Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

Abstract

ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P(3) and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.

OSBP-related protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids

“…In a previous study, we presented data suggesting that overexpressed ORP2 enhances the intracellular transport of cholesterol (21). Therefore, we wanted to study whether ORP2 might, in addition to oxysterols, bind cholesterol.…”

“…LDs were induced in A431 cells by overnight treatment with 350 mM oleic acid-BSA complexes prepared according to (28). Stably transfected ORP2 HeLa TRex cells were cultured and induced with doxycyclin as previously described (21).…”

“…OSBP is targeted to Golgi complex in response to treatment of cells with its high-affinity ligand 25-hydroxycholesterol (25OHC). In several cases, the ORP pleckstrin homology domains are known to mediate the binding of phosphoinositides, but also the ORDs of ORP1, -2, -9, and -10 seem to be capable of binding these negatively charged phospholipids (19)(20)(21). A motif called FFAT (two phenylalanines in an acidic track), which mediates the binding to the ER resident protein VAP-A (for vesicle-associated membrane protein-associated protein), specifies association of several ORPs with the ER membranes (22).…”

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

“…Overexpression of STARD4 also accelerates sterol transport from the PM to the endocytic recycling compartment (ERC) (Mesmin et al 2011), suggesting that STARD4 functions as a sterol transporter in intact cells. ORP2 was also proposed to function as an ER-PM sterol transporter, as its overexpression enhanced efflux of newly synthesized cholesterol from the ER (Hynynen et al 2005).…”

Synthesis and Biosynthetic Trafficking of Membrane Lipids

“…PIP interactions may also take place in the absence of a PH domain, as in Osh4, where a membrane-binding surface of the protein can generate a phosphoinositide binding site (Schulz et al 2009). In mammalian cells, ORP2 was shown to enhance biosynthetic sterol trafficking from the ER to the plasma membrane (Hynynen et al 2005). It can also facilitate the delivery of sterols in the reverse direction, from the plasma membrane to the ER and lipid droplets (Jansen et al 2011).…”