Radiolabeling, Biotinylation, and Immunoprecipitation of MUC1

Confluent cultures of cells in 35-mm dishes were washed once with 1 ml of DME media lacking methionine and cysteine (Met/Cys; ICN, Costa Mesa, CA) and starved for Met and Cys in 1 ml of the same media for 15 min before addition of 50–100 μCi of [³⁵S]Met/Cys (Easy Tag Express-[³⁵S]Protein Labeling Mixture; New England Nuclear, Wilmington, DE) for the indicated time. Labeled cells were chased in normal culture medium. Where specified, media used to starve, pulse, and chase cells was supplemented with either Gal or GalNAc at the levels indicated in each experiment. After the chase period, cells were rapidly chilled on ice for biotinylation of cell surface proteins as previously described (Gottardi and Caplan, 1992). Briefly, cells were washed four times with 1 ml of phosphate buffered saline (PBS; 137 mM NaCl, 2.6 mM KCl, 15.2 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 0.5 mM MgCl₂, and 0.7 mM CaCl₂) and incubated with 0.5 mg/ml sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate-biotin (Pierce, Rockford, IL) in TEA-buffered saline (10 mM triethanolamine, pH 7.6, 137 mM NaCl, and 1 mM CaCl₂) for 10 min. The reaction was quenched by washing the cells three times with normal culture media. Cells were solubilized at room temperature with 0.2 ml of 60 mM n-octyl β-d-glucopyranoside and 0.1% SDS (both from Sigma, St. Louis, MO) in HEPES-buffered saline (HBS, 10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl) for 20 min, and insoluble material was removed by centrifugation in a microcentrifuge at 14,000 rpm for 7 min. Supernatants were recovered and rotated end-over-end overnight at 4°C after addition of protein G immobilized on Sepharose 4B (Sigma) and mouse monoclonal antibodies VU-3-C6 against the tandem repeat domain (Rye et al., 1998) and 232A1 against an extracellular nontandem repeat domain (Oosterkamp et al., 1997). Immunoprecipitates were recovered by brief centrifugation and washed once with 0.5 ml each: 1% Triton X-100 (Boehringer Mannheim, Indianapolis, IN) in HBS, 0.01% SDS in HBS, and finally HBS. Where indicated, the pellets were resuspended in acetate buffer (50 mM sodium acetate, pH 5.5, 0.15 M NaCl, 9 mM CaCl₂) and incubated with or without 1 mU neuraminidase (Vibrio cholera; Calbiochem, LaJolla CA) for 1 h at 37°C. Biotinylated MUC1 was recovered by elution of the immunoprecipitates for 2 min at 90°C in 80 μl 1% SDS in HBS and incubation with 30 μl ImmunoPure Immobilized Avidin (Pierce) after addition of 0.8 ml HBS. After overnight rotation at 4°C, the avidin-conjugated beads were washed with 1 ml each: 1% Triton X-100 in HBS and HBS. The biotinylated MUC1 was recovered by heating for 3.5 min at 90°C in 50 μl Laemmili SDS-sample buffer containing fresh 0.14 M β-mercaptoethanol (Laemmli, 1970). Samples were subjected to SDS-PAGE (all reagents from Bio-Rad, Richmond, CA) on 3–15% acrylamide gradient gels, and radioactive protein bands were imaged and quantitated from the dried gel using a phosphoimager (Bio-Rad). In some instances, fluorography of gels was carried out using BioMax MR film (Eastman Kodak, Rochester, NY).

