhausen I. Gschmeissner S. Schwientek T. Clausen H. Burchell J. Taylor-Papadimitriou J. J. Biol. Chem. 2001; 276: 11007-11015Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 26Muller S. Hanisch F-G. J. Biol. Chem. 2002; 277: 26103-26112Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and our previous studies indicate that changes in MUC1 glycosylation can alter its membrane trafficking. We found that delivery of MUC1 to the cell surface in Chinese hamster ovary (CHO) cells is absolutely dependent on the addition of O-linked glycans to its mucin-like core of tandem repeats (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). In glycosylation-defective CHO cells, only half as much MUC1 was delivered to the cell surface when it was synthesized with shorter glycans (NeuAcGalNAc-) compared with MUC1 with normal glycans (NeuAcGal(NeuAc)GalNAc-), and MUC1 with shorter O-glycans was internalized by clathrin-mediated endocytosis at twice the rate of MUC1 with normal glycans (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). Thus, changes in MUC1 glycosylation can clearly alter its membrane trafficking and potentially its steady state subcellular localization. We have now carried out experiments to better understand how MUC1 clathrin-mediated endocytosis is regulated independent of its heavily glycosylated ectodomain. Many transmembrane proteins at the plasma membrane use cytoplasmic internalization signals such as YXXϕ (where X is any amino acid and ϕ is a bulky hydrophobic residue) or [DE]XXXL[LI] dileucine motifs to bind the well characterized adaptor protein complex AP-2 (28Boehm M. Bonifacino J.S. Mol. Biol. Cell. 2001; 12: 2907-2920Crossref PubMed Scopus (366) Google Scholar, 29Boehm M. Bonifacino J.S. Gene. 2002; 286: 175-186Crossref PubMed Scopus (118) Google Scholar). AP-2 is a cytosolic heterotetramer with additional binding sites for phosphatidylinositol 4,5-bisphosphate, clathrin heavy chain, and a wide range of other adaptor proteins, including the autosomal recessive hypercholesterolemia protein, β-arrestin, and epsin family members (for a review, see Refs. 30Traub L.M. J. Cell Biol. 2003; 163: 203-208Crossref PubMed Scopus (263) Google Scholar and 31Sorkin A. Curr. Opin. Cell Biol. 2004; 16: 392-399Crossref PubMed Scopus (168) Google Scholar). In turn, the autosomal recessive hypercholesterolemia protein recognizes tyrosine-phosphorylated FXNPXY motifs, β-arrestin recognizes phosphorylated G protein-coupled receptors, and the epsin family members recognize ubiquitinylated proteins through their ubiquitin interaction motifs (30Traub L.M. J. Cell Biol. 2003; 163: 203-208Crossref PubMed Scopus (263) Google Scholar, 31Sorkin A. Curr. Opin. Cell Biol. 2004; 16: 392-399Crossref PubMed Scopus (168) Google Scholar). Together these proteins function at the plasma membrane to concentrate cargo in clathrin-coated pits and initiate their invagination. To focus on identification of endocytosis signals in the cytoplasmic domain of MUC1, we replaced its ectodomain with that of Tac (interleukin 2 receptor α-subunit), a protein that has been used previously in chimeric constructs to analyze carboxyl-terminal cytoplasmic targeting signals (32Marks M.S. Roche P.A. van Donselaar E. Woodruff L. Peters P.J. Bonifacino J.S. J. Cell Biol. 1995; 131: 351-369Crossref PubMed Scopus (178) Google Scholar). Our initial analysis of cytoplasmic domain truncation-mutants produced complex results. Our subsequent focus on truncations that inhibited MUC1 endocytosis and tyrosine mutants revealed that MUC1 internalization is dependent on at least two tyrosine residues: one within a previously described binding site for the adaptor protein Grb2, and one within a binding site for the adaptor protein complex AP-2. Recombinant cDNAs and Transfected Cells—The generation of clonal CHO cells expressing human MUC1 with 22 tandem repeats was described previously (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). The cDNA for full-length Tac (interleukin 2 receptor α-subunit) was a gift from Michael S. Marks (University of Pennsylvania, Philadelphia, PA) (32Marks M.S. Roche P.A. van Donselaar E. Woodruff L. Peters P.J. Bonifacino J.S. J. Cell Biol. 1995; 131: 351-369Crossref PubMed Scopus (178) Google Scholar). Nucleotide residues encoding the amino-terminal Tac ectodomain (239 amino acids) and both the carboxyl-terminal human MUC1 transmembrane (23 amino acids) and cytoplasmic (72 amino acids) domains were amplified using PCR and Pfu DNA polymerase (Stratagene, La Jolla, CA). Primers for PCR were designed to include nucleotide restriction sites for ligation