Gastric H؉ -ATPase activity. Thus, amino acid replacement of the phosphorylation site is not tolerated and a stringent structure appears to be required for enzyme activity. When the lysine residue in the fluorescein isothiocyanate binding site (part of ATP binding site) was mutated to arginine, asparagine, or glutamic acid, the SCH 28080-sensitive K ؉ -ATPase activity was eliminated. However, the mutant in which this residue was changed to glutamine had about 30% of the activity, suggesting that amino acid replacement of this site is tolerated to a certain extent.

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.

Ezrin/radixin/moesin (ERM) proteins function as general cross-linkers between plasma membrane proteins and the actin cytoskeleton and are involved in the functional expression of membrane proteins on the cell surface. They also integrate Rho guanosine 5′-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rho-related proteins. They act as protein kinase A (PKA)-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation. Therefore, ERM proteins seem to play important roles in the membrane transport of electrolytes by ion channels and transporters. In this review, we focus on the pathophysiological roles of ERM proteins in in vivo studies and introduce the phenotypes of their knockout and knockdown mice.Key words ezrin/radixin/moesin protein; cytoskeleton; knockout mouse EZRIN/RADIXIN/MOESIN PROTEINS REGULATE CYTOSKELETAL ORGANIZATION BY CROSS-LINK-ING PLASMA MEMBRANES WITH THE ACTIN CY-TOSKELETON AND INTEGRATING RHO GTPASE SIGNALINGThe ezrin, radixin, and moesin (ERM) proteins are general cross-linkers between cortical actin filaments and plasma membranes. They are concentrated at cell surface structures such as microvilli, filopodia, uropods, ruffling membranes, retraction fibers, and cell adhesion sites where actin filaments are associated with plasma membranes, but not along cytoplasmic actin filaments such as stress fibers.1) ERM proteins also integrate Rho guanosine 5′-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rhorelated proteins.2) The apparent molecular mass of ERM is 82, 80, and 75 kDa, respectively. They show high amino acid identity, especially in their amino-and carboxy-terminal domains. Their amino-terminal domains consisting of ca. 300 amino acid residues are termed "the band four point one and ERM (FERM) domain" because their amino acid sequences are conserved within ERM proteins and the erythrocyte band 4.1 protein (Fig. 1). FERM domains are also found in numerous membrane-associated signaling and cytoskeletal proteins such as talin.3) The FERM domains, which are composed of three structural modules (F1, F2, and F3), together form a compact clover-shaped structure 4,5) and bind to integral membrane proteins, [6][7][8][9][10][11][12][13][14][15][16] scaffold proteins, [17][18][19][20] and the Rho-related proteins (such as the Rho-guanosine 5′-diphosphate (GDP)-dissociation inhibitor [Rho-GDI] and Dbl) 21,22) listed in Table 1, as well as to phosphatidylinositol 4,5-bisphosphate (PIP 2 ). 12) The scaffold proteins Na + /H + exchanger regulatory factors (NHERF) 1 and 2, which contain two PDZ (PSD-95, Discs-large, and ZO-1) domains, bind to the FERM domain at the carboxy-terminus. 17,20) These PDZ domains on the NHERFs interact with the PDZ-binding motif of membrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Na 19,25,26) On the other hand, the carboxy-terminal domains, especially 34 amino acid residues, are also h...

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ABSTRACT. We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33°°°°C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39°°°°C). The cells expressed large T-antigen at 33°°°°C but not at 39°°°°C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + + + + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33°°°°C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium.