OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity

Exp Cell Res. 2015 Feb 15;331(2):278-91. doi: 10.1016/j.yexcr.2014.10.019. Epub 2014 Oct 29.


ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and β1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.

Keywords: FFAT motif; Integrin; ORP3; Phosphorylation; R-Ras; VAPA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / metabolism*
  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Endoplasmic Reticulum / metabolism*
  • Enzyme Activation
  • Fatty Acid-Binding Proteins
  • HEK293 Cells
  • Humans
  • Integrin beta1 / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Tetradecanoylphorbol Acetate / analogs & derivatives
  • Tetradecanoylphorbol Acetate / pharmacology
  • Vesicular Transport Proteins / biosynthesis
  • Vesicular Transport Proteins / metabolism*
  • ras Proteins / metabolism*


  • Carrier Proteins
  • Fatty Acid-Binding Proteins
  • Integrin beta1
  • OSBPL3 protein, human
  • VAPA protein, human
  • Vesicular Transport Proteins
  • phorbolol myristate acetate
  • RRAS protein, human
  • ras Proteins
  • Tetradecanoylphorbol Acetate