Localization of RhoA GTPase to Endothelial Caveolae-Enriched Membrane Domains

Gingras, Denis; Gauthier, France; Lamy, Sylvie; Desrosiers, Richard R.; Béliveau, Richard

doi:10.1006/bbrc.1998.8885

Cited by 105 publications

(96 citation statements)

References 24 publications

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“…The mechanism of communication between cholesterol-rich domains such as caveolae and lipid rafts, which harbor the initial, inactive signaling complexes, and focal adhesions that organize active signaling complexes is unclear. It is possible that these two compartments may interact physically via actin-binding proteins or via GTP-binding proteins such as RhoA, which are found in caveolae-enriched membrane domains as well as focal adhesions (27,33). The exact temporal and spatial relationships between cholesterolrich microdomains and focal adhesions in activation of Ang II signaling will require further investigation.…”

Section: Fig 2 Effect Of Cholesterol-binding Agents On Egf-r Phosphmentioning

confidence: 99%

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

Ushio‐Fukai¹,

Hilenski²,

Santanam³

et al. 2001

Journal of Biological Chemistry

187

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Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT 1 receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by ␤-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca 2؉ increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosinephosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.Angiotensin II (Ang II) 1 is a highly pluripotential hormone in vascular smooth muscle cells (VSMCs) and stimulates multiple signaling pathways, including Src family kinases, as well as mitogen-activated protein kinases (MAPKs) and Akt/protein kinase B (PKB), that mediate VSMC hypertrophy and growth via AT 1 receptors (AT 1 Rs). Increasing evidence suggests that transmodulation of the epidermal growth factor receptor (EGF-R), which serves as a scaffold for various signaling molecules, plays an essential role in organizing Ang II-mediated tyrosine kinase signaling pathways. We and others (1, 2) have demonstrated that EGF-R transactivation by Ang II is mediated through Ca 2ϩ , c-Src, and NADPH oxidase-derived reactive oxygen species, leading to activation of downstream signaling such as extracellular signal regulated kinase (ERK) and Akt/ PKB in VSMCs.Relatively little is known of the mechanisms controlling the spatial and temporal organization of AT 1 R signaling in VSMCs or of how specificity is achieved. We showed originally that Ang II signaling in VSMC is biphasic and that internalization or sequestration of the agonist-occupied receptor into a "signaling domain" ...

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Section: Fig 2 Effect Of Cholesterol-binding Agents On Egf-r Phosphmentioning

confidence: 99%

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

Ushio‐Fukai¹,

Hilenski²,

Santanam³

et al. 2001

Journal of Biological Chemistry

187

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“…Association of RHO1, the Saccharomyces cerevisiae homolog of the mammalian RhoA, with cortical actin patches is saturable and requires prenylation (38). In addition, we reported that RhoA and Cdc42 are associated with caveolae-enriched membrane domains in endothelial cells and that RhoA interacts with caveolin-1 in vitro (39). Although the hypothesis of a membrane receptor for the small Rho proteins is supported by several observations, its existence remains to be clearly demonstrated.…”

Section: Discussionmentioning

confidence: 83%

Modulation of Rho and Cytoskeletal Protein Attachment to Membranes by a Prenylcysteine Analog

Desrosiers

Gauthier

Lanthier

et al. 2000

Journal of Biological Chemistry

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The GTPases Rho regulate the assembly of polymerized actin structures. Their C-terminal sequences end with the CAAX motif that undergo a lipidation of the cysteine residue. Analogs to the C-terminal ends of Rho proteins, N-acetyl-S-all-trans,trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, were used to analyze the role of prenylation in their membrane association. Silver-stained gels indicated that Nacetyl-S-all-trans-geranylgeranyl-L-cysteine treatment released only a few proteins of 20, 46, and 60 kDa. Western blot analysis showed that N-acetyl-S-all-trans-geranylgeranyl-L-cysteine released RhoB (10%), RhoA (28%), and Cdc42 (95%) from membranes, whereas N-acetyl-Sall-trans and trans-farnesyl-L-cysteine did not. Rab1, which possesses two geranylgeranyl groups, was also strongly extracted by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, whereas Ras, which is farnesylated, was not. Furthermore, N-acetyl-S-all-trans-geranylgeranyl-L-cysteine was very efficient (95%) in dissociating actin and tubulin from membranes but not integral membrane protein P-glycoprotein and sodium/phosphate cotransporter NaP i -2. The extraction of Rho and cytoskeletal proteins occurred below the critical micellar concentration of N-acetyl-S-all-trans-geranylgeranyl-Lcysteine. Membrane treatments with 0.7 M KI totally extracted actin, whereas 70% of Cdc42 was released. Actin was, however, insoluble in Triton X-100-treated membranes, whereas this detergent extracted (80%) Cdc42. These data show that Rho proteins and actin are not physically bound together and suggest that their extraction from membranes by N-acetyl-S-all-trans-geranylgeranyl-L-cysteine likely occurs via different mechanisms.

