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GPI-anchored proteins are partially resistant to TX-100 extraction in Caco-2 cells. Cells were broken by passing through a 23 G needle. Post-nuclear supernatant was obtained by centrifugation (1,000 g, 10 min, 4°C). After 5 min of extraction with 1% OG or 1% TX-100, soluble (s) and insoluble (l) materials were separated by centrifugation (100,000 g, 1 h, 4°C). Immunodetection of antigens was performed by SDS-PAGE (6% to 15%) and Western blotting with 125 I-Protein A. Black arrowhead indicates CEA180; white arrowhead indicates CEA110. GPI-anchored proteins (PLAP, CEA180) were fully solubilized in OG and only partially solubilized in TX-100.
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In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-oc...
Citations
... In addition, a subapical cell compartment seems to function as a docking platform for vesicles containing functional proteins (98). Parental Caco-2 cells and clones have also been used to identify the mechanisms underlying the sorting and surface delivery of apical and basolateral proteins in human enterocytes (99)(100)(101)(102)(103)(104)(105)(106)(107) and to find out how functional intestinal proteins take their place in cell membrane domains, including brush border-associated functional proteins such as SI (82,84,86,90,94,100,106,(108)(109)(110)(111)(112)(113)(114), AP (110,111), lactasephlorizin hydrolase (108,115), maltase-glucoamylase (108), APN (90,108,111), DPP IV (82,90,100,108,(116)(117)(118)(119)(120)(121), angiotensin I-converting enzyme (108), ␣-glucosidase (122), p-aminobenzoic acid peptide hydrolase (108), SGLT1, GLUT1, GLUT2, GLUT3, and GLUT5 (123)(124)(125), PEPT1 (126), H ϩ -coupled dipeptide transporter (127), NHEs (128,129), Cl/HCO 3 exchanger DRA (130), monocarboxylate transporter 1 (MCT1) (131), cholesteryl ester transfer protein (132), AQP3 and AQP10 (133)(134)(135)(136)(137), Na ϩ -K ϩ ATPase (110), and diamine oxidase (117,138) (Table 1) (Fig. 5G to I). Parental cells and clones have been also used to study the regulatory functions of membrane-bound receptors, including EGF receptor (139), IGF-BP-2, IGF-BP-3, and IGF-BP-4 (139), VIP receptor (52), PAR2 (51), and PPAR gamma (140). ...
... In addition, a subapical cell compartment seems to function as a docking platform for vesicles containing functional proteins (98). Parental Caco-2 cells and clones have also been used to identify the mechanisms underlying the sorting and surface delivery of apical and basolateral proteins in human enterocytes (99)(100)(101)(102)(103)(104)(105)(106)(107) and to find out how functional intestinal proteins take their place in cell membrane domains, including brush border-associated functional proteins such as SI (82,84,86,90,94,100,106,(108)(109)(110)(111)(112)(113)(114), AP (110,111), lactasephlorizin hydrolase (108,115), maltase-glucoamylase (108), APN (90,108,111), DPP IV (82,90,100,108,(116)(117)(118)(119)(120)(121), angiotensin I-converting enzyme (108), ␣-glucosidase (122), p-aminobenzoic acid peptide hydrolase (108), SGLT1, GLUT1, GLUT2, GLUT3, and GLUT5 (123)(124)(125), PEPT1 (126), H ϩ -coupled dipeptide transporter (127), NHEs (128,129), Cl/HCO 3 exchanger DRA (130), monocarboxylate transporter 1 (MCT1) (131), cholesteryl ester transfer protein (132), AQP3 and AQP10 (133)(134)(135)(136)(137), Na ϩ -K ϩ ATPase (110), and diamine oxidase (117,138) (Table 1) (Fig. 5G to I). Parental cells and clones have been also used to study the regulatory functions of membrane-bound receptors, including EGF receptor (139), IGF-BP-2, IGF-BP-3, and IGF-BP-4 (139), VIP receptor (52), PAR2 (51), and PPAR gamma (140). ...
