Membrane Mucin Research Articles

MUC4 expression and localization in gastrointestinal tract and skin of human embryos

MUC4 is a heterodimeric membrane mucin, composed of two tightly linked subunits and implicated in the protection of wet-surfaced epithelia. Although human MUC4 and its rat analogue Muc4/sialomucin complex have been extensively studied in the adult human and in the adult and embryonic rat, respectively, there has been little attention paid to date to the human embryo. Based on studies with our monoclonal antibody 1G8 and commercial tissue arrays, we describe some unexpected features of the expression of MUC4 in human embryonic epithelia. In the human small intestine and colon, MUC4 appears at an earlier relative stage of development, compared to gestation time, than in the rat. Interestingly, MUC4 also appears in the embryo in the skin, then disappears late in gestation, consistent with its absence in adult skin. These results are consistent with an important protective role for MUC4 in the human embryo that is different from that in the rat or in the adult human.

MUC4 Is a Novel Prognostic Factor of Extrahepatic Bile Duct Carcinoma

Many of the patients with extrahepatic bile duct carcinoma (EHBDC) show a poor outcome. We have reported that MUC4 is a novel prognostic factor of pancreatic adenocarcinoma and intrahepatic cholangiocarcinoma. The aim of this study is to evaluate the prognostic significance of MUC4 expression in EHBDC. We examined the expression profile of MUC4 in EHBDC tissues from 70 patients using immunohistochemistry. MUC4 is a membrane mucin like MUC1. In addition, MUC4 is an intramembrane ligand for receptor tyrosine kinase ErbB2 and is related with regulation of p27. We compared the MUC4 expression with MUC1, ErbB2, or p27 expression in EHBDC. MUC4 was expressed in 36 of the 70 patients with EHBDC. There was no significant correlation between the MUC4 expression and MUC1, ErbB2, or p27 expression. The survival of 19 patients with high MUC4 expression (>or=20% of carcinoma cells stained) was significantly worse than that of the 51 patients with low MUC4 expression (under 20% of carcinoma cells stained; P = 0.0072). The univariate analysis showed that high MUC4 expression (P = 0.0072), high MUC1 expression (P = 0.0092), histologic grading (P = 0.0029), surgical margin involvement (P = 0.0137), and nodal metastasis (P = 0.0036) were statistically significant risk factors. The backward stepwise multivariate analysis showed that high MUC4 expression (P = 0.0195) and surgical margin involvement (P = 0.0358) were statistically significant independent risk factors. MUC4 expression in EHBDC is a new independent factor for poor prognosis and predicts the outcome of patients with EHBDC.

Membrane Mucin Muc4 Induces Density Dependent Changes in Erk Activation in Mammary Epithelial and Tumor Cells

The membrane mucin Muc4 has been shown to alter cellular behavior through both anti-adhesive effects on cell-cell and cell-extracellular matrix interactions and its ability to act as an intramembrane ligand for the receptor tyrosine kinase ErbB2. The extracellular-regulated kinase (Erk) pathway is regulated by both cell-matrix and cell-cell adhesion. An analysis of the effects of Muc4 expression on Erk phosphorylation in mammary tumor and epithelial cells which exhibit both adhesion dependent growth and contact inhibition of growth showed that the effects are density dependent, with opposing effects on proliferating cells and on contact-inhibited cells. In these cells, cell-matrix interactions through integrins are required activation of the Erk mitogenesis pathway. However, cell-cell interactions via cadherins inhibit the Erk pathway. Expression of Muc4 reverses both of these effects. In contact-inhibited cells, Muc4 appears to activate the Erk pathway at the level of Raf-1; this activation does not depend on Ras activation. The increase in Erk activity correlates with an increase in cyclin D1 expression in these cells. This abrogation of contact inhibition is dependent on the number of mucin repeats in the mucin subunit of Muc4, indicative of an anti-adhesive effect. The mechanism by which Muc4 disrupts contact inhibition involves a Muc4-induced relocalization of E-cadherin from adherens junctions at the lateral membrane of the cells to the apical membrane. Muc4-induced abrogation of contact inhibition may be an important mechanism by which tumors progress from an early more benign state to invasiveness.

Reduction of mucin-1 gene expression associated with increased Escherichia coli adherence in the canine uterus in the early stage of dioestrus

The relation between adherence of Escherichia coli and expression of mucin-1 (Muc1: an integral membrane mucin) mRNA in the endometrium was studied in beagle bitches at different stages of the oestrous cycle and in those with cystic endometrial hyperplasia/pyometra complex (pyometra). The number of E. coli adhering to the endometrium was low at pro-oestrus and oestrus and increased at the early stage (day 10) of dioestrus, corresponding to the implantation period; it declined thereafter. Adhesion of the organisms to endometrial epithelial cells collected at day 10 of dioestrus was inhibited by the addition of d-mannose. When endometrial epithelial cells collected at pro-oestrus were treated with hyaluronidase, an enzyme that digests mucins, the numbers of E. coli adhering to the cells tended to increase. With polymerase chain reaction analysis it was possible to detect Muc1 gene transcripts in the endometrium at all stages of the oestrous cycle, although the level of Muc1 mRNA decreased by day 10 of dioestrus. The levels of Muc1 mRNA in bitches with a clinical stage of pyometra were low and comparable to those at day 10 of dioestrus. The number of E. coli adhering to the endometrium and Muc1 mRNA levels in the endometrium were inversely correlated ( r = −0.77, P < 0.01). Immunohistochemical analysis showed little staining for Muc1 in the endometrial epithelia at day 10 of dioestrus and in bitches with pyometra. These results suggest that reduction of Muc1 expression is associated with increased E. coli adherence in the canine uterus at the early stage of dioestrus, possibly facilitating the development of pyometra.

Molecular cloning and characterization of bovine P-selectin glycoprotein ligand-1

Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH 2-terminal of PSGL-1, the putative P-selectin binding domain. In the NH 2-terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular P-selectin binding domain.

Glycoprotein contributions to mammary gland and mammary tumor structure and function: Roles of adherens junctions, ErbBs and membrane MUCs

Mammary function is dependent on its three-dimensional organization, which is established and maintained by cell adhesive junctions linked through the membrane to the cell cytoskeleton. These junctions serve not only as structural elements, but also function as initiators and integrators of cell signals. In this review we discuss three types of glycoproteins whose interactions impinge on the function of mammary cell-cell junctions, cadherins, ErbB receptor tyrosine kinases and membrane mucins, as a microcosm of events regulating mammary cell behaviors. Actions of these components are integrated by the critical signaling element beta-catenin. When functioning properly, these glycoproteins, beta-catenin and associated signaling pathways mesh into a highly structured program for development and function of the gland. However, disruption or dysfunction of these glycoproteins or the signaling elements can lead to disorganization of the epithelia and ultimately to neoplasia.

Muc1 affects c-Src signaling in PyV MT-induced mammary tumorigenesis

MUC1 is an integral membrane mucin glycoprotein that is normally expressed on the apical surface of most simple, secretory epithelia and hematopoietic cells. Overexpression of aberrantly glycosylated MUC1 is a hallmark of many carcinomas including 90% of breast carcinomas. MUC1 has been shown to bind to c-Src tyrosine kinase in vitro, whereby c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif. c-Src is an extensively studied nonreceptor tyrosine kinase implicated in mammary tumorigenesis. Previously, mouse mammary tumor virus-driven polyoma middle T-antigen (MMTV-PyV MT) transgenic mice crossed onto a Muc1 null background exhibited a significant delay in tumor progression. c-Src has been shown to interact with PyV MT, and to play an integral and indispensable role in MMTV-PyV MT-induced mammary tumorigenesis. Here, we determine the effect of Muc1 expression on c-Src activation and signaling. Examination of MMTV-PyV MT glands on a wild-type or Muc1 null background demonstrates that Muc1 expression promotes c-Src signaling by influencing its association with known substrates such as the p85 subunit of phosphatidylinositol 3-kinase and beta-catenin. These findings may provide a mechanism for the delay in tumor progression that is observed in the absence of Muc1.

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP-2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non-adhesive luminal surface and to act as an intrinsic protection and survival factor.

