Cardiovascular Research Advance Access originally published online on December 8, 2007
Cardiovascular Research 2008 77(4):791-799; doi:10.1093/cvr/cvm097
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Caveolin-1 interacts and cooperates with the transforming growth factor-β type I receptor ALK1 in endothelial caveolae
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1 Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas (CSIC), and Center for Biomedical Research on Rare Diseases (CIBERER), Ramiro de Maeztu 9, 28040 Madrid, Spain
2 Departamento de Biologia, Universidad Autonoma, Madrid, Spain
3 Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
* Corresponding author. Tel: +34 34 91 8373112 ext. 4246; fax: +34 91 5360432. E-mail address: bernabeu.c{at}cib.csic.es
Aims: Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-β type I membrane receptor restricted almost entirely to endothelial cells (ECs) and involved in vascular remodelling and angiogenesis. Previous reports have shown that the ubiquitous TGF-β type I receptor ALK5 and the type II receptor are located in cholesterol-rich membrane microdomains named caveolae. The aim of this work was to assess the location of ALK1 in endothelial caveolae as well as to study the role of caveolin-1 on the TGF-β/ALK1 signalling pathway.
Methods and results: The subcellular distribution of ALK1 was analysed by confocal microscopy and co-fractionation experiments in human ECs. The association between human ALK1 and caveolin-1 was studied in caveolin-1-deficient human epithelial cells by co-immunoprecipitation. The functional role of caveolin-1 on the ALK1-mediated TGF-β signalling was elucidated using ALK1-specific luciferase reporters in human ECs, caveolin-1–/–mouse embryonic fibroblasts, and rat myoblasts. Confocal microscopy analyses, as well as cholesterol depletion experiments in the presence of cholesterol-depleting agents such as nystatin or methyl-β-cyclodextrin, demonstrated that ALK1 is located in endothelial caveolae. Also, co-immunoprecipitation assays showed that ALK1 associates with the main caveolae component caveolin-1. Mapping of the ALK1/caveolin-1 interaction revealed that the caveolin-1 scaffolding domain and the caveolin-1 binding motif in ALK1 are responsible for this association. Moreover, this hitherto not reported interaction had a functional consequence for the ALK1-dependent signalling. In contrast with the previously published ALK5/caveolin-1 interaction, caveolin-1 enhances the TGF-β/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling pathway.
Conclusion: ALK1 is located in endothelial caveolae where it functionally interacts with caveolin-1 through its scaffolding domain, suggesting a joint contribution of ALK1 and caveolin-1 as key mediators of the TGF-β pathway in angiogenesis.
KEYWORDS Angiogenesis; Caveolae; Growth factors; Endothelial receptors; Signal transduction
Time for primary review: 27 days
Present address. Laboratorio de Biologia Celular, Instituto de Nutrición y Tecnologia de los Alimentos, INTA, Universidad de Chile.
These authors contributed equally to this work.