ORP2 interacts with phosphoinositides and controls the subcellular distribution of cholesterol
ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation.
Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L.
ORP2 showed specific affinity for PI(4,5)P2, PI(3,4,5)P3 and PI(4)P, with suggestive Kd values in the μM range. Also binding of cholesterol by ORP2 was detectable, but a Kd could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions.
Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
a – Minerva Foundation Institute for Medical Research, Biomedicum 2U, FI-00290, Helsinki, Finland
b – Department of Anatomy, Faculty of Medicine, FI-00014, University of Helsinki, Finland
c – Department of Biochemistry and Developmental Biology, Faculty of Medicine, FI-00014, University of Helsinki, Finland
d – Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, FI-00014, University of Helsinki, Finland