... It is well established (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)) that many membrane proteins are organized in clusters to perform their cellular function, rather than diffuse freely on the lipid membrane. A question then naturally arises as to how or why those proteins self-assemble into clusters. ...
... A question then naturally arises as to how or why those proteins self-assemble into clusters. For transmembrane proteins, several physical models have been proposed, and the protein-protein interaction can be specific, homophilic interactions (18) or nonspecific, including depletion interactions due to the lipid osmotic pressure (27) and hydrophobic mismatch (28). Schmidt et al. (29) demonstrated that the cluster formation of transmembrane proteins can be attributed to the effects of hydrophobic mismatch (30,31). ...
... When the hydrophobic domain of transmembrane proteins does not match the thickness of the membrane hydrophobic core, clustering of proteins has been observed. Anchored proteins, such as glycosylphosphatidyl-inositol-anchored proteins, have been validated to form clusters experimentally (18,23); however, the protein-protein interaction and the mechanism of clustering are rarely studied. ...
Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss-both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins.
... Role of furin-processed CD109 in TGF-b signaling S Hagiwara et al GPI-anchored proteins are thought to be recruited to lipid rafts on the plasma membrane. Thus, cells were treated with the non-ionic detergent, n-octyl-b-D-glucopyranoside (nOG), which can dissolve lipid rafts (Garcia et al., 1993). Treatment with increasing concentrations of nOG (0-2%) resulted in the immunoprecipitation of increasing amounts of 25 kDa CD109 with the anti-CD109-2 antibody ( Figure 3c). ...
CD109 is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, whose expression is upregulated in squamous cell carcinomas of the lung, esophagus, uterus and oral cavity. CD109 negatively regulates transforming growth factor (TGF)-beta signaling in keratinocytes by directly modulating receptor activity. In this study, we further characterized CD109 regulation of TGF-beta signaling and cell proliferation. We found that CD109 is produced as a 205 kDa glycoprotein, which is then processed in the Golgi apparatus into 180 kDa and 25 kDa proteins by furin (furinase). 180 kDa CD109 associated with GPI-anchored 25 kDa CD109 on the cell surface and was also secreted into the culture medium. To investigate whether furinase cleavage of CD109 is necessary for its biological activity, we mutated arginine 1273 in the CD109 furinase cleavage motif (amino acid 1270-RRRR-1273) to serine (R1273S). Interestingly, CD109 R1273S neither significantly impaired TGF-beta signaling nor affected TGF-beta-mediated suppression of cell growth, although it was expressed on the cell surface as a 205 kDa protein. Consistent with this finding, the 180 kDa and 25 kDa CD109 complex, but not CD109 R1273S, associated with the type I TGF-beta receptor. These findings indicate that processing of CD109 into 180 kDa and 25 kDa proteins by furin, followed by complex formation with the type I TGF-beta receptor is required for the regulation of TGF-beta signaling in cancer cells and keratinocytes.
... The nondelivery of apical glycoproteins in the absence of galectin-4 suggests that this lectin plays a pivotal role in the quality control of the biogenesis of the brush border membrane of enterocyte-like HT-29 5M12 cells. In the model of enterocytic Caco-2 cells, for which raft-dependent apical trafficking has also been reported (30,31), galectin-4 begins to be expressed after confluence, when the cells begin their enterocytic differentiation with the concomitant presence of an apical brush border and of brush border-associated hydrolases, suggesting that galectin-4 could also play a role in the differentiation process of this cell type (data not shown). ...