Endocytosis Assay for MUC1

Confluent cultures of MUC1-expressing CHO or ldlD cells in Falcon six-well dishes (35-mm wells; Becton Dickinson Labware, Franklin Lakes, NJ) were pulse-labeled with [³⁵S]Met/Cys for 30 min and chased for 90 min before biotinylation of cell surface proteins on ice (described above). Where indicated, GalNAc, with or without Gal, was added to the culture media throughout the radiolabeling protocol. Cells in six-well dishes were rapidly warmed to 37°C on a metal plate in a Precision (Winchester, VA) model 66566 circulating water bath by the addition of prewarmed HEPES-buffered MEM (20 mM HEPES-NaOH, pH 7.4, 0.6% BSA, 4 mM NaHCO₃), incubated to allow endocytosis for 1–30 min and then rapidly cooled on a metal plate on ice by two quick washes with ice-cold PBS. Where indicated, 0.45 M sucrose was included in the culture media to inhibit clathrin-mediated endocytosis (Hansen et al., 1993). Biotin remaining at the cell surface was stripped by three 20-min incubations with 1 ml ice-cold 100 mM sodium 2-mercaptoethanesulfonic acid (MESNA) in 50 mM Trizma-HCl, pH 8.6, 100 mM NaCl, 1 mM EDTA, and 0.2% BSA, and residual MESNA was quenched by a 10-min incubation with ice-cold 120 mM iodoacetic acid in PBS. The internalized biotinylated MUC1 was recovered from cell extracts as described above. The total biotinylated MUC1 was determined for each sugar condition on separate six-well dishes by excluding the MESNA and iodoacetic acid washes. Cells representing the zero time point of endocytosis were incubated in ice-cold HEPES-buffered MEM on ice for 10 min before washing. Percent internalization of MUC1 was calculated by dividing the amount of biotinylated [³⁵S]MUC1 remaining after MESNA treatment by the amount of biotinylated [³⁵S]MUC1 recovered without MESNA treatment at each time point. Background values obtained at t = 0 were subtracted from each time point and were typically 1–5% of total MUC1. Data are presented for individual representative experiments as means ± SD for triplicate samples, allowing for error propagation. Data calculated for combined experiments are presented as the means ± SEM.

Endocytosis Assay for the Polymeric Immunoglobulin Receptor

Endocytosis of the polymeric immunoglobulin receptor (pIgR) in ldlD cells was determined exactly as described for MUC1 above, except that samples were immunoprecipitated using a sheep antibody directed against the lumenal domain of pIgR (5αSC).

Assay for Fluid Phase Endocytosis

Confluent cultures of CHO or ldlD cells expressing MUC1 in 24-well plastic dishes (15-mm wells; Costar, Cambridge, MA) were incubated for 2 h with 1 mM GalNAc, with or without 0.1 mM Gal as indicated, to mimic the conditions for radiolabeling and endocytosis of MUC1. Cells were moved to ice and washed once with cold HEPES-buffered MEM before addition of prewarmed media (with or without 0.45 M sucrose as indicated) containing horseradish peroxidase (HRP type II, Sigma) at 1 mg ml⁻¹ and incubated at 37°C for varying times. Cells were rapidly chilled on ice and washed twice quickly before three 10-min washes with HEPES-buffered MEM and a final rinse with PBS. Cells for the zero time point were incubated on ice with media containing HRP for 15 min. Cells were solubilized at room temperature with 0.2 ml of 60 mM n-octyl β-d-glucopyranoside and 0.1% SDS in HEPES-buffered saline for 20 min and assayed for peroxidase activity using tetramethylbenzidine dihydrochloride (Sigma) as described by the manufacturer. Activity is reported as the means ± SD of triplicate samples.

Expression of Proteins from Recombinant Adenoviruses

The cDNA for pIgR was subcloned into the pAdlox vector and a recombinant adenovirus (AV-pIgR) generated as described in Hardy et al. (1997). A recombinant adenovirus encoding mutant K44A dynamin-1 with an HA epitope tag (AV-K44A) was prepared as described previously (Altschuler et al., 1998). Expression of dynamin from this virus requires coexpression of a tetracycline-repressible transactivating protein encoded by a different adenovirus (AV-TA). Using this system, dynamin expression can be completely blocked by including low levels of doxycycline (DOX; 20 ng/ml) in the postinfection medium. Because initial experiments showed poor infection of CHO cells by adenovirus, a clonal line of MUC1-expressing CHO or ldlD cells stably transfected with hCAR (Bergelson et al., 1997) was used for these studies. Confluent cultures of these cells were washed once with PBS and incubated with AV-TA with or without AV-K44A in 0.8 ml PBS (without calcium) for 6-well dishes or AV-pIgR in 0.3 ml PBS (without calcium) for 24-well dishes (multiplicity of infection ~ 200). After 2 h at 37°C, virus was removed, and cells were washed once with 1 ml of normal culture media and incubated overnight at 37°C in the same media. Controls for the K44A dynamin expression were either cells infected with only AV-TA or cells infected with both viruses but subsequently incubated with DOX. The following day, endocytosis assays were performed as described above. Inhibition of K44A expression by DOX was confirmed by Western blot analysis of cell extracts using the mouse anti-HA monoclonal antibody 12CAS (BabCO, Richmond, CA).