of Tac and MUC1 cDNAs to form the Tac-MUC1 chimera shown in Fig. 1. Mutation of tyrosine residues or placement of stop codons within the Tac-MUC1 cytoplasmic tail was carried out by PCR-based, site-directed mutagenesis using primers with specific nucleotide changes. Clonal lines of CHO cells stably transfected with either the Tac-MUC1 chimera or Tac-MUC1 mutants in pCDNA3(neo) (Invitrogen) were selected by growth in G-418 (0.5 mg/ml). Cells were cultured as described previously (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). Endocytosis Assay—The protocol used to measure endocytosis of MUC1 in CHO cells was previously published (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). In brief, cells were metabolically labeled with [35S]Met/Cys for 30 min and chased in media containing Met/Cys for 90 min before cell surface biotinylation on ice with sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate. Cells were moved to 37 °C for the indicated times before stripping the cell surface biotin with the membrane-impermeant reducing agent MESNA on ice. Biotinylated MUC1 or Tac-MUC1 was recovered from immunoprecipitates using avidin-conjugated beads before SDS-PAGE and quantifying 35S-labeled bands using the BioRad Personal Molecular Imager FX (Bio-Rad) and Quantity One software. The fraction internalized at each time point was calculated after subtraction of background at time zero by using total biotinylated 35S-labeled MUC1 or chimera (without MESNA stripping) as 100%. At least two clonal cell lines expressing either MUC1 with 22 tandem repeats or Tac-MUC1 chimeras were analyzed multiple times as indicated in the figure legends. The mean and S.E.M. for data from each construct are presented in the figures, and the statistical significance of the differences between data obtained with the wild-type Tac-MUC1 and the mutants was calculated using an unpaired Student's t test based on equal variance. Equal variance was determined using Stata Statistical Software (ver. 7.0; Stata Corp., College Station, TX). Antibodies and Immunoblotting—Mouse monoclonal antibody against MUC1 (VU-3-C6) prepared by Jo Hilgers (Free University, Amsterdam, The Netherlands) (33Rye P.D. Price M.R. Hilgers J. Nustad K. Tumor Biology. 1998; 19: 1-151Crossref PubMed Scopus (16) Google Scholar) was obtained from Olivera Finn (University of Pittsburgh, Pittsburgh, PA); Armenian hamster monoclonal antibody against a peptide representing the carboxyl-terminal 17 amino acids of MUC1 (CT2) was obtained from Sandra Gendler (Mayo Clinic, Scottsdale, AZ) (34Schroeder J.A. Thompson M.C. Gardner M.M. Gendler S.J. J. Biol. Chem. 2001; 276: 13057-13064Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar); and mouse monoclonal antibody against Tac (human CD25, clone 7G7B6) was purchased from Ancell Corporation (Bayport, MN). Mouse monoclonal antibody against the AP-2 α subunit (AP.6) was purchased from Affinity BioReagents (Golden, CO), and the rabbit polyclonal antibody (R11–29) against a peptide representing the amino-terminal sequence of the μ2 subunit prepared by Juan Bonifacino (35Aguilar R.C. Ohno H. Roche K.W. Bonifacino J.S. J. Biol. Chem. 1997; 272: 27160-27166Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was obtained from Linton Traub (University of Pittsburgh). Mouse anti-Grb2 monoclonal antibody was from Research Diagnostics, Inc. (Flanders, NJ). Horseradish peroxidase (HRP)-conjugated anti-Armenian hamster antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and HRP-conjugated goat anti-rabbit antibodies were from Kirkegaard and Perry Laboratories (Gaithersburg, MD). AP-2 was immunoprecipitated from detergent extracts of CHO cells using the anti-AP-2 α subunit mouse monoclonal antibody before immunoblotting with an Armenian hamster anti-MUC1 cytoplasmic tail antibody (CT2) and HRP-conjugated second antibody using our published protocol (36Poland P.A. Kinlough C.L. Rokaw M.D. Magarian-Blander J. Finn O.J. Hughey R.P. Glycoconjugate J. 1997; 14: 89-96Crossref PubMed Scopus (17) Google Scholar). Phosphatase inhibitors (mixture set II) and protease inhibitors (mixture set III) were added to the detergent solution as directed by the manufacturer (Calbiochem). The blots were subsequently stripped by incubation in 0.1 m glycine, pH 2.3, for 30 min at room temperature before reblocking and immunoblotting with a rabbit anti-AP-2 μ2 subunit rabbit polyclonal antibody and HRP-conjugated second antibodies. Grb2 was immunoprecipitated from detergent extracts of CHO cells after 3–4 h in serum-free media followed by 20 min with 25 ng/ml betacellulin (R&D