Novel Roles for beta-arrestin2 in TGF-beta mediated Endoglin Signaling

描述（由申请人提供）：转化生长因子-β（TGF-β）是多种细胞过程（包括增殖、血管生成和凋亡）的重要调节因子。尽管TGF-β作用抑制大多数细胞类型的增殖，但内皮细胞是独特的，因为它们可以响应于TGF-β刺激而引发两种不同的途径。一种途径促进血管生成内皮细胞增殖和迁移，主要由内皮细胞特异性TGF-β受体复合物、内皮糖蛋白（III型受体）和ALK-1（I型受体）介导。另一种途径涉及普遍存在的I型TGF-β受体ALK-5，其抑制内皮细胞增殖和迁移。这些受体突变或缺失导致的异常信号传导具有严重的生物学意义，如分别在缺乏ALK-1或内皮糖蛋白的敲除小鼠中观察到的遗传性出血性毛细血管扩张症（HHT）和致死性表型所证明。相反，内皮糖蛋白的表达升高与肿瘤诱导的血管生成相关。确定调节这两个相反的通路的分子机制将为肿瘤发生和严重血管疾病的基础提供见解。基于我们的初步研究，提出了以下假设：ALK-1磷酸化endoglin以促进endoglin与p-arrestin 2的相互作用，导致endoglin和ALK-1内化，并降低ALK-1信号传导和MARK通路的endoglin依赖性信号传导。具体目标是：1）确定内皮糖蛋白/β-arrestin 2相互作用和内皮细胞内化的调节机制。2)确定endoglin与p-arrestin 2的相互作用是否介导endoglin和ALK-1信号传导以调节内皮细胞增殖和迁移。3)确定内皮糖蛋白/β-arrestin 2相互作用是否介导内皮糖蛋白依赖性信号传导至MARK通路以促进内皮细胞增殖和迁移。内皮糖蛋白和β-抑制蛋白2之间相互作用所需的结构元件将通过使用共免疫沉淀和共定位研究来表征。将采用胸苷掺入和细胞迁移/小管形成试验研究内皮糖蛋白/β-抑制蛋白2相互作用的生物学影响。最后，将研究MARK途径主要组分（ERK、JNK/SAPK和P38）响应endoglin/p-arrestin 2相互作用的催化活性。内皮糖蛋白是一种被鉴定为正常血管发育的关键介质之一的蛋白质，但也涉及促进肿瘤发生。确定内皮糖蛋白信号传导如何被适当调节可能对靶向肿瘤形成和发展至关重要。

DESCRIPTION (provided by applicant): Transforming growth factor-p (TGF-P) is an important regulator of diverse cellular processes including proliferation, angiogenesis and apoptosis. Whereas TGF-p actions inhibit proliferation in most cell types, endothelial cells are unique in that they can elicit two divergent pathways in response to TGF-p stimulation. One pathway promotes angiogenic endothelial cell proliferation and migration, mediated by the predominantly endothelial cell-specific TGF-p receptor complex, endoglin (a type III receptor) and ALK-1 (a type I receptor). The other pathway involves the ubiquitous type I TGF-p receptor, ALK-5, which inhibits endothelial cell proliferation and migration. Aberrant signaling as a result of mutations in or loss of these receptors have serious biological implications, as evidenced by the hereditary hemorrhagic telangiectasia (HHT) and the lethal phenotype observed in knockout mice lacking ALK-1 or endoglin, respectively. Conversely, elevated expression of endoglin is associated with tumor-induced angiogenesis. Defining the molecular mechanisms for modulating these two opposing pathways will provide insights to the basis of tumorigenesis and severe vascular disorders. Based upon our preliminary studies the following hypothesis is proposed: ALK-1 phosphorylates endoglin to promote the interaction of endoglin with p-arrestin2, resulting in endoglin and ALK-1 internalization and decreased ALK-1 signaling, and endoglin-dependent signaling to MARK pathways. The specific aims are: 1) Define the mechanisms which regulate the endoglin/p-arrestin2 interaction and internalization in endothelial cells. 2) Determine whether the interaction of endoglin with p-arrestin2 mediates endoglin and ALK-1 signaling to regulate endothelial cell proliferation and migration. 3) Determine whether the endoglin/p-arrestin2 interaction mediates endoglin-dependent signaling to MARK pathways to promote endothelial cell proliferation and migration. The structural elements required for the interaction between endoglin and p-arrestin2 will be characterized through the use of co- immunoprecipitation and co-localization studies. The biological impact of the endoglin/p-arrestin2 interaction will be investigated by employing thymidine incorporation and cell migration/tubule formation assays. Finally, the catalytic activities of the major components of the MARK pathways (ERK, JNK/SAPK and P38) will be studied in response to endoglin/p-arrestin2 interaction. Endoglin is a protein identified as one of the key mediators of normal vascular development, but has also been implicated in promoting tumorigenesis. Determining how endoglin signaling is properly regulated may be crucial to targeting tumor formation and development.