Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M409360200 on October 7, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53071-53077, December 17, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
279/51/53071    most recent
M409360200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kinlough, C. L.
Right arrow Articles by Hughey, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kinlough, C. L.
Right arrow Articles by Hughey, R. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

MUC1 Membrane Trafficking Is Modulated by Multiple Interactions*

Carol L. Kinlough, Paul A. Poland, James B. Bruns, Keri L. Harkleroad, and Rebecca P. Hughey{ddagger}

From the Laboratory of Epithelial Cell Biology, Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, August 16, 2004 , and in revised form, September 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 YXX{phi} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
MUC1 is a mucin-like type 1 transmembrane protein normally expressed on the apical surface of epithelial cells (for review, see Refs. 1 and 2). 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 (3). 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 (4). In carcinomas, cells often lose polarity, and MUC1 is found on all surfaces of the plasma membrane. In general, MUC1 overexpression in tumors from breast, lung, kidney, and thyroid correlates with an aggressive tumor and increased metastasis (5–11). However, a recent immunohistological study of 71 breast carcinomas, coupled with a review of the literature, indicates that it is actually the aberrant localization of intracellular MUC1 or MUC1 in a non-apical pattern that is associated with a worse prognosis for the patient (11).

The reason for the intracellular (cytoplasmic) MUC1 staining in breast carcinomas is not clear. Schroeder et al. (12) found a tumor-specific complex between MUC1 and {beta}-catenin in the cytoplasm and nucleus in metastatic lesions of breast cancer patients; aberrant cytoplasmic and nuclear levels of activated {beta}-catenin in breast tumors also correlates with a poor prognosis for the patient (13). {beta}-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),1 protein kinase C{delta}, and glycogen synthase kinase 3{beta} (14–18). Although trafficking of the MUC1 cytoplasmic tail and {beta}-catenin (or {gamma}-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 (18–20). The large subunit of MUC1 is not found with {beta}-catenin in the nucleus, but immunohistochemical staining with subunit-specific antibodies indicates that both MUC1 subunits are present in the cytoplasm of breast carcinomas (18, 20, 21). Thus, it is quite likely that trafficking of the MUC1/{beta}-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 (22–26), 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 (27). 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 (27). 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 YXX{phi} (where X is any amino acid and {phi} is a bulky hydrophobic residue) or [DE]XXXL[LI] dileucine motifs to bind the well characterized adaptor protein complex AP-2 (28, 29). 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, {beta}-arrestin, and epsin family members (for a review, see Refs. 30 and 31). In turn, the autosomal recessive hypercholesterolemia protein recognizes tyrosine-phosphorylated FXNPXY motifs, {beta}-arrestin recognizes phosphorylated G protein-coupled receptors, and the epsin family members recognize ubiquitinylated proteins through their ubiquitin interaction motifs (30, 31). 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 {alpha}-subunit), a protein that has been used previously in chimeric constructs to analyze carboxyl-terminal cytoplasmic targeting signals (32). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant cDNAs and Transfected Cells—The generation of clonal CHO cells expressing human MUC1 with 22 tandem repeats was described previously (27). The cDNA for full-length Tac (interleukin 2 receptor {alpha}-subunit) was a gift from Michael S. Marks (University of Pennsylvania, Philadelphia, PA) (32). 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 (27).



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 1.
Structure of the Tac-MUC1 chimera. The amino-terminal ectodomain of Tac was fused to the carboxyl-terminal transmembrane (TM) domain and cytoplasmic tail (CT) of MUC1 as indicated. The amino acid sequence of the cytoplasmic tail is presented in single letter code. The full-length tail of 72 amino acids (WT 72), the truncation mutations within the tail (X10, X25, X38, X49, and X59), and tyrosine residues mutated in the context of the full-length tail (Y8, Y20, Y46, and Y60) are underlined.

 
Endocytosis Assay—The protocol used to measure endocytosis of MUC1 in CHO cells was previously published (27). 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) (33) 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) (34); and mouse monoclonal antibody against Tac (human CD25, clone 7G7B6) was purchased from Ancell Corporation (Bayport, MN). Mouse monoclonal antibody against the AP-2 {alpha} 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 (35) 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 {alpha} 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 (36). 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 (36). 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.



