Chapple J Paul, Grayson Celene, Hardcastle Alison J, Bailey Tracey A, Matter Karl, Adamson Peter, Graham Catriona H, Willison Keith R, Cheetham Michael E
Division of Pathology, Institute of Ophthalmology, University College London, UK.
Biochem J. 2003 Jun 1;372(Pt 2):427-33. doi: 10.1042/BJ20021475.
Mutations in the retinitis pigmentosa protein gene RP2 account for up to 15% of X-linked retinitis pigmentosa. RP2 is a novel protein of unknown function, which is targeted to the plasma membrane by dual N-terminal acyl-modification. Dual-acylated proteins are targeted to lipid rafts, and some are subject to polarized sorting. Therefore we investigated the organization of RP2 on the plasma membrane. Endogenous RP2 protein was predominantly localized at the plasma membrane, and exogenously expressed green-fluorescent-protein-tagged protein was also targeted to the membrane in a wide range of cultured cells. High levels of endogenous RP2 protein were present in HeLa cells and in the retinal pigment epithelium-derived cell line ARPE19. A significant proportion of RP2 in cultured neuroblastoma cells was associated with detergent-resistant membranes (DRMs), but much less than other dually acylated proteins (e.g. Lyn and Fyn). In contrast, the RP2-interacting protein Arl3 (ADP-ribosylation factor-like 3) was not found to be associated with DRMs. The association of RP2 with DRMs was cholesterol-dependent. In polarized epithelial cells in culture and in vivo, RP2 was present in both the apical and basolateral domains of the plasma membrane. These data show that RP2 is not specific to either domain, unlike some other dually acylated proteins. Interestingly, the level of RP2 protein increased in the epithelial cell line Caco-2 with differentiation and polarization. These data show that RP2 is present on the membrane of all cell types examined both in vitro and in vivo, and that RP2 associates with lipid rafts, suggesting a potential role for the protein in signal transduction.
视网膜色素变性蛋白基因RP2的突变在X连锁视网膜色素变性中占比高达15%。RP2是一种功能未知的新型蛋白质，通过双N端酰基修饰定位于质膜。双酰化蛋白定位于脂筏，其中一些还会经历极性分选。因此，我们研究了RP2在质膜上的组织形式。内源性RP2蛋白主要定位于质膜，在外源表达的绿色荧光蛋白标记的蛋白在多种培养细胞中也定位于膜上。HeLa细胞和视网膜色素上皮衍生细胞系ARPE19中存在高水平的内源性RP2蛋白。培养的神经母细胞瘤细胞中相当一部分RP2与抗去污剂膜（DRM）相关，但比其他双酰化蛋白（如Lyn和Fyn）少得多。相比之下，未发现与RP2相互作用的蛋白Arl3（ADP核糖基化因子样3）与DRM相关。RP2与DRM的结合依赖于胆固醇。在培养的和体内的极化上皮细胞中，RP2存在于质膜的顶端和基底外侧结构域。这些数据表明，与其他一些双酰化蛋白不同，RP2对任何一个结构域都不具有特异性。有趣的是，上皮细胞系Caco-2中RP2蛋白的水平随着分化和极化而增加。这些数据表明，RP2在体外和体内所有检测的细胞类型的膜上均有存在，并且RP2与脂筏相关，提示该蛋白在信号转导中可能发挥作用。