We have previously reported that silencing of galectin-4 expression in polarized HT-29 cells perturbed apical biosynthetic trafficking and resulted in a phenotype similar to the inhibitor of glycosylation, 1-benzyl-2-acetamido-2-deoxy-beta-d-galactopyranoside (GalNAcalpha-O-bn). We now present evidence of a lipid raft-based galectin-4-dependent mechanism of apical delivery of glycoproteins in these cells. First, galectin-4 recruits the apical glycoproteins in detergent-resistant membranes (DRMs) because these glycoproteins were depleted in DRMs isolated from galectin-4-knockdown (KD) HT-29 5M12 cells. DRM-associated glycoproteins were identified as ligands for galectin-4. Structural analysis showed that DRMs were markedly enriched in a series of complex N-glycans in comparison to detergent-soluble membranes. Second, in galectin-4-KD cells, the apical glycoproteins still exit the Golgi but accumulated inside the cells, showing that their recruitment within lipid rafts and their apical trafficking required the delivery of galectin-4 at a post-Golgi level. This lectin that is synthesized on free cytoplasmic ribosomes is externalized from HT-29 cells mostly in the apical medium and follows an apical endocytic-recycling pathway that is required for the apical biosynthetic pathway. Together, our data show that the pattern of N-glycosylation of glycoproteins serves as a recognition signal for endocytosed galectin-4, which drives the raft-dependent apical pathway of glycoproteins in enterocyte-like HT-29 cells.
... apikalen Domäne verwendet werden, wie es bereits früher vorgeschlagen worden ist (Hirokawa et al., 1983). Ferner ist bekannt, dass Mikrotubuli für einen effizienten apikalen Transport notwendig sind (Musch, 2004) (Fath et al., 1994;Tuxworth und Titus, 2000;Apodaca, 2001;Krendel und Mooseker, 2005;Delacour und Jacob, 2006 (Garcia et al., 1993;Danielsen, 1995;. (Gottlieb et al., 1993;McConnell und Tyska, 2007 smaller. ...
... transmembrane proteins are resistant, at least partially, to Triton extraction ( Garcia et al., 1993;Danielsen, 1995;Aït Slimane et al., 2003), the best examples being the influenza virus proteins hemagglutinin ( ) and neuraminidase ( ). Furthermore, several apical transmembrane proteins that are readily soluble in Triton X-100 are resistant to milder detergents such as Lubrol WX ( Aït Slimane et al., 2003), and a recent comparison showed that several apical proteins are resistant to Tween 20 extraction whereas basolateral proteins are fully solubilized ( Alfalah et al., 2005). ...
... Par exemple, dans les cellules intestinales, la maltase-glucoamylase, l'aminopeptidase N, l'aminopeptidase A, la dipeptidyl-peptidase IV et la saccharase-isomaltase montrent une insolubilité dans le Triton de 20% à 48%, tandis que la lactase-phlorizine hydrolase est complètement soluble (Danielsen, 1995). Une insolubilité partielle de la saccharase- isomaltase a également été rapportée dans les cellules Caco-2 ( Garcia et al., 1993), de même que pour la dipeptidyl-peptidase IV dans les cellules MDCK et HepG2 cells (Slimane et al., 2001;. Dans les cellules MDCK, la protéine endogène gp114 est complètement soluble dans le Triton, mais elle devient insoluble après pontage par des anticorps ( Verkade et al., 2000). ...
