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

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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.