Systems, Minneapolis, MN). Grb2 immunoprecipitates were immunoblotted with an Armenian hamster anti-MUC1 cytoplasmic tail antibody (CT2) followed by HRP-conjugated second antibody using our published protocol (36Poland P.A. Kinlough C.L. Rokaw M.D. Magarian-Blander J. Finn O.J. Hughey R.P. Glycoconjugate J. 1997; 14: 89-96Crossref PubMed Scopus (17) Google Scholar). Phosphatase inhibitors (mixture sets I and II) and protease inhibitors (mixture set III) were added to the detergent solution as directed by the manufacturer (Calbiochem). Tac-MUC1 expressed in each cell line (1%) was immunoprecipitated as a control with mouse anti-Tac antibody and blotted with hamster CT2 antibody. Bands on film were analyzed with a Microtek 8700 scanner and Bio-Rad Quantity One software for Fig. 5. Bands in Fig. 6 were directly quantified with a Bio-Rad Versadoc and Quantity One software.Fig. 6Mutation of Tyr60 inhibits binding of Grb2. A, Grb2 was immunoprecipitated (IP) from detergent extracts of CHO cells stably expressing either Tac-MUC1 (WT) or Tac-MUC1 with either the Tyr20 or Tyr60 mutated, using the anti-Grb2 mouse monoclonal antibody before immunoblotting (IB) with an Armenia hamster anti-MUC1 cytoplasmic tail antibody (CT2). B, detergent cell extract was incubated with anti-Tac monoclonal antibodies as a control to compare expression levels. The ratio of chimera in the co-IP (A) compared with that in the total IP (B) is expressed as a ratio (C). The immunoblots and data are representative of two separate experiments. Note that two different clones of the Tyr60 mutant (Y60a and Y60b) expressing very different levels of chimera gave similar ratios of co-IP/total IP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results from our previous studies indicated that MUC1 endocytosis was enhanced 2-fold when it was modified with truncated rather than full-length O-glycans in a glycosylation-defective CHO cell line (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar). Because this effect on MUC1 clathrin-mediated endocytosis was most probably caused by a decrease in either steric hindrance or interaction of the extended mucin-like ectodomain with other proteins, a chimera was prepared with the ectodomain of Tac, a cell surface type 1 transmembrane glycoprotein that has been used previously in chimeric constructs to analyze carboxyl-terminal cytoplasmic targeting signals (see Tac-MUC1 chimera design in Fig. 1) (32Marks M.S. Roche P.A. van Donselaar E. Woodruff L. Peters P.J. Bonifacino J.S. J. Cell Biol. 1995; 131: 351-369Crossref PubMed Scopus (178) Google Scholar). Replacement of the MUC1 mucin-like ectodomain of 819 amino acids with the Tac ectodomain of 239 amino acids dramatically altered the level of endocytosis (see representative profiles and data in Fig. 2, A–C). Comparison of data from multiple experiments, using levels of 35S-labeled MUC1 or chimera internalized after 10 min, indicates that this 2.7-fold increase in endocytosis that we observe in Fig. 2D, as a result of the exchange of the ectodomains, is statistically significant (p < 0.001). Thus, the mucin-like ectodomain of MUC1 severely inhibits internalization of this cell surface molecule. Because our previous data indicated that MUC1 internalization proceeds by clathrin-mediated endocytosis (27Altschuler Y. Kinlough C.L. Poland P.A. Bruns J.B. Apodaca G. Weisz O.A. Hughey R.P. Mol. Biol. Cell. 2000; 11: 819-831Crossref PubMed Scopus (148) Google Scholar), we predicted that truncation of the MUC1 cytoplasmic tail would block internalization. Binding sites for clathrin adaptor complexes are consistently found in the cytoplasmic domain of cell surface proteins. As shown in Fig. 3, removal of all but 10 amino acids in the cytoplasmic tail of Tac-MUC1 slowed endocytosis by ∼40%. Comparison of data from multiple experiments indicated that this difference in endocytosis caused by the truncation of the cytoplasmic tail is statistically significant (compare Tac-MUC1 mutant X10 to Tac-MUC1 with a full-length tail, WT 72, p < 0.01). It is interesting that when endocytosis of WT 72 was compared with other truncation mutants, the results were consistent with the presence of multiple signals for endocytosis within the MUC1 cytoplasmic tail. For example, removal of 13 amino acids (mutant X59) also inhibited Tac-MUC1 endocytosis by ∼40% (p < 0.05), but removal of 23 amino acids (mutant X49) did not alter endocytosis, and removal of 34 amino acids (mutant X38) or 47 amino acids (mutant X25) actually stimulated endocytosis (p < 0.01). Truncation of cytoplasmic domains can reveal cryptic endocytosis signals