View larger version (62K):
[in this window]
[in a new window]
  		FIG. 5.
Mutation of Tyr20 inhibits binding of the AP-2 adaptor complex. A, AP-2 complexes were 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-AP-2 {alpha} subunit mouse monoclonal antibody before immunoblotting (IB) with an Armenian hamster anti-MUC1 cytoplasmic tail antibody (CT2). The blots were subsequently stripped and reprobed with a rabbit anti-AP-2 µ2 subunit rabbit polyclonal antibody (D). Detergent cell extract was incubated with anti-Tac monoclonal antibodies as a control to compare expression levels (B). 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 three separate experiments. Note that two different clones of the Tyr20 mutant (Y20a and Y20b) expressing very different levels of chimera gave the same ratio of co-IP/total IP.

 



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 6.
Mutation 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.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (27). 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) (32). 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.



View larger version (60K):
[in this window]
[in a new window]
  		FIG. 2.
MUC1 endocytosis is affected by features in the ectodomain. The internalization of 35S-labeled MUC1 (A) or Tac-MUC1 chimera (B) from the surface of stably transfected CHO cells was followed for the indicated times as described under "Experimental Procedures." C, triplicate samples on SDS-gels were analyzed with the BioRad Personal Molecular Imager FX, and the mean (± S.D.) levels internalized at each time point are plotted for this representative experiment; the percentage of 35S-labeled MUC1 (6.9%) or Tac-MUC1 (15.8%) internalized at 10 min was calculated from the total immunoprecipitate (Total) after subtracting the background at time zero. D, the replacement of the MUC1 ectodomain with Tac significantly increased endocytosis when data were analyzed from multiple experiments. *, p < 0.0001.

 
Because our previous data indicated that MUC1 internalization proceeds by clathrin-mediated endocytosis (27), 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 (37), 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.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 3.
Tac-MUC1 endocytosis is dependent on two domains in the cytoplasmic tail. The endocytosis of Tac-MUC1 chimera with either the full-length (WT 72) or a truncated tail (X10, X25, X38, X49, or X59) were studied in stably transfected CHO cells using the assay described under "Experimental Procedures." Experiments were carried out multiple times (n = 14 for X10, n = 9 for X25, n = 8 for X38, n = 5 for X49, n = 7 for X59, and n = 34 for WT 72) with at least two different clonal cell lines in each case. Percent internalized after 10 min is presented as mean and S.E. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.01) between endocytosis of mutants and WT 72.

 
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 YXX{phi} 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 YXX{phi}, 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 (38). 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 YXX{phi}-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.



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 4.
Two tyrosine motifs are required for efficient endocytosis of Tac-MUC1. The endocytosis of Tac-MUC1 chimeras with the full-length tail (WT 72) or with mutated tyrosine residues in the full-length tail (Y8, Y20, Y46, Y60, or Y20,60) was measured in stably transfected CHO cells using the assay described under "Experimental Procedures." Experiments were carried out multiple times (n = 4 for Y8N, n = 11 for Y20N, n = 5 for Y46A, n = 12 for Y60N, n = 12 for the double mutant Y20,60N, and n = 34 for WT) with at least two different clonal cell lines in each case. Percentage internalized after 10 min is presented as mean ± S.E. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.01; ***, p < 0.001) between endocytosis of mutants and WT 72.

 
To determine whether Y20HPM represents a binding site for AP-2, the adaptor protein complex was immunoprecipitated with anti-AP-2 {alpha} 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-{alpha} 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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 3{beta}, Src-family kinases, {beta}- or {gamma}-catenin, and Grb2 (14–19, 34, 38). We have now defined two new sites in this complex cytosolic tail that are essential for efficient clathrin-mediated endocytosis of MUC1. The Y20HPM site clearly represents a YXX{phi} type motif recognized by AP-2 that links cargo to clathrin, whereas Grb2 binding at tyrosine phosphorylated Y60TNP has the potential to recruit additional adaptors or Cbl through interaction with its SH3 binding domains. We have also presented new data showing that MUC1 internalization is retarded by its mucin-like ectodomain. Thus, MUC1 membrane trafficking is modulated by features of both its intracellular and extracellular domains.

The Role of the MUC1 Ectodomain in Modulating Its Endocytosis—The MUC1 ectodomain is composed primarily of a variable number of tandem repeats of 20 amino acids, an inherited polymorphism in which 40 and 80 repeats are most common (39, 40). The tandem repeats form an extended polyproline {beta}-turn helix that displays the immunodominant peptide epitope (APTDR), and any O-glycans on the five serine and threonine residues, at regular intervals (41, 42). This extended structure means that MUC1 could extend 200–500 nm from the cell surface, and packaging into clathrin-coated vesicles (~150 nm) is likely to be affected by this anomalous length as well as by the number and size of any O-glycans. In fact, our previous characterization of MUC1 endocytosis revealed that its rate of internalization was enhanced ~2-fold by truncation of the O-glycans. Thus, the 2.7-fold increase in endocytosis caused by replacement of the MUC1 ectodomain (with 22 repeats) with the ectodomain of Tac was not unexpected (Fig. 2). Moreover, the replacement of the MUC1 ectodomain with Tac also enhanced our ability to characterize any critical residues in the MUC1 cytoplasmic domain by site-directed mutagenesis independent of any subsequent effects on MUC1 glycosylation.