Summary The plasmic membrane of the polarized epithelial cells comprises two distinct domains, the apical domain and the basolateral domain. Each domain has a determined composition in lipids and proteins, enabling them to ensure of the specific functions. The molecular mechanisms responsible for the sorting and the targeting of transmembrane proteins towards the apical pole are still badly known. The apical membrane is enriched in glycosphingolipides and cholesterol which form microdomains called “rafts”. In experiments, the rafts can be isolated in the form of DRM (detergent-resistant membranes) defined by their resistance to a nonionic detergent, the Triton X-100. It was proposed that the rafts recruit apical proteins on the level of the trans-golgien network and are used as platform for their targeting with the apical pole. Indeed the proteins anchored by the glycosylphosphatidyl-inositol are resistant to the Triton and are in general localised with the apical membrane. On the other hand, the majority of apical transmembrane proteins are soluble in the Triton, although they are resistant to the action of mild detergents like Lubrol WX. The objective of work of thesis was to study the role of the rafts in the apical transmembrane protein targeting and to include/understand the differential effect of the Triton and Lubrol on their solubilization. The nucleotides pyrophosphatases NPP1 (basolateral) and NPP3 (apical) expressed in a stable way in cells MDCK were used as models. NPP3 is insoluble in Lubrol and partially insoluble in the Triton, while NPP1 is primarily solubilized. The study of the localization and the sensitivity to the detergents of mutants and chimeraes a combining of the cytoplasmic, transmembrane and extracellular domains of NPP3 and NPP1, showed that there was not strict correlation between apical targeting and resistance to the detergents. The resistance of NPP3 to solubilization by Lubrol is acquired precociously during its biosynthesis, independently of its final destination. This resistance depends on amino acids charged positively located in the cytoplasmic tail, close to the membrane. In order to include/understand the selectivity of the Triton and Lubrol in the extraction of proteins and the membrane lipids, the lipidic composition of the DRM obtained after extraction by the Triton and Lubrol were compared. The DRM extracted by the Triton and Lubrol are cholesterol enriched what corresponds to the definition of the rafts. However, the DRM Triton are impoverished in lipids of the internal layer while the DRM Lubrol are enriched in phosphatidylethanolamine. The DRM Lubrol are also enriched in protein associated with the internal layer with the membrane. In conclusion, this work shows that the resistance of apical protein NPP3 to the extraction by Lubrol, and partly by the Triton, is an intrinsic property which probably corresponds to an adaptation of protein to the lipidic composition of the apical domain, but that this property does not determine its polarized targeting. Moreover, this work shows that the detergents are very interesting tools to study the interactions between the membrane proteins and lipids, but that there probably does not exist detergent able to insulate in a strict way of the membrane microdomains such as are defined the rafts. Our results suggest that the layer interns rafts is enriched in phosphatidylethanolamine and cholesterol, that it is partly solubilized by the Triton, which would destabilize transmembrane proteins and would involve their extraction. Keywords : detergent, raft,, apical targeting, cholesterol, membrane microdomain, inner membrane leaflet, phosphatidylethanolamine.
... In the case of the chimeric LI-cadherin molecules fused to a GPI-anchor clustering is very likely, since GPIanchored proteins are associated with raft-like membrane microdomains. 42,43 Indeed, T-cadherin, the only known GPI-anchored cadherin, 44 is capable of mediating Ca 2+ -dependent cell-cell adhesion 45 and was found to be located in distinct plasma membrane "rafts". 46 In two quantitative cell-cell adhesion assays based on stably transfected CHO cells, LI-cadherin and E-cadherin exhibited almost identical aggregation indices. ...
Cadherins are Ca(2+)-dependent transmembrane glycoproteins that mediate cell-cell adhesion and are important for the structural integrity of epithelia. LI-cadherin and the classical E-cadherin are the predominant two cadherins in the intestinal epithelium. LI-cadherin consists of seven extracellular cadherin repeats and a short cytoplasmic part that does not interact with catenins. In contrast, E-cadherin is composed of five cadherin repeats and a large cytoplasmic domain that is linked via catenins to the actin cytoskeleton. Whereas E-cadherin is concentrated in adherens junctions, LI-cadherin is evenly distributed along the lateral contact area of intestinal epithelial cells. To investigate if the particular structural properties of LI-cadherin result in a divergent homotypic adhesion mechanism, we analyzed the binding parameters of LI-cadherin on the single molecule and the cellular level using atomic force microscopy, affinity chromatography and laser tweezer experiments. Homotypic trans-interaction of LI-cadherin exhibits low affinity binding with a short lifetime of only 1.4 s. Interestingly, LI-cadherin binding responds to small changes in extracellular Ca(2+) below the physiological plasma concentration with a high degree of cooperativity. Thus, LI-cadherin might serve as a Ca(2+)-regulated switch for the adhesive system on basolateral membranes of the intestinal epithelium.