Characterization of human PA2.26 antigen (T1α-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1alpha-2, podoplanin), a small mucin-type transmembrane glycoprotein originally identified as a cell-surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E-cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell-surface protrusions and reduced Ca(2+)-dependent cell-cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.

PA2.26 antigen (T1alpha, podoplanin), a membrane mucin associated with cancer which is involved in epithelial-mesenchymal transitions

PA2.26 antigen (T1alpha, podoplanin), a membrane mucin associated with cancer which is involved in epithelial-mesenchymal transitions

The Trypanosoma cruzi membrane mucin AgC10 inhibits T cell activation and IL-2 transcription through L-selectin.

Trypanosoma cruzi infection is associated with a severe T cell unresponsiveness to antigens and mitogens characterized by a decreased IL-2 synthesis and by nitric oxide (NO) production. Although spleen cell unresponsiveness to ConA was less severe in infected IFN-gammaR-/- or inducible nitric oxide synthase (iNOS)-/- mice than in control littermates, IL-2 inhibition was as severely impaired. Ag C10, a T. cruzi mucin, inhibited T cell proliferation as well as IL-2 secretion and IL-2 mRNA induction in response to mitogens and to anti-CD3. This effect took place at the transcriptional level since Ag C10 was able to inhibit IL-2 promoter-driven transcription. Moreover, the transcription of reporter genes controlled by CD28RE, NFAT or AP-1, but not by NF-kappaB sites, were inhibited by AgC10 to different degrees, although the greatest effect was observed for NFAT. In agreement with that, overexpression of NFAT significantly reverted Ag C10 inhibition of IL-2 transcription. AgC10 also inhibited early steps of T cell activation as tyrosine phosphorylation of the tyrosine kinase ZAP-70 and the adapter protein SLP-76. AgC10 binds to T cell surface through CD62L, and antibodies to CD62L inhibited T cell proliferation and IL-2 secretion and transcription as efficiently as AgC10. Indeed, AgC10 did not inhibit activation by T cells from CD62L-deficient mice (Sell-/-). Our results suggest that Ag C10, through binding to L-selectin, was able to inhibit different activation pathways that lead to inhibition of IL-2 secretion and T cell proliferation. This was independent of NO and IFN-gamma.

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function.

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.

MUC4 and ErbB2 expression in squamous cell carcinoma of the upper aerodigestive tract: correlation with clinical outcomes.

Expression of the membrane mucin MUC4 has been associated with a variety of malignancies, including squamous cell carcinoma of the upper aerodigestive tract. MUC4 modulates cell signaling pathways as an intramembrane ligand of ErbB2. The hypotheses of the study were that MUC4 expression would correlate with ErbB2 expression and that MUC4 expression would correlate with clinical outcomes in squamous cell carcinoma of the upper aerodigestive tract. Retrospective chart review was combined with immunohistochemical analysis of paraffin-embedded tumor specimens from patients treated with initial definitive surgical resection at an academic tertiary care medical center. MUC4 and ErbB2 receptor expression was localized by immunohistochemical studies using archival formalin-fixed and paraffin-embedded tissue. A limited number of fresh-frozen tissues were further analyzed by Western blot. Clinical outcomes and histopathological parameters were determined by retrospective chart review and correlated with immunohistochemical findings. One hundred fifty-four patients were analyzed with a median follow-up of 12 months among 54 patients who died and 49 months among 100 surviving patients. Membrane expression of MUC4 and ErbB2 was seen in 12% and 13% of tumors, respectively. MUC4 expression was not correlated with pathological grade. A significant correlation was found between MUC4 expression and ErbB2 expression. Multivariate survival analyses revealed that patients whose tumors exhibited MUC4 membrane expression had statistically significant improvement in survival and longer time to recurrence compared with patients whose tumors did not express MUC4 as defined by immunohistochemical staining patterns. No correlations between ErbB2 expression and survival or recurrence were observed. Patients with tumors that retain MUC4 expression exhibit improved survival and decreased recurrence in squamous cell carcinoma of the upper aerodigestive tract. Correlations between MUC4 expression patterns and ErbB2 expression are also observed, suggesting that MUC4-ErbB2 mediated cell signaling pathways may provide insights into this clinical result.