The Role of Muc1 Cytosolic Tail in Modulating Its Endocytosis—The extended ectodomain of MUC1 clearly has antiadhesive properties because of its length, as well as adhesive properties as a result of presentation of specific glycan structures (43–53); however, MUC1 functions as far more than a membrane-tethered mucin because of its cytosolic tail-dependent association with members of the EGF receptor family (ErbB1–4) and many of the same cytosolic binding partners that interact with these receptors (14–19, 34, 38). Characterization of MUC1 cytoplasmic interactions by several groups has revealed that MUC1 plays a role in signal transduction and may function as either a cell surface sensor or a modifier of growth factor receptor signaling (for review, see Ref. 54). The MUC1 cytosolic tail can be phosphorylated on 1) Ser44 by glycogen synthase kinase 3{beta}, 2) Tyr46 by Src-family kinases or EGFR, 3) Thr41 by protein kinase C{delta}, and 4) Tyr60 by an unknown kinase. The STDRS44 sequence is a binding site for glycogen synthase kinase 3{beta}, and tyrosine-phosphorylated Y46EKV and Y60TNP are binding sites for Src-family kinases and the SH2 binding domain of Grb2, respectively. Phosphorylation at Ser44, Tyr46, and Thr41 differentially regulates {beta}-catenin binding to MUC1 at the SXXXXXSSLS59 motif and most probably affects any subsequent colocalization of the MUC1 cytosolic tail with {beta}-catenin (or {gamma}-catenin) in the nucleus (18–20). Neither the function nor the mechanism of this nuclear localization of the MUC1 cytosolic tail is presently understood, but it is likely to reflect a new role for MUC1 in transcriptional regulation partly as a result of the stabilization of {beta}-catenin. Wang et al. (55) also found phosphorylation of Tyr20, Tyr29, Tyr46, and Tyr60 when anti-CD8 antibodies were used to cross-link a chimera comprising the CD8 ectodomain and transmembrane domain with the cytoplasmic tail of MUC1. Phosphorylation of Tyr20 and Tyr29 would produce binding sites for the p85 adaptor of type 1a phosphatidylinositol 3-kinase (pY20HPM, consensus pYxxM) and for the adaptor Shc (pY29HTH), respectively (56–59). Sites for p85 and Shc binding are also found on EGF receptor family members, and receptor-bound Shc also recruits the adaptor protein Grb2 (56). However, evidence for binding of either p85 or Shc to MUC1 has not been reported.

The results of our present study indicate that two of seven tyrosine residues in the MUC1 cytoplasmic tail are essential for efficient clathrin-mediated endocytosis. Mutation of Tyr20 or Tyr60 partially inhibited chimera endocytosis, whereas mutation of both Tyr20 and Tyr60 blocked 77% of chimera endocytosis. The residual endocytosis of the double mutant is probably caused by constitutive internalization of plasma membrane. It is interesting that truncation of the MUC1 tail to just 10 residues (CQCRRKNYGQ10) blocked endocytosis by only 40%. This anomaly in endocytosis of the mutant X10 could be caused by the loss of a signal for retention at the plasma membrane, such as interactions with ErbB2 or -4 present in CHO cells (60) or association with a specific lipid microdomain, thereby increasing the availability for endocytosis. Lipid microdomains enriched in cholesterol and glycosphingolipids form a liquid-ordered phase that is poorly solubilized in cold detergents; in many cell types, these domains constitute rafts or platforms on the plasma membrane for association of proteins involved in signal transduction (61–64). In fact, Handa et al. (65) recently reported that MUC1 is insoluble in cold Brij 58 and is enriched in a low density membrane fraction with both T cell-specific transducer molecules Lck and CD45, and Src-family kinases Yes and Fyn. Transmembrane proteins in detergent-resistant membranes are often acylated, and in recent studies, we found that MUC1 is palmitoylated at the CQC motif found at the junction of the transmembrane and cytoplasmic domains.2 Thus, inefficient palmitoylation of the mutant chimera X10 or its association with other proteins in lipid microdomains could explain its anomalous rate of endocytosis in the absence of the two critical tyrosines at Tyr20 and Tyr60.

