Abstract
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.
Highlights
Transforming growth factor- (TGF-)1 superfamily ligands, including TGF-s, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function [1]
Identification of Proteins Interacting with ACVRL1, Endoglin and TGFBR2 by LUMIER Assay—Using the LUMIER assay [10, 29], we identified proteins interacting with ACVRL1 (ALK1), ENG, and TGFBR2 (TGF- receptor 2) and implicated in the regulation of vascular functions
The LUMIER assay [10, 29] allowed us to test a total of 2052 potential interactions with endoglin, ACVRL1 and TGFBR2 receptors
Summary
Cell Culture—HEK-293T cells obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium with 100 IU/ml penicillin, 100 g/ml streptomycin, and 10% FCS (Invitrogen, Carlsbad, CA). HEK-293T cells were cotransfected in 96-well plates with 640 Flag-tagged cDNA plasmid preys and a Renilla luciferase-tagged receptor bait (ACVRL1, ENG, and TGFBR2 respectively). Bioinformatics and Data Analysis—Data were analyzed by three independent methods to generate a list of potential candidate preys interacting with the receptor baits. The LUMIER intensity ratio (LIR) was calculated as X/n, where X is the raw luminescence value in a given well, and n the mean of negative controls within a plate [10] These were ranked from high to low and those greater than five, were retained. Comparison of experimentally determined interactions that passed our stringent analysis criteria to those of literature-curated protein– protein interaction data were performed as described in supplemental Material. Comparison of Experimentally Determined to Literature-curated Protein–Protein Interactions—Literature-curated protein-protein interaction data were retrieved from the iRefWeb resource [23]. In cases in which interactions were expected from the literature, a t test with Welch correction for unequal variances was applied. p Ͻ 0.05 was considered significant
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