Determination of MUC1 Half-life

CHO and ldlD cells expressing MUC1 were pulse-labeled for 30 min with [³⁵S]Met/Cys and chased in normal media for 90 min as described above. Where indicated, GalNAc with or without Gal were added to the culture media throughout the radiolabeling protocol. Cells were returned to culture for either 0 or longer times (3.5–20 h) before solubilization, immunoprecipitation, and recovery of [³⁵S]MUC1 for SDS-PAGE and phosphoimager analysis. The half-life of MUC1 was calculated from the levels of radioactivity using the formula t_1/2 = 0.693/k_d.

MUC1 Internalization Is Affected by Glycosylation

The reduced levels of underglycosylated MUC1 found at the cell surface could be due either to decreased delivery to the cell surface or to more rapid internalization from the plasma membrane. To test whether underglycosylation of MUC1 would affect its endocytosis, we developed an assay to measure MUC1 internalization. CHO and ldlD cells expressing MUC1 were starved for Met/Cys, pulsed-labeled with [³⁵S]Met/Cys for 30 min and chased for 90 min in the presence of GalNAc, with or without Gal, before chilling the cells and biotinylating the cell surface with sulfo-NHS-SS-biotin. This chase period is sufficient to deliver both underglycosylated and mature newly synthesized MUC1 to the cell surface (see Figure ​Figure3).3). Internalization of [³⁵S]MUC1 was initiated by rapid warming of the cells to 37°C for 15 or 30 min. At each time point, vesicular traffic was stopped by rapidly cooling the cells on ice. Biotin remaining on cell surface proteins was removed with the membrane-impermeant reducing agent MESNA. Internalized biotinylated [³⁵S]MUC1 (protected from MESNA) was recovered after cell lysis by incubation of immunoprecipitated MUC1 with avidin-conjugated beads. The percent of surface MUC1 internalized at a given time point was calculated by comparison to control plates of cells not washed with MESNA. As shown in Figure ​Figure4,4, the level of internalized [³⁵S]MUC1 was near maximal at 15 min for all glycosylated forms of MUC1. Interestingly, the level of underglycosylated MUC1 (synthesized in ldlD + 500GN or + 1000GN) that was internalized over this time period was approximately twice that of mature MUC1 (synthesized in CHO cells or in ldlD cells + G/GN). However, the intracellular accumulation of poorly glycosylated [³⁵S]MUC1 (synthesized in ldlD + 100GN) was not enhanced, indicating that there is a threshold of reactivity associated with the number of truncated O-glycans on the MUC1.

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Figure 4

MUC1 internalization from the cell surface is affected by its glycosylation state. CHO and ldlD cells expressing MUC1 were pulse labeled for 30 min and chased for 90 min before cell surface biotinylation on ice as described in MATERIALS AND METHODS. Varying levels of Gal (G, 100 μM) and GalNAc (GN, 100, 500, or 1000 μM) were included in the starvation, pulse and chase media as indicated. Cells were rapidly warmed to 37°C for the indicated times to allow internalization of surface MUC1 and rapidly cooled on ice, and remaining cell surface biotin was stripped with MESNA. Some samples were not treated with MESNA in order to determine the total amount of biotinylated MUC1 at t = 0. Internalized biotinylated MUC1 was recovered from the MUC1 immunoprecipitates with avidin-conjugated beads, and [³⁵S]MUC1 was analyzed after SDS-PAGE using a phosphoimager. Data are plotted as the percent of total biotinylated [³⁵S]MUC1 internalized at each time point and are presented as means ± SD of triplicate samples. Similar results were obtained in six experiments.

