MPP5 recruits MPP4 to the CRB1 complex in photoreceptors
MPP5 recruits MPP4 to the CRB1 complex in photoreceptors
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DOI：
10.1167/iovs.04-1417
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发表时间：
2005-06-01
影响因子：
4.4
通讯作者：
Wijnholds, J
中科院分区：
文献类型：
--
作者：
Kantardzhieva, A;Gosens, I;Wijnholds, J
目的.人类Crumbs同源物1（CRB 1）基因突变是Leber先天性黑蒙（LCA）和各种形式的视网膜色素变性的常见原因。CRB 1被认为在视网膜中组织细胞内蛋白质支架，其参与光感受器极性。本研究的重点是鉴定，亚细胞定位，并与CRB 1连接的蛋白质支架的新成员的结合特性。为了剖析与CRB 1连接的蛋白质支架，使用酵母双杂交方法来筛选相互作用的蛋白质。谷胱甘肽S-转移酶（GST）下拉分析和免疫沉淀用于验证蛋白质-蛋白质相互作用。通过免疫组织化学和共聚焦显微镜对人视网膜和免疫电镜对小鼠视网膜的蛋白质的亚细胞定位可视化。与CRB 1连接的支架的一个新成员，称为膜棕榈酰化蛋白（MPP）亚家族成员4（MPP 4），一种膜相关鸟苷酸激酶（MAGUK）蛋白，被鉴定出来。MPP 4通过与MPP亚家族成员MPP 5（PALS 1）直接相互作用而与CRB 1形成复合物。3D同源性建模提供了一种机制的证据，该机制调节MPP 4和-5蛋白的同源和异源二聚体向复合物的募集。视网膜定位研究显示CRB 1、MPP 5和MPP 4共定位于视网膜外界膜（OLM）。这些数据暗示MPP 4和MPP 5在光感受器极性中具有作用，并且通过与CRB 1相关联，确定同源基因为遗传性视网膜病的功能候选基因。
PURPOSE. Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of Leber congenital amaurosis (LCA) and various forms of retinitis pigmentosa. CRB1 is thought to organize an intracellular protein scaffold in the retina that is involved in photoreceptor polarity. This study was focused on the identification, subcellular localization, and binding characteristics of a novel member of the protein scaffold connected to CRB1.METHODS. To dissect the protein scaffold connected to CRB1, the yeast two-hybrid approach was used to screen for interacting proteins. Glutathione S-transferase (GST) pull-down analysis and immunoprecipitation were used to verify protein-protein interactions. The subcellular localization of the proteins was visualized by immunohistochemistry and confocal microscopy on human retinas and immunoelectron microscopy on mouse retinas.RESULTS. A novel member of the scaffold connected to CRB1, called membrane palmitoylated protein (MPP) subfamily member 4 (MPP4), a membrane-associated guanylate kinase (MAGUK) protein, was identified. MPP4 was found to exist in a complex with CRB1 through direct interaction with the MPP subfamily member MPP5 (PALS1). 3D homology modeling provided evidence for a mechanism that regulates the recruitment of both homo- and heterodimers of MPP4 and -5 proteins to the complex. Localization studies in the retina showed that CRB1, MPP5, and MPP4 colocalize at the outer limiting membrane (OLM).CONCLUSIONS. These data imply that MPP4 and -5 have a role in photoreceptor polarity and, by association with CRB1, pinpoint the cognate genes as functional candidate genes for inherited retinopathies.