Activation of the PI3K-Akt Pathway by Activated Protein C Occurs Via a Novel Receptor, Apolipoprotein E Receptor 2 (ApoER2)
Authors: Yang Xia V. , Banerjee Yajnavalka , Fernandez Jose A. , Deguchi Hiroshi , Xu Xiao , Mosnier Laurent O. , Urbanus Rolf T , deGroot Phillip G , White Tara C , McCarty Owen J.T. , Griffin John H. 
Published: October 2019

Abstract
Recombinant Activated Protein C (APC) is an FDA-approved agent for reducing death in severe sepsis patients, and it is currently the subject of an NIH-sponsored clinical trial for therapy of ischemic stroke. Independent of its long-known anticoagulant activity, APC binds to cells and triggers a variety of cytoprotective activities that suppress apoptosis, inflammation and endothelial barrier disruption. APC protects not only endothelial cells but also neurons from stress. One current paradigm for APC’s cell signaling mechanisms emphasizes APC binding to endothelial cell protein C receptor (EPCR) followed by subsequent protease activated receptor 1 (PAR1) activation and GPCR pathway signaling. However, several findings imply that the APC-EPCR-PAR1 signaling paradigm is not always applicable or might not be sufficient to explain APC’s multiple actions and that alternative signaling mechanisms and/or receptors might be important. A recent report of platelet interactions with immobilized APC indicated that APC binds to apolipoprotein E receptor 2 (ApoER2) (aka low density lipoprotein receptor-related protein 8, LRP8) and to glycoprotein Ibα. Thus, to evaluate cell signaling functional significance for APC binding to ApoER2, we used human monocytic-like U937 cells and determined the nature of ApoER2-dependent signaling by APC. First, we characterized the forms of ApoER2 mRNA in U937 cells and identified two isoforms, both with deletions of exons 5 and 18 and one with an additional deletion of exon 15 that codes for the extracellular O-linked sugar region. Western blotting confirmed the presence of two predicted forms of ApoER2. Second, we determined whether APC caused phosphorylation of signaling members of the Reelin-ApoER2 pathway. APC initiated rapid Tyr220 phosphorylation of the adaptor protein, disabled-1 (Dab1), and of Ser473 in Akt. APC also induced phosphorylation of Ser9 in glycogen synthase kinase 3 beta (GSK3β) which was blocked by the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002. Receptor-associated protein (RAP), an antagonist for ApoER2 ligand binding, inhibited APC-induced phosphorylation of Dab1 and GSK3β, whereas anti-EPCR or anti-PAR1 blocking antibodies did not, suggesting APC signals via a Reelin-like pathway. Third, Surface Plasmon Resonance (SPR) analysis indicated that soluble (s) ApoER2 was bound with apparently higher affinity to APC (34 nM) than was sEPCR (195 nM), and that RAP completely blocked binding of sApoER2 to APC but had no effect on APC binding of sEPCR. No binding of another Reelin signaling pathway receptor, sVLDLR, to APC was detected by SPR. Thus, RAP blocks high affinity binding of APC to sApoER2, consistent with the hypothesis that the RAP-sensitive, APC-induced signaling in U937 cells is caused by APC ligation of ApoER2 without binding of APC to VLDLR. Fourth, RAP and the blocking anti-EPCR RCR252 antibody ablated binding of cells to immobilized APC but not to immobilized fibronectin, suggesting that both a RAP-sensitive receptor (eg., ApoER2) and EPCR were required for high affinity binding of U937 cells to immobilized-APC after vigorous washing. Fifth, we pretreated cells with various blocking antibodies or RAP and then treated cells with LPS and APC. APC diminished LPS-induced procoagulant activity due to tissue factor as expected. Either RAP alone or the blocking anti-EPCR RCR252 antibody alone totally ablated APC’s effects. However, two antibodies (ATAP2 and WEDE15) that block APC activation of PAR1 had no effect on APC’s ability to blunt LPS-induction of tissue factor activity. Thus, both a RAP-sensitive receptor (eg., ApoER2) and EPCR were required for APC to down-regulate tissue factor procoagulant activity on LPS-stimulated cells. In summary, these data indicate that ligation of ApoER2 on cells by APC, like ligation of ApoER2 on neurons by Reelin, causes signaling involving Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3β by mechanisms that are independent of EPCR and PAR1. As ApoER2 and Dab1 are present on endothelium as well as neurons, we hypothesize that ApoER2 is a novel functional receptor for APC that provides a direct pathway for activation of the PI3K-Akt signaling pathway by APC which could act in concert with GPCR-induced signaling induced by EPCR-dependent activation of PAR1 to mediate APC’s beneficial effects on cells.