Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

Xia V. Yang, Yajnavalka Banerjee, José A. Fernández, Hiroshi Deguchi, Xiao Xu, Laurent O. Mosnier, Rolf T. Urbanus, Phillip G. De Groot, Tara C. White-Adams, Owen McCarty, John H. Griffin

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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 3β (GSK3β), 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 GSK3β, 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 Kd, ≈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 GSK3β, thereby contributing to APC's beneficial effects on cells.

Original languageEnglish (US)
Pages (from-to)274-279
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number1
DOIs
StatePublished - Jan 6 2009

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U937 Cells
Protein C
Ligation
Glycogen Synthase Kinase 3
Phosphorylation
Proteins
Phosphatidylinositol 3-Kinases
Protein Binding
low density lipoprotein receptor-related protein 8
PAR-1 Receptor
Immobilized Proteins
Coagulants
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Aptitude
Blocking Antibodies
Surface Plasmon Resonance
LDL Receptors
Thromboplastin
Endotoxins
Small Interfering RNA

Keywords

  • Akt
  • Endothelial protein C receptor
  • Monocyte
  • PI3K
  • Reelin

ASJC Scopus subject areas

  • General

Cite this

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells. / Yang, Xia V.; Banerjee, Yajnavalka; Fernández, José A.; Deguchi, Hiroshi; Xu, Xiao; Mosnier, Laurent O.; Urbanus, Rolf T.; De Groot, Phillip G.; White-Adams, Tara C.; McCarty, Owen; Griffin, John H.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 1, 06.01.2009, p. 274-279.

Research output: Contribution to journalArticle

Yang, XV, Banerjee, Y, Fernández, JA, Deguchi, H, Xu, X, Mosnier, LO, Urbanus, RT, De Groot, PG, White-Adams, TC, McCarty, O & Griffin, JH 2009, 'Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells', Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 1, pp. 274-279. https://doi.org/10.1073/pnas.0807594106
Yang, Xia V. ; Banerjee, Yajnavalka ; Fernández, José A. ; Deguchi, Hiroshi ; Xu, Xiao ; Mosnier, Laurent O. ; Urbanus, Rolf T. ; De Groot, Phillip G. ; White-Adams, Tara C. ; McCarty, Owen ; Griffin, John H. / Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 1. pp. 274-279.
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AU - Mosnier, Laurent O.

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