This increased intracellular accumulation of underglycosylated MUC1 could reflect an increased initial rate of endocytosis, a decreased recycling rate, or both. We therefore compared the initial rate of endocytosis of surface biotinylated underglycosylated and mature [³⁵S]MUC1 in ldlD cells (Figure ​(Figure5).5). Endocytosis of both mature (G+GN) and underglycosylated (1000GN) MUC1 was evident within 1 min and proceeded in a linear manner for at least 7 min. However, we consistently found a twofold greater initial rate of endocytosis for the MUC1 with truncated O-glycans, which was consistent with the levels of MUC1 accumulating after 15–30 min (Figure ​(Figure4).4). Although it is possible that underglycosylated MUC1 with smaller glycans could exhibit higher levels of biotinylation that could affect its structure and endocytosis, this is unlikely since the repetitive sequences with the most O-glycans do not contain lysine residues. Although we attempted to modify our assay to compare the initial rate of return of internalized MUC1 to the cell surface (recycling), we did not obtain reproducible results using this approach.

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Figure 5

The initial rate of MUC1 endocytosis is enhanced by altered glycosylation. (A) The rate of endocytosis of [³⁵S]MUC1 in ldlD cells, synthesized in the presence of either 1000 μM GalNAc (1000GN) with or without 100 μM Gal (G+GN), was characterized as described in MATERIALS AND METHODS and the legend to Figure ​Figure4.4. The mean of the percent endocytosis ± SEM for each time point for three experiments is shown. By paired Student's t test analysis, only the data at 7 min was significantly different (p = 0.015) between the two sugar conditions. Regression lines are drawn for each data set and indicate a 2.2-fold difference in the rate of endocytosis (p < 0.05). (B) A representative SDS-gel pattern for internalized [³⁵S]MUC1 from a single experiment is shown, where n = 3 at each time point.

Increased Internalization Does Not Lead to Increased Degradation of MUC1

One explanation for the increased internalization of underglycosylated MUC1 might be recognition by quality control machinery at the cell surface leading to MUC1 retrieval and degradation. To determine whether enhanced internalization is linked to increased degradation of underglycosylated MUC1, the half-life of MUC1 in CHO and ldlD cells grown under various culture conditions was determined as described in MATERIALS AND METHODS. As shown in Table ​Table1,1, the MUC1 synthesized in ldlD cells in the presence of 1000 μM GalNAc, with or without 100 μM Gal, had the same half-life as the mature MUC1 synthesized in CHO cells. The degradation of MUC1 in CHO cells was blocked by including an inhibitor of lysosomal acidification (the weak base ammonium chloride) in the culture media (unpublished observations), but not by the presence of a proteosome inhibitor (the peptide aldehyde MG-132) (Rock et al., 1994). Because MUC1 in these cells is not shed into the media (unpublished observations), this would indicate that MUC1 turns over by degradation in lysosomes. Thus, increased intracellular accumulation of underglycosylated MUC1 in ldlD cells is the direct result of increased endocytosis and is not linked to increased degradation.

Table 1

Enhanced endocytosis of MUC1 is not linked to increased degradation

Cells and culture conditions	Half-life (h)
CHO MUC (n = 8)	29.9 ±6.6
ldlD MUC + G/GN (n = 5)	26.1 ±3.5
ldlD MUC + 1000GN (n = 5)	20.7 ±5.2

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Values are means ± SEM. CHO and ldlD cells were pulse labeled for 30 min and chased for 90 min as described in MATERIALS AND METHODS. Where indicated, GalNAc, with or without Gal, were included in the starvation, pulse, and chase media (abbreviations are described in legend to Figs. ​Figs.11–6). Cells were either solubilized immediately or returned to culture; immunoprecipitates of [³⁵S]MUC1 were subjected to SDS-PAGE and phosphoimager analysis. The half-life of [³⁵S]MUC1 was calculated as described in MATERIALS AND METHODS. 

