RESUMEN
Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/fisiología , Quimiotaxis de Leucocito/fisiología , Células Endoteliales/metabolismo , Animales , Antígenos CD/química , Bencetonio/análogos & derivados , Cadherinas/química , Técnica del Anticuerpo Fluorescente , Técnicas de Sustitución del Gen , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Fosforilación , Tirosina/metabolismoRESUMEN
Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.
Asunto(s)
Peroxisomas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Peroxinas , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Endothelial cells express two different classical cadherins, vascular endothelial (VE) cadherin and neural (N) cadherin, having distinct functions in the vascular system. VE-cadherin is specific to endothelial adherens junctions and is strictly necessary for vascular morphogenesis. On the contrary, N-cadherin shows diffuse localization on the cell surface and interacts with mural cells for vessel stabilization. In this study, we sought to clarify the cellular mechanisms leading to the distinct cellular locations and functions of the two cadherins in the endothelium. VE-cadherin has been shown to be responsible for the junctional exclusion of N-cadherin. Using several endothelial models, we demonstrate that this property is dependent on VE-cadherin binding to p120 catenin (p120(ctn)). Moreover, although in the absence of VE-cadherin N-cadherin can localize to cell contacts, angiogenesis remains impaired, demonstrating that endothelial junction formation is not sufficient for normal vessel development. Interestingly, we show that VE-cadherin, but not N-cadherin, is partially associated with cholesterol-enriched microdomains. Lipid raft-associated-VE-cadherin is characterized by a very high level of p120(ctn) association, and this association is necessary for VE-cadherin recruitment into lipid rafts. Altogether, our results indicate a critical role for p120(ctn) in regulating the membrane distribution of endothelial cadherins with functional consequences in terms of cadherin stabilization and intracellular signaling.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Cateninas/fisiología , Endotelio Vascular/metabolismo , Animales , Western Blotting , Adhesión Celular , Membrana Celular/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Uniones Intercelulares , Microdominios de Membrana , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Venas Umbilicales , Catenina deltaRESUMEN
We have recently shown that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial membrane protein, associates with VE-cadherin and is required for optimal VE-cadherin function and endothelial cell contact integrity. The dissociation of VE-PTP from VE-cadherin is triggered by vascular endothelial growth factor (VEGF) and by the binding of leukocytes to endothelial cells in vitro, suggesting that this dissociation is a prerequisite for the destabilization of endothelial cell contacts. Here, we show that VE-cadherin/VE-PTP dissociation also occurs in vivo in response to LPS stimulation of the lung or systemic VEGF stimulation. To show that this dissociation is indeed necessary in vivo for leukocyte extravasation and VEGF-induced vascular permeability, we generated knock-in mice expressing the fusion proteins VE-cadherin-FK 506 binding protein and VE-PTP-FRB* under the control of the endogenous VE-cadherin promoter, thus replacing endogenous VE-cadherin. The additional domains in both fusion proteins allow the heterodimeric complex to be stabilized by a chemical compound (rapalog). We found that intravenous application of the rapalog strongly inhibited VEGF-induced (skin) and LPS-induced (lung) vascular permeability and inhibited neutrophil extravasation in the IL-1ß inflamed cremaster and the LPS-inflamed lung. We conclude that the dissociation of VE-PTP from VE-cadherin is indeed required in vivo for the opening of endothelial cell contacts during induction of vascular permeability and leukocyte extravasation.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/fisiología , Células Endoteliales/fisiología , Leucocitos/fisiología , Neumonía/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Migración Transendotelial y Transepitelial/fisiología , Animales , Células COS , Chlorocebus aethiops , Cartilla de ADN/genética , Células Endoteliales/metabolismo , Técnicas de Sustitución del Gen , Immunoblotting , Inmunoprecipitación , Lipopolisacáridos , Pulmón/metabolismo , Ratones , Proteínas Recombinantes de Fusión/metabolismo , Tacrolimus/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)-cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885-4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin-mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor alpha-activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, beta-catenin, and plakoglobin. Surprisingly, only plakoglobin but not beta-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not beta-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not beta-catenin.