Santibanez Juan F, Blanco Francisco J, Garrido-Martin Eva M, Sanz-Rodriguez Francisco, del Pozo Miguel A, Bernabeu Carmelo
Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, Madrid, Spain.
Cardiovasc Res. 2008 Mar 1;77(4):791-9. doi: 10.1093/cvr/cvm097. Epub 2007 Dec 8.
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.
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.
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.
激活素受体样激酶（ALK）1是一种转化生长因子（TGF）-βI型膜受体，几乎完全局限于内皮细胞（EC），并参与血管重塑和血管生成。先前的报道表明，普遍存在的TGF-βI型受体ALK5和II型受体位于名为小窝的富含胆固醇的膜微区中。这项工作的目的是评估ALK1在内皮小窝中的定位，并研究小窝蛋白-1对TGF-β/ALK1信号通路的作用。
通过共聚焦显微镜和共分级实验分析了人内皮细胞中ALK1的亚细胞分布。通过共免疫沉淀在小窝蛋白-1缺陷的人上皮细胞中研究了人ALK1与小窝蛋白-1之间的关联。使用人内皮细胞、小窝蛋白-1（-/-）小鼠胚胎成纤维细胞和大鼠成肌细胞中的ALK1特异性荧光素酶报告基因，阐明了小窝蛋白-1对ALK1介导的TGF-β信号的功能作用。共聚焦显微镜分析以及在制霉菌素或甲基-β-环糊精等胆固醇消耗剂存在下的胆固醇消耗实验表明，ALK1位于内皮小窝中。此外，共免疫沉淀分析表明ALK1与小窝的主要成分小窝蛋白-1相关联。ALK1/小窝蛋白-1相互作用的定位显示，小窝蛋白-1支架结构域和ALK1中的小窝蛋白-1结合基序负责这种关联。此外，这种迄今未报道的相互作用对ALK1依赖性信号传导具有功能影响。与先前发表的ALK5/小窝蛋白-1相互作用相反，小窝蛋白-1增强了TGF-β/ALK1信号通路，促进了ALK1特异性报告基因的活性。相反，小窝蛋白-1的特异性抑制消除了ALK1信号通路。
ALK1位于内皮小窝中，在那里它通过其支架结构域与小窝蛋白-1发生功能相互作用，这表明ALK1和小窝蛋白-1作为血管生成中TGF-β途径的关键介质共同发挥作用。