Although there are three potential tyrosine-based YXX{phi} internalization motifs in the MUC1 cytosolic tail (Y8GQL, Y20HPM, and Y46EKV), we found that only Tyr20 was essential for efficient endocytosis of MUC1. It is interesting that the context of Tyr20 (RDTY20HPM) is also optimal for µ2 binding; Arg at Y-3, Asp at Y-2, Thr at Y-1, His at Y+1, and Pro at Y+2 are all favored residues for µ2 binding, whereas the context of Tyr8 (RKNY8GQL) and Tyr46 (RSPY46EKV) are less optimal (see Fig. 1) (66). Ohno et al. (67) also found that shortening the distance to eight residues between the endocytosis motif (SDYQRL) and the transmembrane domain in TGN38 caused a decrease of more than 10-fold in interactions with the µ2 subunit of AP-2 in an in vitro binding assay. Thus, Y8GQL in MUC1 might be sterically hindered from binding µ2. Tyrosine phosphorylation also blocks binding of the µ2 subunit (67); it is interesting that both Tyr20 and Tyr46 were identified by Wang et al. (55) as being phosphorylated in response to cell surface antibody cross-linking. Thus, the inability of Y46EKV to act as a YXX{phi} endocytosis motif could be the result of a primary role as an SH2 binding site for Shc. Tyr60 was also required for efficient endocytosis of the Tac-MUC1 chimera, but residues in the SLSYT60NP are highly disfavored for binding to µ2. We did find that the Tyr60 mutation decreased AP-2 binding of the chimera by 33%, whereas the Tyr20 mutation decreased AP-2 binding by 75%. Because the tyrosine phosphorylated Y60TNP was previously identified as a site for Grb2 binding of full-length MUC1 (38), we tested the role of Tyr60 in Grb2 binding to the Tac-MUC1 chimera. Because our results clearly showed that the Y60N mutation blocked Tac-MUC1 co-immunoprecipitation with Grb2 by 80–85%, our data indicate that Grb2 binding is also required for efficient incorporation of MUC1 into clathrin-coated pits.

The Role of Grb2 in Endocytosis and Down-regulation of Cell Surface Proteins—Although AP-2 has been well characterized as the major cytoplasmic link between the clathrin-heavy chain and many internalized proteins, the role of Grb2 in clathrin-mediated endocytosis is best characterized for down-regulation (internalization and degradation) of the EGFR. AP-2 coimmunoprecipitation with EGFR was dependent on a YXX{phi} AP-2-binding motif (Y974RAL) in the receptor; however, down-regulation of the mutant Y974A receptor was normal (68). The importance of AP-2 interactions in EGFR endocytosis is controversial. Several reports indicate that disruption of AP-2 function by either overexpression of adaptor-associated kinase or knockdown of the µ2 or {alpha} subunit of AP-2 or the clathrin heavy chain had no effect on EGFR endocytosis (69–71). However, Huang et al. (72) more recently reported that knockdown of clathrin heavy chain, dynamin II, or subunits of AP-2 ({alpha}, {beta}2, or µ2) significantly reduced EGFR internalization, as measured by 125I-EGF internalization under more physiological conditions.

Several groups have now shown that receptor association with Grb2 and ubiquitination by Cbl are key to EGFR internalization, although the exact mechanism is not clear (37, 73–76). Grb2 has a central SH2 binding domain flanked by two SH3 binding domains. Upon receptor binding of the EGF ligand, Grb2 is recruited to an SH2 site on the receptor and thereby links the ubiquitin ligase Cbl to the receptor through interaction with its SH3 binding site (31, 75–77). Stang et al. (78) recently showed that a Grb2-Cbl complex binding to EGF-stimulated EGFR caused receptor recruitment to the edge of clathrin-coated pits along with the adaptor Eps-15. However, entry into the clathrin-coated pit required ubiquitination by Cbl because entry was blocked by overexpression of either dominant-negative ubiquitin (lacking two carboxyl-terminal Gly and thereby blocking ubiquitin-interacting domains on adaptor proteins) or the Cbl-binding/regulatory protein hSpry2 (78, 79). Because MUC1 exhibits sites on its cytoplasmic tail for many of the same binding partners as the EGFR, it is highly probable that Cbl will be recruited through Grb2 to MUC1.

