The epithelial Na ؉ channel (ENaC) is a tetramer of two ␣-, one ␤-, and one ␥-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone ␣-(95 kDa), ␤-(96 kDa), and ␥-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of ␣␤␥ENaC subunits revealed a second band for each subunit (65 kDa for ␣, 110 kDa for ␤, and 75 kDa for ␥) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [ 3 H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed ␣-and ␥-subunits is also consistent with proteolytic cleavage. By using ␣-and ␥-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the ␣-and ␥-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for ␣ and 18 kDa for ␥) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is composed of three structurally related subunits, termed ␣-, ␤-, and ␥-ENaC. The three subunits exhibit limited amino acid sequence identity (30 -40%) but are structurally similar with two membrane-spanning domains and cytosolic amino and carboxyl termini. We and others have shown that ENaC expressed in Xenopus oocytes has a subunit stoichiometry of two ␣-, one ␤-, and one ␥-subunit (1, 2

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.

MUC1 is normally expressed on the apical surface of epithelial cells, where its highly extended mucin-like structure serves a protective role by modulating clearance or retention of secreted mucins and by providing a scaffold for the presentation of glycans that are recognized by bacteria and viruses (1-9). In tumors of epithelial origin, cell polarity is lost, and MUC1 expression on all cell surfaces contributes to an aggressive tumor phenotype; the extended peptide core inhibits cell-cell and cell-matrix interactions, whereas the presence of specific glycan structures such as sialyl-Le X and sialyl-Le a can act as ligands for selectin-like molecules on endothelial cells and thereby enhance metastasis (10 -13).The anti-adhesive property of MUC1 is also enhanced by its ability to compete with the cell adhesion molecule E-cadherin for binding of cytoplasmic ␤-catenin, an important link in the maintenance of actin interactions with the adherins junctions of epithelia (14, 15). In fact, loss of E-cadherin and aberrant localization of both ␤-catenin and MUC1 correlate with an aggressive tumor phenotype and a poor prognosis for the patient (16, 17). The binding of ␤-catenin to MUC1 is regulated by phosphorylation at adjacent sites by glycogen synthase kinase-3␤, Src family kinases, the epidermal growth factor receptor, or protein kinase C␦ (15, 18 -22). MUC1 is autocatalytically cleaved within the SEA (sea urchin sperm protein, enterokinase, and agrin) domain in the endoplasmic reticulum, but the larger mucin-like subunit remains tightly associated with the small transmembrane subunit (23-25). Using antibodies directed against a peptide corresponding to the MUC1 small subunit C terminus, researchers have reported that (i) treatment of human ZR-75-1 breast cancer cells with the ErbB ligand heregulin targets a complex of ␥-catenin and MUC1 to the nucleus; (ii) overexpression of MUC1 in pancreatic cancer cell lines targets ␤-catenin and MUC1 to the nucleus; (iii) activation of Lyn kinase in multiple myeloma cells with interleukin-7 targets a complex of ␤-catenin and MUC1 to the nucleus; and (iv) MUC1 is targeted to mitochondria when HCT116 colon carcinoma cells overexpressing MUC1 are stimulated with heregulin (20, 26 -28). Although nuclear and cytoplasmic ␤-catenins were observed by one research group in breast cancer patients (17), others found ␤-catenin and MUC1 only in the cytoplasm and plasma membrane in both human breast cancer samples and a spontaneous mouse model of breast cancer (29 -31).The mechanism for nuclear or mitochondrial targeting of the MUC1 small subunit is unknown, but delivery of the subunit to any intracellular compartment is likely dependent on its endocytosis from the cell surface. We have reported previously that MUC1 is internalized faster with shorter glycans (32), a feature of MUC1 expressed in several human breast tumor cell lines (33-36). Replacement of the extended ectodomain of MUC1 with that of Tac (interleukin-2 receptor ␣-subunit) also enhances endocytosis; and using site-specific mut...

Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo posttranslational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein ␣A-crystallin, have recently been shown to play a role in this process. We have now found that ␣A-crystallin is expressed in cultured mouse collecting duct cells, where apical Na ؉ transport is mediated by epithelial Na ؉ channels (ENaC). ENaC-mediated Na ؉ currents in Xenopus oocytes were reduced by co-expression of ␣A-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of ␣A-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that ␣A-crystallin and ENaC coimmunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation.Like most other integral membrane proteins, newly synthesized epithelial Na ϩ channel (ENaC) 4 subunits translocate cotranslationally into the endoplasmic reticulum (ER), where folding and post-translational modifications occur. Within the ER, proteins are also subjected to quality control checkpoints to ensure that only properly folded proteins mature beyond the ER. If folding is inefficient, the misfolded protein may be degraded by proteasomes via a process known as ER-associated degradation (ERAD). This prevents the accumulation of abnormal proteins in the ER, which, left unchecked, may form toxic protein aggregates. Molecular chaperones, which can bind to exposed, hydrophobic motifs in unfolded proteins, play a key role in selecting substrates for this process.ENaC is expressed at the apical membranes of Na ϩ absorptive epithelia. There, in conjunction with the basolateral Na ϩ /K ϩ ATPase, ENaC facilitates transepithelial Na ϩ transport (1). ENaC is found in a variety of tissues, including the lung airway and alveoli and the distal nephron, where ENaC influences mucociliary clearance and extracellular fluid Na ϩ and volume regulation, respectively (1-3). ENaC is comprised of three homologous subunits, ␣, ␤, and ␥, although the stoichiometry of the functional channel remains controversial (4 -7). Each subunit has short cytoplasmic amino-and carboxyl-terminal domains (50 -110 residues), two transmembrane segments, and a large extracellular domain (ϳ450 residues) (8 -10). Like most other molecular chaperones, small heat shock proteins (sHsps) can bind unfolded, aggregation-prone substrates to retain them in solution. In addition, sHsps have been implicated in pro...

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 [35 S]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 Tyr 20 and Tyr 60 were both required for efficient endocytosis. Mutation of Tyr 20 significantly blocked coimmunoprecipitation of the chimera with AP-2, indicating that Y 20 HPM is recognized as a YXX motif by the 2 subunit. The tyrosinephosphorylated Y 60 TNP was previously identified as an SH2 site for Grb2 binding, and we found that mutation of Tyr 60 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 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 expression 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 ␤-catenin in the cytoplasm and nucleus in metastatic lesions of breast cancer patients; aberrant cytoplasmic and nuclear levels of activated ␤-catenin in breast tumors also correlates with a poor prognosis for the patient (13). ␤-Catenin binding to the cytop...