Cardiovascular Research Advance Access [Accepted Manuscript] published online on December 8, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm097
CAVEOLIN-1 INTERACTS AND COOPERATES WITH THE TRANSFORMING GROWTH FACTOR-β TYPE I RECEPTOR ALK1 IN ENDOTHELIAL CAVEOLAE
a 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
b Departamento de Biologia, Universidad Autonoma, Madrid, Spain
c Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
Corresponding author: Centro de Investigaciones Biologicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, SPAIN. Telephone: 34 91 8373112 x 4246; Fax: 34-91-5360432; e-mail: bernabeu.c{at}cib.csic.es
Aim: Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-β type I membrane receptor restricted almost entirely to endothelial cells 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: The subcellular distribution of ALK1 was analyzed by confocal microscopy and co-fractionation experiments in human endothelial cells. 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 signaling was elucidated using ALK1-specific luciferase reporters in human endothelial cells, caveolin-1–/-mouse embryonic fibroblasts, and rat myoblasts.
Results: 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 signaling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signaling pathway.
Conclusions: 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
* Current address: Laboratorio de Biologia Celular, Instituto de Nutrición y Tecnología de los Alimentos, INTA, Universidad de Chile, Santiago, Chile.
# These authors contributed equally to this work
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