MUC1 Is Internalized by Dynamin-dependent, Clathrin-mediated Endocytosis

To determine whether normal and enhanced internalization of MUC1 proceed via the same mechanism or by different pathways, endocytosis of mature and underglycosylated MUC1 was measured under conditions that are known to inhibit clathrin-mediated endocytosis. As shown in Figure ​Figure6A,6A, endocytosis of normal MUC1 in CHO cells and underglycosylated MUC1 in ldlD cells (synthesized in the presence of 1000 μM GalNAc) was completely inhibited by hypertonic media. By contrast, fluid phase endocytosis of HRP, which proceeds through both clathrin-mediated and clathrin-independent pathways was inhibited only 50% (Figure ​(Figure6B).6B). Thus, the increased internalization of MUC1 resulting from its altered glycosylation does not occur via an alternate route but is likely to result directly from increased clathrin-mediated endocytosis of MUC1.

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Figure 6

MUC1 internalization is inhibited by hypertonic media. (A) CHO and ldlD cells expressing MUC1 were pulse labeled for 30 min and chased for 90 min. GalNAc (1000 μM) was included in the starvation, pulse, and chase media for the ldlD cells. After biotinylation on ice, cells were rapidly warmed to 37°C in the presence of normal or hypertonic media (supplemented with 0.45 M sucrose) for the indicated times. The level of internalized MUC1 was determined as described in MATERIALS AND METHODS and the legend to Figure ​Figure4.4. (B) ldlD cells expressing MUC1 were preincubated for 2 h in media with 1000 μM GalNAc, with or without 100 μM Gal. Endocytosis of the fluid phase marker HRP, carried out in the presence (+ sucrose) or absence of hypertonic media, was determined as described in MATERIALS AND METHODS.

Additional experiments were carried out to confirm that MUC1 internalization occurs via clathrin-coated pits. Because the actual budding of clathrin-coated endocytic vesicles requires the 100-kDa GTPase dynamin-1, CHO cells expressing MUC1 were infected with recombinant adenovirus encoding the dominant-negative mutant (K44A) of dynamin-1 (Altschuler et al., 1998). Expression of K44A dynamin in this system is under control of the tetracycline operon and can be blocked by inclusion of DOX in the postinfection medium (Figure ​(Figure7C).7C). As shown in Figure ​Figure7A,7A, expression of dominant-negative dynamin K44A inhibits the internalization of MUC1 by ~ 80%, whereas fluid phase endocytosis of HRP is inhibited only 20% (Figure ​(Figure7B).7B). This inhibition of MUC1 endocytosis was directly due to the expression of the mutant dynamin, because normal endocytosis levels were observed either in cells infected only with AV-TA or in cells cultured in the presence of DOX. Finally, ultrathin cryosections of CHO cells expressing MUC1 were stained with the anti-MUC1 antibody VU-3-C6 and protein A-conjugated 5 nm gold to assess the subcellular localization of the MUC1 in these cells. Consistent with our biochemical data, MUC1 was found evenly distributed on the plasma membrane, including the microvillar protrusions (Figure ​(Figure8,8, A and C). Most notably, MUC1 was observed in both coated pits (Figure ​(Figure8B)8B) and coated vesicles (Figure ​(Figure8,8, A and C), consistent with clathrin-mediated endocytosis of MUC1; however, MUC1 was not found in any noncoated pits on the plasma membrane. In addition, MUC1 was also found in Golgi-associated vesicles (Figure ​(Figure8D),8D), although there is no way to know if this is within the recycling pathway or the pathway of de novo synthesis. Together, these experiments demonstrate that internalization of both mature and underglycosylated MUC1 is mediated by the classical clathrin- and dynamin-dependent endocytic machinery.