In conclusion, we report that replacement of the highly extended ectodomain of MUC1 with that of Tac stimulated endocytosis, confirming our previous hypothesis that the ectodomain interferes with clathrin-mediated endocytosis of MUC1. We have also identified two new interactions of the complex cytoplasmic tail of MUC1 that are required for its efficient endocytosis. Although AP-2 is probably the major link between MUC1 and the clathrin heavy chain, Grb2 binding to MUC1 clearly plays a major role in incorporation of MUC1 into clathrin-coated vesicles. The most likely role for Grb2 is in the recruitment of Cbl to MUC1, and future experiments will test this possibility.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK54787, P50-CA9044, and P50-DK56490 and by the Pennsylvania American Lung Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 412-383-8949; Fax: 412-383-8956; E-mail: hughey{at}msx.dept-med.pitt.edu.

1 The 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. Back

2 R. P. Hughey, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Olivera Finn, Sandra Gendler, and Linton Traub for providing antibodies and Michael Marks for Tac cDNA. We thank Kevin Ho for help with the statistical analysis of the data, Ora Weisz for providing helpful suggestions and critically reviewing the manuscript, and Rick Strempler for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hanisch, F.-G., and Muller, S. (2000) Glycobiology 10, 439–449[Free Full Text]
  2. Gendler, S. J. (2001) J. Mammary Gland. Biol. Neoplasia 6, 339–353[CrossRef][Medline] [Order article via Infotrieve]
  3. Ligtenberg, M. J. L., Kruijshaar, L., Buijs, F., van Meijer, M., Litvinov, S. V., and Hilkens, J. (1992) J. Biol. Chem. 267, 6171–6177[Abstract/Free Full Text]
  4. Julian, J., and Carson, D. D. (2002) Biochem. Biophys. Res. Commun. 293, 1183–1190[CrossRef][Medline] [Order article via Infotrieve]
  5. McGuckin, M. A., Walsh, M. D., Hohn, B. G., Ward, B. G., and Wright, R. G. (1995) Hum. Pathol. 26, 432–439[CrossRef][Medline] [Order article via Infotrieve]
  6. Bièche, I., Ruffet, E., Zweibaum, A., Vildé, F., Lidereau, R., and Franc, B. (1997) Thyroid 7, 725–731[Medline] [Order article via Infotrieve]
  7. Guddo, F., Giatromanolaki, A., Koukourakis, J. I., Reina, C., Vignola, A.-M., Chlouverakis, G., Hilkens, J., Gatter, K. C., Harris, A. L., and Bonsignore, G. (1998) J. Clin. Pathol. 51, 667–671[Abstract]
  8. Guddo, F., Giatromanolaki, A., Patriarca, C., Hilkens, J., Reina, C., Alfano, R. M., Vignola, A.-M., Koukourakis, J. I., Gambacorta, M., Pruneri, G., Coggi, G., and Bonsignore, G. (1998) Anitcancer Res. 18, 1915–1920
  9. Fujita, K., Denda, K., Yamamoto, M., Matsumoto, T., Fujime, M., and Irimura, T. (1999) Br. J. Cancer 80, 301–308[CrossRef][Medline] [Order article via Infotrieve]
  10. Chu, J. S., and Chang, K. J. (1999) Cancer Lett. 142, 121–127[CrossRef][Medline] [Order article via Infotrieve]
  11. Rahn, J. J., Dabbagh, L., Pasdar, M., and Hugh, J. C. (2001) Cancer 91, 1973–1982[CrossRef][Medline] [Order article via Infotrieve]
  12. Schroeder, J. A., Adriance, M. C., Thompson, M. C., Camenisch, T. D., and Gendler, S. J. (2003) Oncogene 22, 1324–1332[CrossRef][Medline] [Order article via Infotrieve]
  13. Lin, S.-Y., Xiz, W., Wang, J. C., Kwong, K. Y., Spohn, B., Wen, Y., Pestell, R. G., and Hung, M.-C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4262–4266[Abstract/Free Full Text]
