Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin

Mirre, C.; Monlauzeur, Laure; Garcia, Martine; Delgrossi, Marie-Hélène; Bivic, André Le

doi:10.1152/ajpcell.1996.271.3.c887

Cited by 84 publications

(79 citation statements)

References 13 publications

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“…This lends support to the notion that one important role of the stalk region in the overall structure of pro-SI is to serve as a link between the globular protein and the membrane. An association of O-glycans with lipid rafts has been demonstrated to be the driving sorting mechanism of pro-SI to the apical membrane (17,35) whereby this association is disrupted in the absence of O-glycans (19). The data presented here define structural determinants required for this association and demonstrate that O-glycans alone are neither sufficient for an association of pro-SI with lipid rafts nor for its apical targeting.…”

Section: Discussionmentioning

confidence: 78%

“…Other Procedures-Digestion of 35 S-labeled immunoprecipitates with endo-␤-N-acetylglucosaminidase H (Endo H) and endo-␤-N-acetylglucosaminidase F/glycopeptidase F (Endo F/GF) was performed as described previously (32). ⌬ST , and Pro-SI ⌬MA in MDCK Cells-To investigate the influence of the stalk and the membrane anchoring domain on the sorting of pro-SI in polarized cells, two constructs of pro-SI were generated.…”

Section: Materials Andmentioning

confidence: 99%

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Structural Determinants Required for Apical Sorting of an Intestinal Brush-border Membrane Protein

Jacob¹,

Alfalah²,

Grünberg³

et al. 2000

Journal of Biological Chemistry

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The distinct protein and lipid constituents of the apical and basolateral membranes in polarized cells are sorted by specific signals. O-Glycosylation of a highly polarized intestinal brush-border protein sucrase isomaltase is implicated in its apical sorting through interaction with sphingolipid-cholesterol microdomains. We characterized the structural determinants required for this mechanism by focusing on two major domains in pro-SI, the membrane anchor and the Ser/Thr-rich stalk domain. Deletion mutants lacking either domain, pro-SI ⌬ST (stalk-free) and pro-SI ⌬MA (membrane anchorfree), were constructed and expressed in polarized Madin-Darby canine kidney cells. In the absence of the membrane anchoring domain, pro-SI ⌬MA does not associate with lipid rafts and the mutant is randomly delivered to both membranes. Therefore, the O-glycosylated stalk region is not sufficient per se for the high fidelity of apical sorting of pro-SI. Pro-SI ⌬ST does not associate either with lipid rafts and its targeting behavior is similar to that of pro-SI ⌬MA . Only wild type pro-SI containing both determinants, the stalk region and membrane anchor, associates with lipid microdomains and is targeted correctly to the apical membrane. However, not all sequences in the stalk region are required for apical sorting. Only O-glycosylation of a stretch of 12 amino acids (Ala 37 -Pro 48 ) juxtapose the membrane anchor is required in conjunction with the membrane anchoring domain for correct targeting of pro-SI to the apical membrane. Other O-glycosylated domains within the stalk (Ala 49 -Pro 57 ) are not sufficient for apical sorting. We conclude that the recognition signal for apical sorting of pro-SI comprises O-glycosylation of the Ala 37 -Pro 48 stretch and requires the presence of the membrane anchoring domain.

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Section: Discussionmentioning

confidence: 78%

Section: Materials Andmentioning

confidence: 99%

Structural Determinants Required for Apical Sorting of an Intestinal Brush-border Membrane Protein

Jacob¹,

Alfalah²,

Grünberg³

et al. 2000

Journal of Biological Chemistry

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“…In this pathway, Shc is phosphorylated by the tyrosine kinase Fyn, and this phosphorylation is dependent on the presence of caveolin (9). While there is some controversy in the literature over whether Caco-2 cells contain caveolin (27,41,42), which has been detected in intestinal epithelium in vivo (41), Western blotting analysis failed to detect caveolin in the Caco-2 cells used in these experiments (not shown). Since Western blotting analysis indicated that Fyn (not shown), Shc, and ␣ 5 ␤ 1 integrin (Fig.…”

Section: Caco-2 Cell Erk Activation By Collagen IV Requires Fakmentioning

confidence: 85%

Collagen IV-dependent ERK Activation in Human Caco-2 Intestinal Epithelial Cells Requires Focal Adhesion Kinase