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Figure 7

MUC1 internalization is dynamin dependent. (A) Clonal CHO cells expressing both MUC1 and hCAR were infected with adenoviruses expressing the tetracycline-repressible transactivating protein (AV-TA) and the dominant-negative mutant of dynamin-1 (+K44A, ). Controls (−K44A, ●) for the experiments were either cells infected with only AV-TA or cells infected with both viruses but incubated subsequently with the tetracyline analogue doxycycline (DOX) in the media. The following day, [³⁵S]MUC1 endocytosis was determined as described in MATERIALS AND METHODS and the legend to Figure ​Figure5.5. The mean of the percent endocytosis ± SEM for each time point for six (t = 5 min) and seven (t = 10 min) experiments is shown. By paired Student's t test analysis, MUC1 endocytosis under the two conditions was significantly different at both time points (p ≤ 0.01). (B) CHO cells expressing MUC1 and infected as described above for (A) were grown overnight with (−K44A) or without (+K44A) DOX in the media before assay of HRP uptake as described in MATERIALS AND METHODS. Data are a representative experiment with the mean ± SD of triplicate samples at each time point. (C) Identical aliquots of adenoviral-infected cells from the experiment described in (B) were grown overnight with (+DOX) or without (−DOX) doxycycline and subjected to Western blot analysis with an anti-HA tag antibody to visualize HA-tagged dynamin-1 (K44A) under the two culture conditions.

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Figure 8

Distribution of MUC1 in transfected CHO cells. Ultrathin cryosections of CHO cells expressing MUC1 were sequentially incubated with anti-MUC1 monclonal antibody VU-3-C6 and protein A conjugated to 5 nm colloidal gold. MUC1 was observed at the cell surface (A–C), in coated pits (B), and in coated invaginations and coated vesicles (marked with arrows in A and C, respectively). (D) MUC1 was also found in Golgi-associated vesicles. G, Golgi; LY, lysosome; M, mitochondria.

MUC1 Endocytosis Is Clathrin Mediated and Dynamin Dependent

To characterize the regulation of MUC1 endocytic trafficking in CHO cells, we first developed a sensitive biotin protection assay to follow the internalization of the de novo [³⁵S]MUC1 synthesized with varying levels of glycosylation. Although the underglycosylated MUC1 in ldlD cells (+1000 μM GalNAc) was internalized at twice the rate of the normally glycosylated MUC1 (Figure ​(Figure5),5), this is not a generalized effect on endocytosis of all proteins in the ldlD cells, because neither pIgR nor its ligand was endocytosed differently under the two glycosylation states (Figure ​(Figure99).

This glycosylation-dependent enhancement of MUC1 endocytosis in ldlD cells (Figure ​(Figure5)5) led us to determine whether there were alternate routes for basal and stimulated MUC1 endocytosis. Hypertonic media is known to inhibit clathrin-mediated endocytosis (Hansen et al., 1993), with lesser effects on fluid phase endocytosis (Oka et al., 1989), and we found that both the normal and stimulated uptake of MUC1 were completely inhibited by inclusion of hypertonic media in the endocytosis assay (Figure ​(Figure6).6). Fluid phase endocytosis was inhibited 50% under these same conditions, which is comparable to levels of inhibition previously reported (Oka et al., 1989). Thus, it appears that the altered glycosylation of MUC1 results in a direct stimulation of its endocytosis through clathrin-coated pits. The role of clathrin-mediated endocytosis in the internalization of MUC1 was confirmed by the observation that expression of the dominant-negative mutant of the GTPase dynamin-1 (K44A), which plays a direct role in the fission of deeply invaginated clathrin-coated pits (Schmid, 1997), also inhibits MUC1 endocytosis by 80% (Figure ​(Figure7).7). By comparison, fluid phase endocytosis was inhibited only 20% by expression of dynamin-1 (K44A), comparable to inhibition levels previously reported in Hela cells for the expression of dominant-negative dynamin-1 mutants (Damke et al., 1995; Skretting et al., 1999).