  14. Li, Y., Bharti, A., Chen, D., Gong, J., and Kufe, D. (1998) Mol. Cell. Biol. 18, 7216–7224[Abstract/Free Full Text]
  15. Li, Y., Kuwahara, H., Ren, J., Wen, G., and Kufe, D. (2001) J. Biol. Chem. 276, 6061–6064[Abstract/Free Full Text]
  16. Li, Y., Ren, J., Yu, W., Li, Q., Kuwahara, H., Yin, L., Carraway, K. L. I., and Kufe, D. (2001) J. Biol. Chem. 276, 35239–35242[Abstract/Free Full Text]
  17. Ren, J., Li, Y., and Kufe, D. (2002) J. Biol. Chem. 277, 17616–17622[Abstract/Free Full Text]
  18. Li, Y., Chen, W., Ren, J., Yu, W., Li, Q., Yoshida, K., and Kufe, D. (2003) Cancer Biol. Ther. 2, 37–43
  19. Li, Y., Yu, W., Ren, J., Chen, W., Huang, L., Kharbanda, S., Loda, M., and Kufe, D. (2003) Mol. Cancer Res. 1, 765–775[Abstract/Free Full Text]
  20. Wen, Y., Caffrey, T. C., Wheelock, M. J., Johnson, K. R., and Hollingsworth, M. A. (2003) J. Biol. Chem. 278, 38029–38039[Abstract/Free Full Text]
  21. Croce, M. V., Isla-Larrain, M. T., Rua, C. E., Rabassa, M. E., Gendler, S. J., and Segal-Eiras, A. (2003) J. Histochem. Cytochem. 51, 781–788[Abstract/Free Full Text]
  22. Hull, S. R., Bright, A., Carraway, K. L., Abe, M., Hayes, D. F., and Kufe, D. W. (1989) Cancer Commun. 1, 261–267[Medline] [Order article via Infotrieve]
  23. Brockhausen, I., Yang, J.-M., Burchell, J., Whitehouse, C., and Taylor-Papadimitriou, J. (1995) Eur. J. Biochem. 233, 607–617[Medline] [Order article via Infotrieve]
  24. Whitehouse, C., Burchell, J., Gschmeissner, S., Brockhausen, I., Lloyd, K. O., and Taylor-Papadimitriou, J. (1997) J. Cell Biol. 137, 1229–1241[Abstract/Free Full Text]
  25. Dalziel, M., Whitehouse, C., McFarlane, I., Brockhausen, I., Gschmeissner, S., Schwientek, T., Clausen, H., Burchell, J., and Taylor-Papadimitriou, J. (2001) J. Biol. Chem. 276, 11007–11015[Abstract/Free Full Text]
  26. Muller, S., and Hanisch, F.-G. (2002) J. Biol. Chem. 277, 26103–26112[Abstract/Free Full Text]
  27. Altschuler, Y., Kinlough, C. L., Poland, P. A., Bruns, J. B., Apodaca, G., Weisz, O. A., and Hughey, R. P. (2000) Mol. Biol. Cell 11, 819–831[Abstract/Free Full Text]
  28. Boehm, M., and Bonifacino, J. S. (2001) Mol. Biol. Cell 12, 2907–2920[Abstract/Free Full Text]
  29. Boehm, M., and Bonifacino, J. S. (2002) Gene 286, 175–186[CrossRef][Medline] [Order article via Infotrieve]
  30. Traub, L. M. (2003) J. Cell Biol. 163, 203–208[Abstract/Free Full Text]
  31. Sorkin, A. (2004) Curr. Opin. Cell Biol. 16, 392–399[CrossRef][Medline] [Order article via Infotrieve]
  32. Marks, M. S., Roche, P. A., van Donselaar, E., Woodruff, L., Peters, P. J., and Bonifacino, J. S. (1995) J. Cell Biol. 131, 351–369[Abstract/Free Full Text]
  33. Rye, P. D., Price, M. R., Hilgers, J., and Nustad, K. (1998) Tumor Biology 19, 1–151[Medline] [Order article via Infotrieve]
  34. Schroeder, J. A., Thompson, M. C., Gardner, M. M., and Gendler, S. J. (2001) J. Biol. Chem. 276, 13057–13064[Abstract/Free Full Text]
  35. Aguilar, R. C., Ohno, H., Roche, K. W., and Bonifacino, J. S. (1997) J. Biol. Chem. 272, 27160–27166[Abstract/Free Full Text]
  36. Poland, P. A., Kinlough, C. L., Rokaw, M. D., Magarian-Blander, J., Finn, O. J., and Hughey, R. P. (1997) Glycoconjugate J. 14, 89–96[CrossRef][Medline] [Order article via Infotrieve]
  37. Jiang, X., Huang, F., Marusyk, A., and Sorkin, A. (2003) Mol. Biol. Cell 14, 858–870[Abstract/Free Full Text]
  38. Pandey, P., Kharbanda, S., and Kufe, D. (1995) Cancer Res. 55, 4000–4003[Abstract/Free Full Text]