Sanders¹,

Basson²

2000

Journal of Biological Chemistry

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Integrin-initiated extracellular signal-regulated kinase (ERK) activation by matrix adhesion may require focal adhesion kinase (FAK) or be FAK-independent via caveolin and Shc. This remains controversial for fibroblast and endothelial cell adhesion to fibronectin and is less understood for other matrix proteins and cells. We investigated Caco-2 intestinal epithelial cell ERK activation by collagen I and IV, laminin, and fibronectin. Collagens or laminin, but not fibronectin, stimulated tyrosine phosphorylation of FAK, paxillin, and p130 cas and activated ERK1/2. Shc, tyrosine-phosphorylated by matrix adhesion in many cells, was not phosphorylated in Caco-2 cells in response to any matrix. Caveolin expression did not affect Caco-2 Shc phosphorylation in response to fibronectin. FAK, ERK, and p130 cas tyrosine phosphorylation were activated after 10-min adhesion to collagen IV. FAK activity increased for 45 min after collagen IV adhesion and persisted for 2 h, while p130 cas phosphorylation increased only slightly after 10 min. ERK activity peaked at 10 min, declined after 30 min, and returned to base line after 1 h. Transfection with FAK-related nonkinase, but not substrate domain deleted p130 cas , strongly inhibited ERK2 activation in response to collagen IV, indicating Caco-2 ERK activation is at least partly regulated by FAK.Cell-matrix interactions activate extracellular signal-regulated kinases (ERKs) 1 in several cell types (1-5). Integrins, the principal cellular receptors for the extracellular matrix, have been theorized to initiate this activation by at least two different pathways. In one proposed mechanism, ␣ 5 ␤ 1 integrin activation activates ERK1 and -2 via association of Src with tyrosine-phosphorylated focal adhesion kinase (FAK) and subsequent activation of the Ras/Raf/MAP kinase kinase (MEK) pathway (6, 7). In contrast, a FAK-independent mechanism has been described for ␣ 1 ␤ 1 , ␣ 5 ␤ 1 , and ␣ v ␤ 3 integrins. In this pathway, extracellular matrix activates ERK via association of these integrins with the integral membrane protein caveolin and subsequent tyrosine phosphorylation of the adaptor protein Shc by the tyrosine kinase Fyn in some cell types (8,9). Other studies have also indicated Ras and FAK-independent activation of ERK in response NIH 3T3 cell adhesion to fibronectin (10, 11). Much of this work has focused on fibroblast and endothelial cell lines adherent to fibronectin. Less is known about mechanisms of ERK activation in other cell types or initiated by other matrix proteins such as collagens or laminin.Intestinal epithelial cells in vivo exist on a basement membrane rich in laminin and type IV collagen, while type I collagen and tissue fibronectin are present in the interstitial matrix below the basement membrane. Several investigators, including our own group, have described regulation of intestinal epithelial cell adhesion, spreading, migration, and brush border enzyme activity on these matrix proteins (Refs. 12-18; reviewed in Refs. 19 -23). While in vivo and in vitro studies...

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“…Wild-type Caco_2 cells, which do not express caveolin_1 ( Fig. 1 and see also Mirre et al 1996), generate only a small membrane current when exposed to a 25% hypotonic stimulus (HTS) (Fig. 2A).…”

Section: Immunofluorescencementioning

confidence: 99%

Caveolin‐1 modulates the activity of the volume‐regulated chloride channel

Trouet

Nilius

Jacobs

et al. 1999

The Journal of Physiology

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Caveolae are Ù-shaped invaginations of the plasma membrane with a diameter of 50-100 nm that are present in many different cell types (Anderson, 1998). Caveolae form discrete lipid microdomains within the plasma membrane due to the preferential packing of sphingolipids and cholesterol. The principal protein component of caveolae is caveolin, a 21-24 kDa membrane protein that inserts into the caveolar membrane via an intramembrane hairpin loop. Four different isoforms encoded by three genes have been described: caveolin_1á, _1â, _2 and _3 (Parton, 1996). Caveolins form homo-and hetero-oligomeric complexes (Song et al. 1997) that bind to cholesterol and sphingolipids, which seems to be an important step in the formation of caveolae (Li et al. 1996). Heterologous expression of caveolin_1 in lymphocytes or CaCo_2 cells that lack both caveolin_1 and morphological caveolae induces the appearance of caveolae in the plasma membrane (Fra et al. 1995;Mirre et al. 1996;Vogel et al. 1998). It has recently emerged that caveolae form specialised membrane compartments involved in signal transduction with caveolin_1 forming a molecular scaffold onto which signalling proteins such as the endothelial NO synthase, the c-src protein tyrosine kinase or small GTPases such as Ras and Rho are assembled in their inactive configuration (Okamoto et al. 1998).

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Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin

Cited by 84 publications

References 13 publications

Structural Determinants Required for Apical Sorting of an Intestinal Brush-border Membrane Protein

Structural Determinants Required for Apical Sorting of an Intestinal Brush-border Membrane Protein

Collagen IV-dependent ERK Activation in Human Caco-2 Intestinal Epithelial Cells Requires Focal Adhesion Kinase

Caveolin‐1 modulates the activity of the volume‐regulated chloride channel

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