Most microvillar proteins are excluded from clathrin-coated pits (Bretscher et al., 1980; Rodman et al., 1986). The simplest explanation for this observation is that both the microvillar GPI-anchored proteins and transmembrane hydrolases lack cytoplasmic signals for endocytosis. However, MUC1 is unique because it is localized primarily on microvilli but does enter clathrin-coated pits, as shown by immunoelectron microscopy in Figure ​Figure9.9. This localization in clathrin-coated pits suggests that one of the seven tyrosine motifs in the MUC1 cytoplasmic domain may bind adaptors involved in recruitment of clathrin to the plasma membrane (Kirchhausen et al., 1997). In fact, both the first (YGQL) and sixth (YEKV) tyrosine motif from the membrane fit the consensus sequence (YXX) for binding adaptors, and characterization of their role in MUC1 endocytosis is in progress.

Potential Mechanisms for Stimulation of MUC1 Endocytosis

The simplest explanation for the increased endocytosis of MUC1 with truncated O-glycans when compared with mature MUC1 is that more of the underglycosylated MUC1 can fit into a coated pit because there is less steric hindrance between MUC1 molecules. Future experiments will be directed at testing this possibility using MUC1 with larger glycans in CHO cells expressing the core 2 β-1,6-GlcNAc transferase. In addition, the secondary structure and length of this highly extended protein is likely to be modulated by the number and size of O-glycans (Fontenot et al., 1995), which in turn could affect budding of clathrin-coated pits into coated vesicles. It is predicted that the fully extended MUC1 with 30–90 tandem repeats would be 200–500 nm long (Hilkens et al., 1992), whereas the diameter of a clathrin-coated vesicle is ~150 nm. Thus, a mechanism must exist for the compaction of MUC1 within intracellular vesicles and the presence of smaller O-glycans on MUC1 should enhance this step.

We also considered the possibility that the presence of more MUC1 molecules in each clathrin-coated pit could enhance budding into vesicles by recruitment of cytoplasmic proteins regulating endocytosis. For example, there have been reports that the adaptor protein Grb2, which binds the PRD domain of dynamin through its SH3 sites (Barylko et al., 1998), can also bind a phophorylated tyrosine motif in the cytoplasmic domain of the MUC1 through its SH2 site (Pandey et al., 1995). Thus, increased MUC1 accumulation within the clathrin-coated pit due to the truncated O-glycans could have the side effect of recruiting more dynamin and enhancing the rate of coated-vesicle formation. This hypothesis also would predict that clathrin-mediated endocytosis of other proteins such as pIgR, which use clathrin-mediated endocytosis, would also be enhanced when coexpressed with MUC1 under these stimulated conditions. This possibility was tested by following the endocytosis of pIgR when coexpressed with MUC1. However, culture conditions that produce enhanced endocytosis of MUC1 did not show any change in the internalization of pIgR or its ligand. Thus, the enhanced endocytosis of MUC1 resulting from its expression with truncated O-glycans is specific for MUC1 and does not seem to affect endocytosis of other proteins using the clathrin-mediated endocytosis pathway. Future experiments will be directed at understanding the regulation of MUC1 phosphorylation and whether its interaction with Grb2 might affect its entry into clathrin-coated pits.

We thank Jennifer Henkel for help with the dimeric IgA endocytosis experiments; W. Geovany Ruiz for preparation of ultrathin cryosections for immunoelectron microscopy; Jeffrey M. Bergelson (Children's Hospital, Philadelphia, PA) and Robert W. Finberg (Harvard Medical School, Boston, MA) for the hCAR cDNA and anti-hCAR antibodies; John Hilkens (The Netherlands Cancer Institute, Amsterdam) for 232A1 anti-MUC1 antibody; Olivera J. Finn (University of Pittsburgh, Pittsburgh, PA) for VU-3-C6 anti-MUC1 antibody obtained from Jo Hilgers (Free University, Amsterdam, The Netherlands); and Keith Mostov (UCSF, San Francisco, CA) for the pIgR cDNA. This work was supported by fellowship DAMD17–1-97 from the Department of Defense (DOD-Army Breast Cancer Fellowship to Y.A.); National Institutes of Health grants DK51970 (to G.A.), DK54407 (to O.A.W.), and DK26012 (to R.P.H.); and the Dialysis Clinic, Inc.