  39. Gendler, S., Lancaster, C., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E. N., and Wilson, D. (1990) J. Biol. Chem. 265, 15285–15293
  40. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2320–2323[Abstract/Free Full Text]
  41. Fontenot, J. D., Tjandra, N., Bu, D., Ho, C., Montelaro, R. C., and Finn, O. J. (1993) Cancer Res. 53, 5386–5394[Abstract/Free Full Text]
  42. Fontenot, J. D., Mariappan, S. V. S., Catasti, P., Domenech, N., Finn, O. J., and Gupta, G. (1995) J. Biomol. Struct. Dynam. 13, 245–260[Medline] [Order article via Infotrieve]
  43. Ligtenberg, M. J. L., Buijs, F., Vos, H. L., and Hilkens, J. (1992) Cancer Res. 52, 2318–2324[Abstract/Free Full Text]
  44. van de Wiel-van Kemenade, E., Ligtenberg, M. J. L., de Boer, A. J., Buijs, F., Vos, H. L., Melief, C. J. M., Hilkens, J., and Figdor, C. G. (1993) J. Immunol. 151, 767–776[Abstract]
  45. Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A., and Hilkens, J. (1995) J. Cell Biol. 129, 255–265[Abstract/Free Full Text]
  46. Hudson, M. J. H., Stamp, G. W. H., Hollingsworth, M. A., Pignatelli, M., and Lalani, E. (1996) Am. J. Pathol. 148, 951–960[Abstract]
  47. Wesseling, J., van der Valk, S. W., and Hilkens, J. (1996) Mol. Biol. Cell 7, 565–577[Abstract]
  48. Zhang, K., Baeckström, D., Brevinge, H., and Hansson, G. C. (1996) J. Cell Biol. 60, 538–549
  49. Arcasoy, S. M., Latoche, J., Gondor, M., Watkins, S. C., Henderson, R. A., Hughey, R., Finn, O. J., and Pilewski, J. M. (1997) Am. J. Respir. Cell Mol. Biol. 17, 422–435[Abstract/Free Full Text]
  50. Kam, J. L., Regimbald, L. H., Hilgers, J. H., Hoffman, P., Krantz, M. J., Longenecker, B. M., and Hugh, J. C. (1998) Cancer Res. 58, 5577–5581[Abstract/Free Full Text]
  51. Arcasoy, S. M., Desai, S. S., Latoche, J. L., and Pilewski, J. M. (1998) Am. J. Respir. Crit. Care Med. 157, A582
  52. Lillehoj, E. P., Hyun, S. W., Kim, B. T., Zhang, X. G., Lee, D. I., Rowland, S., and Kim, K. C. (2001) Am. J. Physiol. 280, L181–L187
  53. Lillehoj, E. P., Kim, B. T., and Kim, K. C. (2002) Am. J. Physiol. 282, L751–L756
  54. Carraway, K. L., Ramasauer, V. P., Haq, B., and Carothers Carraway, C. A. (2002) Bioessays 25, 66–71
  55. Wang, H., Lillehoj, E. P., and Kim, K. C. (2003) Biochem. Biophys. Res. Commun. 310, 341–346[CrossRef][Medline] [Order article via Infotrieve]
  56. Olayioye, M. A., Neve, R. M., Lane, H. A., and Hynes, N. E. (2000) EMBO J. 19, 3159–3167[CrossRef][Medline] [Order article via Infotrieve]
  57. Djordjevic, S., and Driscoll, P. C. (2002) Trends Biochem. Sci. 27, 426–432[CrossRef][Medline] [Order article via Infotrieve]
  58. Foster, F. M., Traer, C. J., Abraham, S. M., and Fry, M. J. (2003) J. Cell Sci. 116, 3037–3040[Free Full Text]
  59. Ravichandran, K. S. (2001) Oncogene 20, 6322–6330[CrossRef][Medline] [Order article via Infotrieve]
  60. Xu, X., Kelleher, K. F., Liao, J., Creek, K. E., and Pirisi, L. (2000) Oncogene 19, 3172–3181[CrossRef][Medline] [Order article via Infotrieve]
  61. Brown, D. A., and London, E. (2000) J. Biol. Chem. 275, 17221–17224[Free Full Text]
  62. Simons, K., and Toomre, D. (2000) Nat. Rev. Mol. Cell. Biol. 1, 31–39[CrossRef][Medline] [Order article via Infotrieve]
  63. Fullekrug, J., and Simons, K. (2004) Ann. N. Y. Acad. Sci. 1014, 164–169[CrossRef][Medline] [Order article via Infotrieve]
  64. Simons, K., and Vaz, W. L. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 269–295[CrossRef][Medline] [Order article via Infotrieve]
