Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.
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Author contributions: G.X., M.B., M.T., and M.L. designed research; G.X., M.B., M.J., S.V., D.V., and M.T. performed research; M.B. and J.L.W. contributed new reagents or analytic tools; G.X., M.J., A.L.T., R.N., D.V., M.T., and M.L. analyzed data; G.X. and M.L. wrote the paper; G.X. prepared several figures; M.B. and M.T. edited the paper; A.L.T. prepared all network figures; R.N. performed stats and prepared figure 1a; J.L.W. reviewed the paper.
Conflict of interest: The authors declare no competing financial interest.
G. Xu was recipient of a fellowship from The Heart and Stroke Foundation of Canada. This research was supported by grants from the Canadian Institutes of Health Research (CIHR) to M. Letarte; Ontario Research Fund GL2 to J Wrana; CIHR grant and Ontario Research Fund to A. L. Turinsky; Le Fonds Québécois de la Recherche sur la Nature et les Technologies to R. Nadon; National Natural Science Foundation of China and Shanghai Committee of Science and Technology to G. Xu; BIDMC Commitment Fund and Klarman Family Foundation to M. Toporsian.