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“…It has been reported, instead, that caveola domains may be involved in potocytosis, signal transduction and cholesterol transport through caveolin-1, Rho, nNOS, IP ! -receptor and so on (Parton, 1996;Anderson, 1998;Gingras et al, 1998;Fujimoto et al, 1998;Aoki et al, 1999). In this connection, the colocalization of dystrophin is significant in terms of signal transduction as suggested for the dystrophin complex in skeletal muscle fibers (Yoshida et al, 2000).…”

Section: Discussionmentioning

confidence: 86%

Localization of Plectin and Other Related Proteins along the Sarcolemma in Smooth Muscle Cells of Rat Colon.

Tanaka

Hijikata

Murakami

et al. 2001

Cell Struct. Funct.

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ABSTRACT. Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments. To better understand the functional roles of plectin in smooth muscle cells, we examined the distribution of plectin and other related proteins in rat colon smooth muscles by confocal laser and electron microscopy. The sarcolemma of smooth muscle cells exhibits two ultrastructurally distinct domains, domains associated with dense plaques and caveola-rich domains. Staining with anti-plectin and anti-desmin antibodies showed that plectin was localized along the sarcolemma in an intermittent manner and desmin was distributed in the sarcoplasm and intermittently at the cell periphery where it was codistributed with desmin. Plectin exhibited complementary and non-overlapping distribution to caveolin-1 and dystrophin, components of caveola domains, whereas plectin was codistributed with vinculin, talin and integrin > > > >1, components of dense plaques. Plectin was also codistributed with > > > >2-chain laminin but not with > > > >1-chain laminin. Electron microscopic observations on the sarcolemma revealed close association of intermediate filaments with dense plaques. Correlated confocal and electron microscopy clearly demonstrated that anti-plectin fluorescence corresponded to dense plaques but not to caveola domains in electron microscopic images. These findings indicate that plectin is confined to dense plaques to which desmin intermediate filaments may be anchored in rat colon smooth muscle cells.Key words: smooth muscle/plectin/dense plaques/confocal laser microscopy/electron microscopy/caveolin-1 Plectin, a member of the plakin family, is a versatile cytoskeletal linker protein and occurs in a wide variety of tissues and cell types, in which plectin codistributes with various types of intermediate filament proteins, such as vimentin, cytokeratins, desmin and glial fibrillary acidic (GFA) protein (Wiche et al., 1983Hieda et al., 1992;Errante et al., 1994;Yaoita et al., 1996;Svitkina et al., 1996;Eger et al., 1997;Foisner et al., 1988Foisner et al., , 1991. Plectin is located at the intermediate filament-plasma membrane interface, such as hemidesmosomes, desmosomes, and cardiac muscle intercalated discs. Plectin appears to act as molecular bridges between intermediate filaments and actin filaments or microtubules, as shown by whole-mount electron microscopy (Wiche et al., 1982(Wiche et al., , 1983. More recently, plectin has been shown to link intermediate filaments to the Z-discs in skeletal muscle fibers (Hijikata et al., 1999). Consistent with these variable locations, plectin possesses the binding capacity to multiple cytoskeletal proteins, such as =-spectrin (fodrin), high molecular mass microtubule-associated proteins and integrin >4 (Hermann and Wiche, 1987;Svitkina et al., 1996;Rezniczek et al., 1998). The strategic localization and versatile binding property of plectin suggest that it plays significant functional roles in the structural inte...

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Localization of RhoA GTPase to Endothelial Caveolae-Enriched Membrane Domains

Cited by 105 publications

References 24 publications

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

Cholesterol Depletion Inhibits Epidermal Growth Factor Receptor Transactivation by Angiotensin II in Vascular Smooth Muscle Cells

Modulation of Rho and Cytoskeletal Protein Attachment to Membranes by a Prenylcysteine Analog

Localization of Plectin and Other Related Proteins along the Sarcolemma in Smooth Muscle Cells of Rat Colon.

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