that are not functional in the full-length protein (37Jiang X. Huang F. Marusyk A. Sorkin A. Mol. Biol. Cell. 2003; 14: 858-870Crossref PubMed Scopus (255) Google Scholar), so the stimulation of endocytosis observed for mutants X38 and X25 is likely to be irrelevant. Because endocytosis was inhibited when residues 11 to 25 or residues 60 to 72 were removed, this is consistent with binding of adaptor proteins within these domains. The MUC1 cytosolic tail contains seven tyrosine residues. Sequences around three of the tyrosines in the MUC1 tail (Y8GQL, Y20HPM, and Y46EKV) are consistent with the unpublished specificity of binding to the μ2 subunit of AP-2. Because only the tyrosine residue within the YXXϕ motif is absolutely essential for μ2 binding, residues Tyr8, Tyr20, and Tyr46 were mutated individually within the context of the full-length Tac-MUC1 chimera tail and stably expressed in CHO cells. No YXXϕ, NPXY, or dileucine endocytosis motifs are present within the domain between residues 60 and 72, but a tyrosine-phosphorylated motif (pY60TNP) in this domain has been shown previously to be a binding site for the SH2 domain of the adaptor protein Grb2 (38Pandey P. Kharbanda S. Kufe D. Cancer Res. 1995; 55: 4000-4003PubMed Google Scholar). Therefore, full-length Tac-MUC1 chimera with the Y60N mutation was also stably expressed in CHO cells. As shown in Fig. 4, the Y20N mutation reduced Tac-MUC1 endocytosis by 30% (p < 0.05), consistent with the presence of a YXXϕ-type endocytosis motif (Y20HPM) in the domain between residues 11 and 25, whereas mutation of neither Tyr8 or Tyr46 had no significant affect on chimera endocytosis. However, the Y60N mutation inhibited chimera endocytosis by 50% (p < 0.01), consistent with a role for Grb2 binding in MUC1 internalization. Because neither the Y20N nor the Y60N mutation alone fully blocked Tac-MUC1 endocytosis, both mutations were simultaneously introduced into the full-length Tac-MUC1. As shown in Fig. 4, the Y20,60N double mutation reduced MUC1 endocytosis by 77% (p < 0.001) compared with WT Tac-MUC1. To determine whether Y20HPM represents a binding site for AP-2, the adaptor protein complex was immunoprecipitated with anti-AP-2 α subunit antibodies and immunoblotted for both the Tac-MUC1 WT chimera and the μ2 subunit of AP-2 (Fig. 5, A and D). Although similar levels of the μ2 subunit appeared in all anti-α subunit immunoprecipitates, as unexpected, levels of the mutant chimera in the coimmunoprecipitates were decreased compared with the WT chimera. When normalized against the total level of chimera expressed in each cell line, the ratio of chimera associated with AP-2 in two different clonal cell lines expressing the Y20N mutant was decreased by 75% compared with the WT chimera (Fig. 5, B and C). Thus, Tac-MUC1 interaction with the AP-2 adaptor is mediated by the Y20HPM motif. It is interesting that the Y60N mutation reduced chimera co-immunoprecipitation by 33% compared with the WT chimera consistent with the decreased level of endocytosis observed for this mutant in Fig. 4. This indicates that AP-2 can still interact with the chimera even in the absence of Grb2 binding, but Grb2 binding apparently enhances the association of MUC1 with AP-2 by an unknown mechanism. To determine whether Grb2 was binding to the MUC1 cytoplasmic tail when Tac-MUC1 was expressed in CHO cells, Grb2 was immunoprecipitated from CHO cells expressing WT or mutant Tac-MUC1 using anti-Grb2 antibodies and immunoblotted for Tac-MUC1 (Fig. 6). When normalized against the total level of chimera expressed in each cell line, it is clear that the Y60N mutation blocks 80–85% of the interaction of Tac-MUC1 with Grb2. It is interesting that the Y20N mutation that blocked μ2 binding by 75% also blocked Grb2 binding to the same extent, indicating that Grb2 binding to the MUC1 cytoplasmic tail may require prior interaction with AP-2. MUC1 interaction with members of the EGF receptor family is dependent on the MUC1 cytoplasmic tail that is phosphorylated at several residues and provides docking sites for numerous proteins such as glycogen synthase kinase 3β, Src-family kinases, β- or γ-catenin, and Grb2 (14Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar, 15Li Y. Kuwahara H. Ren J. Wen G. Kufe D. J. Biol. Chem. 2001; 276: 6061-6064Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Li Y. Ren J. Yu W. Li Q. Kuwahara H. Yin L. Carraway K.L.I. Kufe D. J. Biol. Chem. 2001; 276: 35239-35242Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Ren J. Li Y. Kufe D. J. Biol. Chem. 2002; 277: 17616-17622Abstract Full Text Full Text PDF