Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization

Pozo, Miguel Á. del; Balasubramanian, Nagaraj; Alderson, Nazilla; Kiosses, William B.; Grande-García, A.; Anderson, Richard G. W.; Schwartz, Martin A.

doi:10.1038/ncb1293

Cited by 382 publications

(447 citation statements)

References 31 publications

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“…5C). Recent progress in understanding the function of integrins has revealed a complicated regulation mechanism among integrins, the glycosphingolipid domain, and endocytosis (51,52). Considering that CTX A3 binds to integrin and the glycosphingolipid domain in the plasma membrane and that it can also be internalized into mitochondria via a still unknown mechanism, future investigations on CTX A3-induced CHO cell death should shed light on the cell signaling process involving integrins and the lipid domain.…”

Section: Resultsmentioning

confidence: 99%

Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption

Lee

Chuang

et al. 2006

Journal of Biological Chemistry

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Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656 -667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to ␣v␤3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble ␣v␤3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble ␣v␤3 bound to CTX A5 with an apparent affinity of ϳ0.3 M. Calf pulmonary artery endothelial cells, which constitutively express ␣v␤3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble ␣v␤3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in ␣v␤3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine ␣v␤3 integrin in osteoclasts and that CTX A5 co-localized with ␤3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.Severe tissue necrosis and inflammation with a retarded wound healing process are the major symptoms of a victim surviving from a cobra bite (1-4). Cobra cardiotoxins (CTXs) 3 are the major toxin peptides and constitute ϳ50% of the weight of cobra venom. CTXs are believed to play a critical role in cobra venom toxicity (5). We have shown that CTXs bind to glycosaminoglycans with specificity and are retained on the membrane surface for action (6 -8). Interestingly, one of the CTXs, CTX A3, interacts with sulfatide to form a pore and becomes internalized to further target mitochondria in cardiomyocytes and H9C2 myoblasts (9 -12). The mechanisms for CTX-induced perturbation of the wound healing process and severe tissue damage are unknown. It is not clear whether there are cellular receptors for CTXs, although other cobra venom components such as secretory phospholipase A 2 are known to have diverse targets for their actions by involving glycosaminoglycans, protein receptors, and membrane lipids (13,14). CTXs are all ␤-sheet basic polypeptides of 60 -62 amino acid residues with a three-fingered loop-folding topology and are members of the Ly-6 protei...

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Section: Resultsmentioning

confidence: 99%

Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption

Lee

Chuang

et al. 2006

Journal of Biological Chemistry

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“…This construct works by blocking phosphorylation of caveolin-1 at Y14 (27), a site far away from the caveolin scaffolding that inhibits eNOS function (2). Interestingly, we observed cell line-dependent differences with this construct.…”

Section: Discussionmentioning

confidence: 99%

Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

Sánchez

Rana²,

Kim³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by plateletactivating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant-and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECVCD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.caveolae ͉ endothelial nitric oxide synthase ͉ endothelial permeability ͉ inflammation ͉ protein traffic N itric oxide (NO) is the signal for several outcomes in the control of circulation. Its main source in the cardiovascular system is endothelial (e)NOS. The enzyme is modified by N-myristoylation and palmitoylation, which targets it to the caveolae in the plasma membrane where it is kept in a basal state by binding to caveolin-1 through a consensus site (1, 2). Agonists activate eNOS through multiple mechanisms: phosphorylation/ dephosphorylation of specific residues, interaction with different proteins, S-nitrosylation, and specific subcellular localization (1, 3-7). Agonists such as platelet-activating factor (PAF) and VEGF phosphorylate eNOS via Akt (8, 9). Despite advances in our understanding of the biochemistry of eNOS, the mechanisms by which these molecular modifications determine the functional outcome of eNOS activation remain unexplored.NO derived from eNOS is a key mediator in the hyperpermeability response to PAF and VEGF (7,10,11). We previously showed that eNOS internalization (endocytosis) is a required step in the signaling cascade leading to PAF-induced hyperpermeability (7). In this study, we tested the hypothesis that caveolar internalization of eNOS is a requirement for localized NO production and NO delivery to a subcellular target to cause hyperpermeability. To address this hypothesis, we used ECV-304 cells (...

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“…Cell adhesion can also control Rac, a GTPbinding protein that regulates actin structure, which translocates to the DRMs at the cell surface in response to adhesion. Both Rac and caveolin contribute to changes in cell migration and adhesion, with Rac promoting lamellipodia assembly and phospho-caveolin promoting integrin internalization [11]. A more detailed explanation of how Rac and caveolin modulate the cell adhesion response can be found in a recent review by del Pozo and Schwartz [12].…”

Section: Introductionmentioning

confidence: 99%

“…For example, caveolin, a cholesterolbinding protein commonly associated with DRMs and GM1, is internalized along with GM1 after cell detachment [11]. Moreover, phospho-caveolin has been shown to localize to focal adhesion sites [11], in agreement with the finding that adhesion sites have a higher lipid order than the cell membrane as a whole [9]. Cell adhesion can also control Rac, a GTPbinding protein that regulates actin structure, which translocates to the DRMs at the cell surface in response to adhesion.…”

Section: Introductionmentioning

confidence: 99%

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

Alvares

Dunn

Brown

et al. 2008

Biochimica et Biophysica Acta (BBA) - General Subjects

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Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and DetergentResistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals-possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior.

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Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization

Cited by 382 publications

References 31 publications

Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption

Non-cytotoxic Cobra Cardiotoxin A5 Binds to αvβ3 Integrin and Inhibits Bone Resorption

Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

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