Changes in mucin‐type O‐glycosylation in breast cancer: implications for the host immune response

The major epithelial mucin, which is expressed by more than 90% of breast carcinomas and epithelial breast cancer cell lines, is MUC1. The MUC1 membrane mucin has a relatively simple structure, where a tandem repeat (TR) region carrying multiple O‐glycans forms the major part of the extracellular domain. The gene shows a size polymorphism, due to variation in the number of tandem repeats, each of which has five potential glycosylation sites. MUC1 has been found to be a good model for studying mucin‐type O‐glycosylation both in vitro and in vivo.Changes in the pattern of glycosylation of MUC1 have been observed in breast cancer (Burchell et al. 2001) and have led to an interest in MUC1 as a possible target antigen for immunotherapeutic intervention (Taylor‐Papadimitriou et al. 2000). The mucin expressed at high levels in the normal lactating mammary gland carries mainly core 2‐based O‐glycans (2–3 per tandem repeat). In breast cancers, the level of the ST3 Gal 1 enzyme is elevated (Burchell et al. 1999) and this is associated with increased numbers of Sialyl T O‐glycans being added, as predicted from in vitro studies, showing competition for the core 1 substrate between this sialyl transferase and the relevant core 2 enzyme (Dalziel et al. 2001). Moreover, the TR domain is more densely glycosylated. While the Sialyl T O‐glycan is commonly found on normal cells (e.g. resting T cells), it is cancer associated in the mammary gland. However, several lines of evidence indicate that this MUC1 glycoform, as expressed on breast cancers, can be immunosuppressive, maybe reflecting an inhibitory interaction with a lectin, possibly a siglec (Nath et al. 1999), on immune effector cells. In 25–30% of breast cancers, O‐glycosylation is terminated very early so that GalNAc (Tn) or NeuAc α2,6 GalNAc (Sialyl Tn) glycans appear. Sialyl Tn is highly tumour‐specific and is detected immunohistochemically only when the enzyme ST6GalNAcI is expressed. The Tn and Sialyl Tn will bind to different lectins, including MGL and could stimulate the immune response. Preliminary studies in mouse models suggest that an immunization protocol which includes MUC1‐carrying Sialyl Tn O‐glycans can protect against tumour growth, while clinical studies in breast cancer patients using immunogens based on the Sialyl Tn glycan are underway.

AgC10, a mucin from Trypanosoma cruzi, destabilizes TNF and cyclooxygenase-2 mRNA by inhibiting mitogen-activated protein kinase p38.

Secretion of proinflammatory mediators by activated macrophages plays an important role in the immune response to Trypanosoma cruzi. We have previously reported that AgC10, a glycosylphosphatidylinositol-anchored mucin from T. cruzi, inhibits TNF secretion by activated macrophages (de Diego, J., Punzon, C., Duarte, M. and Fresno, M., Alteration of macrophage function bya Trypanosoma cruzi membrane mucin. J. Immunol. 1997. 159: 4983-4989). In this report we have further investigated the molecular mechanisms underlying this inhibition. AgC10 inhibited TNF, IL-10 and cyclooxygenase-2 (COX-2) synthesis by macrophages activated with LPS or LPS plus IFN-gamma in a dose-dependent manner. AgC10 did not affect other aspects of macrophage activation induced by LPS, such as inducible nitric oxide synthase (iNOS) expression. AgC10 also had no effect on TNF or COX-2 transcription or the induction of their promoters but inhibited the stability of TNF and COX-2 mRNA, which are regulated post-transcriptionally by the mitogen-activated protein kinase (MAPK) p38 pathway. AgC10 was found to inhibit both the activation and the activity of p38 MAPK, since MAPK activated protein kinase-2 (MAPKAP-K2 or MK-2) phosphorylation was also strongly inhibited. This led to TNF and COX-2 mRNA destabilization. In contrast, AgC10 did not affect p38 activation induced by TNF. Furthermore, AgC10 inhibition must lie upstream in the MAPK activation pathway by LPS, since this mucin also inhibited extracellularly regulated kinase (ERK) and Jun kinase (JNK)activation.

Evidence for a second peptide cleavage in the C-terminal domain of rodent intestinal mucin Muc3.

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G downward arrow SIVV (where G downward arrow S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623-631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse-chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand-receptor and signal transduction events for enterocyte function in vivo.