  65. Handa, K., Jacobs, F., Longenecker, B. M., and Hakomori, S. I. (2001) Biochem. Biophys. Res. Commun. 285, 788–794[CrossRef][Medline] [Order article via Infotrieve]
  66. Ohno, H., Aguilar, R. C., Yeh, D., Taura, D., Saito, T., and Bonifacino, J. S. (1998) J. Biol. Chem. 273, 25915–25921[Abstract/Free Full Text]
  67. Ohno, H., Fournier, M. C., Poy, G., and Bonifacino, J. S. (1996) J. Biol. Chem. 271, 29009–29015[Abstract/Free Full Text]
  68. Sorkin, A., Mazzotti, M., Sorkina, T., Scotto, L., and Beguinot, L. (1996) J. Biol. Chem. 271, 13377–13384[Abstract/Free Full Text]
  69. Conner, S. D., and Schmid, S. L. (2003) J. Cell Biol. 162, 773–779[Abstract/Free Full Text]
  70. Motley, A., Bright, N. A., Seaman, M. N., and Robinson, M. S. (2003) J. Cell Biol. 162, 909–918[Abstract/Free Full Text]
  71. Hinrichsen, L., Harborth, J., Andrees, L., Weber, K., and Ungewickell, E. J. (2003) J. Biol. Chem. 278, 45160–45170[Abstract/Free Full Text]
  72. Huang, F., Khvorova, A., Marshall, W., and Sorkin, A. (2004) J. Biol. Chem. 279, 16657–16661[Abstract/Free Full Text]
  73. Wang, Z., and Moran, M. F. (1996) Science 272, 1935–1939[Abstract]
  74. Thien, C. B., Walker, F., and Langdon, W. Y. (2001) Mol. Cell 7, 355–365[CrossRef][Medline] [Order article via Infotrieve]
  75. Waterman, H., Katz, M., Rubin, C., Shtiegman, K., Lavi, S., Elson, A., Jovin, T., and Yarden, Y. (2002) EMBO J. 21, 303–313[CrossRef][Medline] [Order article via Infotrieve]
  76. Jiang, X., and Sorkin, A. (2003) Traffic 4, 529–543[Medline] [Order article via Infotrieve]
  77. Waterman, H., and Yarden, Y. (2001) FEBS Lett. 490, 142–152[CrossRef][Medline] [Order article via Infotrieve]
  78. Stang, E., Blystad, F. D., Kazazic, M., Bertelsen, V., Brodahl, T., Raiborg, C., Stenmark, H., and Madshus, I. H. (2004) Mol. Biol. Cell 15, 3591–3604[Abstract/Free Full Text]
  79. Wong, E. S., Fong, C. W., Lim, J., Yusoff, P., Low, B. C., Langdon, W. Y., and Guy, G. R. (2002) EMBO J. 21, 4796–4808[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
C. S. M. Ferreira, M. C. Cheung, S. Missailidis, S. Bisland, and J. Gariepy
Phototoxic aptamers selectively enter and kill epithelial cancer cells
Nucleic Acids Res., February 1, 2009; 37(3): 866 - 876.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. S Lau and J. W Dennis
N-Glycans in cancer progression
Glycobiology, October 1, 2008; 18(10): 750 - 760.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
R. Singh, U. Samant, S. Hyland, P. R. Chaudhari, W. S. Wels, and D. Bandyopadhyay
Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors
Mol. Cancer Ther., February 1, 2007; 6(2): 562 - 569.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. L. Kinlough, R. J. McMahan, P. A. Poland, J. B. Bruns, K. L. Harkleroad, R. J. Stremple, O. B. Kashlan, K. M. Weixel, O. A. Weisz, and R. P. Hughey
Recycling of MUC1 Is Dependent on Its Palmitoylation
J. Biol. Chem., April 28, 2006; 281(17): 12112 - 12122.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. Engelmann, C. L. Kinlough, S. Muller, H. Razawi, S. E. Baldus, R. P. Hughey, and F.-G. Hanisch
Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles
Glycobiology, November 1, 2005; 15(11): 1111 - 1124.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
279/51/53071    most recent
M409360200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kinlough, C. L.
Right arrow Articles by Hughey, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kinlough, C. L.
Right arrow Articles by Hughey, R. P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement