title: Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells.
abstract: Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APC's signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3beta (GSK3beta), which was blocked by the PI3K inhibitor LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3beta, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent K(d), approximately 30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APC's ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3beta, thereby contributing to APC's beneficial effects on cells.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Xia V
authors.initials: XV
authors.lastname: Yang
authors.forename: Yajnavalka
authors.initials: Y
authors.lastname: Banerjee
authors.forename: José A
authors.initials: JA
authors.lastname: Fernández
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Deguchi
authors.forename: Xiao
authors.initials: X
authors.lastname: Xu
authors.forename: Laurent O
authors.initials: LO
authors.lastname: Mosnier
authors.forename: Rolf T
authors.initials: RT
authors.lastname: Urbanus
authors.forename: Phillip G
authors.initials: PG
authors.lastname: de Groot
authors.forename: Tara C
authors.initials: TC
authors.lastname: White-Adams
authors.forename: Owen J T
authors.initials: OJ
authors.lastname: McCarty
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001779:Blood Coagulation Factors; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D026502:LDL-Receptor Related Proteins; D009000:Monocytes / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein / Q000378:metabolism*; D015398:Signal Transduction; D020298:U937 Cells
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001779:Blood Coagulation Factors; C115785:DAB1 protein, human; D026502:LDL-Receptor Related Proteins; D009419:Nerve Tissue Proteins; D011486:Protein C; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; C091963:activated protein C receptor; C098773:low density lipoprotein receptor-related protein 8; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3