PA2.26 antigen (T1α/podoplanin): a small mucin-like transmembrane glycoprotein associated with cell migration and cancer

PA2.26 antigen was originally identified as a small mucin-type glycoprotein induced during chemical carcinogenesis on mouse skin. Cloning and sequencing of the murine and human PA2.26 cDNAs revealed that the sequence was identical to other reported proteins termed T1α (a marker for alveolar type I epithelial cells) and podoplanin (a glycoprotein expressed in glomerular podocytes). In this review, we summarise the current knowledge on the biology of PA2.26 (T1α/podoplanin). Although the physiological function of this protein is not well established as yet, several characteristics make this molecule unique among mucin-like glycoproteins. For example, PA2.26 (T1α/podoplanin) appears to play an important role in morphogenesis during development, and it is a marker for the lymphatic endothelium. In cancer, PA2.26 is induced in tumours derived from squamous epithelia, and is associated with cell migration and malignant progression. These findings are discussed in the context of cancer-associated membrane mucins in which PA2.26 antigen, together with a newly-discovered small mucin called dysadherin, form a special group.

Initiation of Transcription of the MUC3A Human Intestinal Mucin from a TATA-less Promoter and Comparison with the MUC3B Amino Terminus

Human intestinal mucin genes MUC3A and MUC3B are members of a membrane mucin gene family residing at chromosome 7q22. In this paper, we utilized genomic and cDNA cloning to elucidate the sequence of the 5'-region of the MUC3A gene including the gene promoter and the amino terminus coding sequence. Following its 21-residue signal peptide, the amino terminus of the mucin consists of a 233-residue Thr-, Ser-, and Pro-rich nonrepetitive sequence that is contiguous with its hypervariable domain of 375-residue repeats. RNase protection analysis and 5'-GeneRacer PCR indicated that MUC3A gene transcripts initiate from multiple start sites along a region spanning approximately 180 bases. The 5'-flanking region of the gene had promoter activity when fused to a luciferase reporter gene in all of the tested cell lines. This region contained binding sites for several transcription factors, including those implicated in the regulation of intestinal genes, but lacked a cognate TATA box. These features of the gene promoter may enable the gene to be expressed at variable levels in several cell types with different repertoires of transcription factors. We also utilized 5'-GeneRacer PCR to determine the sequence of the 5'-terminus of the MUC3B message. The amino termini of the MUC3A and MUC3B mucins are 91% conserved at the amino acid level. Thus, MUC3A and MUC3B have highly conserved amino and carboxyl termini, suggesting a recent duplication of the entire ancestral gene. It remains to be determined whether other members of the 7q22 membrane mucin gene family have amino-terminal domains similar to MUC3A and MUC3B.

Nuclear Association of the Cytoplasmic Tail of MUC1 and β-Catenin

MUC1, an integral membrane mucin associated with the metastatic phenotype, is overexpressed by most human carcinoma cells. The MUC1 cytoplasmic tail (CT) is postulated to function in morphogenetic signal transduction via interactions with Grb2/Sos, c-Src, and beta-catenin. We investigated intracellular trafficking of the MUC1 CT, using epitope-tagged constructs that were overexpressed in human pancreatic cancer cell lines S2-013 and Panc-1. The MUC1 CT was detected at the inner cell surface, in the cytosol, and in the nucleus of cells overexpressing MUC1. Fragments of the MUC1 CT were associated with beta-catenin in both cytoplasm and nuclei. Overexpression of MUC1 increased steady state levels of nuclear beta-catenin but decreased nuclear levels of plakoglobin (gamma-catenin). There was no detectable association between plakoglobin and the MUC1 CT. Coimmunoprecipitation experiments revealed that the cytoplasmic and nuclear association of MUC1 CT and beta-catenin was not affected by disruption of Ca2+-dependent intercellular cadherin interactions. These results demonstrate nuclear localization of fragments of MUC1 CT in association with beta-catenin and raise the possibility that overexpression of the MUC1 CT stabilizes beta-catenin and enhances levels of nuclear beta-catenin during disruption of cadherin-mediated cell-cell adhesion.

SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3.

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine downward arrow serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.