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 K_d values in the μM range. Also binding of cholesterol by ORP2 was detectable, but a K_d 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.

ORP2是一种固醇结合蛋白，在脂质和葡萄糖代谢，Akt信号转导，类固醇生成，细胞粘附，迁移和增殖中具有已记录的功能。

在这里，我们通过表面等离振子共振（SPR）研究了ORP2与磷酸肌醇（PIP）的相互作用，通过下拉测定法对胆固醇的亲和力及其在体外转移固醇的能力。此外，我们确定野生型（wt）ORP2和具有减弱的PIP结合的突变体ORP2（mHHK）对胆固醇亚细胞分布的影响，并分析ORP2与相关胆固醇转运蛋白ORP1L的相互作用。

ORP2对PI（4,5）P ₂，PI（3,4,5）P ₃和PI（4）P表现出特异性亲和力，暗示的K _d值在μM范围内。还可以检测到ORP2与胆固醇的结合，但无法确定K _d。Wt ORP2在HeLa细胞中主要在胞浆，内质网，晚期内体中检测到，偶尔在脂滴（LDs）中检测到，而ORP2（mHHK）显示出增强的LD定位。wt ORP2的过表达使D4H胆固醇探针从内体移开，而ORP2（mHHK）引起探针的内体积聚。尽管ORP2未能在体外转移脱氢麦角固醇在OSBP处于活跃状态的检测中，如D4H和菲律宾探针所检测到的，其敲除导致胆固醇在晚期内吞区室中积聚。有趣的是，显示ORP2与O​​RP1L（ER-内体内体交界处的胆固醇转运蛋白/传感器）相互作用并部分共定位于晚期内体。

我们的数据表明，ORP2结合几个磷酸肌醇，PI（4）P和多重磷酸化的物种。ORP2依赖于其PIP结合能力来调节胆固醇的亚细胞分布。ORP2与O​​RP1L的相互作用表明两个ORP的协同作用。