Caveolin-1 interacts and cooperates with the transforming growth factor-beta type I receptor ALK1 in endothelial caveolae.
OBJECTIVE
Activin receptor-like kinase (ALK)1 is a transforming growth factor (TGF)-beta 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-beta 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-beta/ALK1 signalling pathway.


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-beta 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-beta-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-beta/ALK1 signalling pathway, promoting the activity of the ALK1-specific reporters. Conversely, specific suppression of caveolin-1 abrogated the ALK1 signalling 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-beta pathway in angiogenesis.
Santibanez
Juan F
JF
Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, Madrid, Spain.
Blanco
Francisco J
FJ
Garrido-Martin
Eva M
EM
Sanz-Rodriguez
Francisco
F
del Pozo
Miguel A
MA
Bernabeu
Carmelo
C
D030301:Activin Receptors, Type II / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000172:deficiency; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D018919:Neovascularization, Physiologic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051381:Rats; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; C495537:Cav1 protein, rat; D051242:Caveolin 1; D053773:Transforming Growth Factor beta1; D002784:Cholesterol; C425364:ACVRL1 protein, human; D030301:Activin Receptors, Type II