... For this purpose, we studied the solubilization profiles of SI with several nonionic detergents, Triton X-100, CHAPS, Brij 96, and Tween 20, in biosynthetically labeled Caco-2 cells or MDCK cells stably expressing SI (4). As shown in Fig. 1A the complex glycosylated mature form of SI (SI c ) was found to be associated with the Triton X-100insoluble fraction, confirming previous data on the association of this form with cholesterol/sphingolipid-rich microdomains in the TGN (48,49). An essentially similar pattern of extractability was obtained with CHAPS suggestive of similar solubilization properties of Triton X-100 and CHAPS and implying that the insoluble microdomains revealed upon solubilization with either one of these detergents are similar in their composition. ...
One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.
... Protein association with lipid rafts also could occur during intracellular transport. For example, the GPI-linked protein, PLAP, fully soluble in Triton X-100 after biosynthesis and for the time of residence in the ER compartment, becomes insoluble traveling between the ER and medial Golgi (Garcia et al., 1993). This suggests that insolubility is not an inherent property of GPIlinked and other proteins, but is acquired after exposure to the lipid environment that proteins encounter entering the Golgi complex, where liquid ordered rafts begin to form. ...
Adhesion molecules are known to mediate cell-cell interactions, particularly those between T cells and antigen-presenting or target cells. Recent studies identified ICAM-1 as a co-stimulatory ligand that binds to lymphocyte function associated antigen-1 (LFA-1), thereby promoting the activation of T cells. As ICAM-1 is expressed on virtually any cell, it becomes a crucial molecule for the activation of CD8(+) T cells in the absence of co-stimulation provided by CD80 and CD86 molecules. In addition, ICAM-1 might function as cell-surface receptor, capable of initiating intracellular signaling. ICAM-1 is associated with other cell molecules, including MHC-I proteins, and our recent data show that productive engagement of ICAM-1 on target cells leads to recruitment of the MHC-I proteins to the contact area and enhances presentation of cognate peptide MHC-I complexes to cytotoxic T cells.
... These data suggest that MHC-I and ICAM-1 are associated in both detergent-soluble and detergentinsoluble membranes, and that the MHC-I-ICAM-1 interactions are not necessarily supported by the structure of rafts. Consistent with this, we have found that exposure of isolated rafts to 1% N-octylglucoside, conditions in which raft integrity is no longer preserved, 33,34 led to only an insignificant decrease of the amount of co-immunoprecipitated HLA-A2 and ICAM-1 (Fig. 2a,b). ...
Polarization and segregation of the T-cell receptor (TCR) and integrins upon productive cytotoxic T-lymphocyte (CTL) target cell encounters are well documented. Much less is known about the redistribution of major histocompatibility complex class I (MHC-I) and intercellular adhesion molecule-1 (ICAM-1) proteins on target cells interacting with CTLs. Here we show that human leucocyte antigen-A2 (HLA-A2) MHC-I and ICAM-1 are physically associated and recovered from both the raft fraction and the fraction of soluble membranes of target cells. Conjugation of target cells with surrogate CTLs, i.e. polystyrene beads loaded with antibodies specific for HLA-A2 and ICAM-1, induced the accumulation of membrane rafts, and beads loaded with ICAM-1-specific antibodies caused the selective recruitment of HLA-A2 MHC-I at the contact area of the target cells. Disruption of raft integrity on target cells led to a release of HLA-A2 and ICAM-1 from the raft fraction, abatement of HLA-A2 polarization, and diminished the ability of target cells bearing viral peptides to induce a Ca(2+) flux in virus-specific CTLs. These data suggest that productive engagement of ICAM-1 on target cells facilitates the polarization of MHC-I at the CTL-target cell interface, augmenting presentation of cognate peptide-MHC (pMHC) complexes to CTLs. We propose that ICAM-1-MHC-I association on the cell membrane is a mechanism that enhances the linkage between antigen recognition and early immunological synapse formation.
