Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium.
Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium.
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发表时间：
2001-12
影响因子：
4.4
通讯作者：
V. Bonilha;E. Rodriguez-Boulan
中科院分区：
文献类型：
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作者：
V. Bonilha;E. Rodriguez-Boulan
目的鉴定与视网膜色素上皮细胞顶端微绒毛和基底外侧内折叠的形态发生有关的肌动蛋白结合蛋白ezrin的结合伴侣。方法采用免疫荧光显微镜和免疫印迹技术对大鼠眼、大鼠原代RPE、大鼠RPE J细胞系和转染人ezrin cDNA的RPE J细胞克隆系进行分析。两个ezrin结合蛋白的免疫荧光定位进行了冷冻切片的大鼠眼睛的不同年龄和单层洗涤剂Triton X-100提取和固定在多聚甲醛。通过SDS-PAGE和免疫印迹分析这两种蛋白与ezrin和谷胱甘肽-S-转移酶（GST）-ezrin融合蛋白的相互作用。结果免疫荧光显微镜观察到视网膜色素上皮细胞顶侧微绒毛上的50 kDa磷酸化蛋白（EBP 50）和基底侧表面的97 kDa突触相关蛋白（SAP 97）与埃兹蛋白（ezrin）相重叠。这两种PDZ（突触后密度蛋白[PSD-95]/盘大[DLG]-A/ZO-1）结构域蛋白具有相似的极化分布和对去污剂可提取性的高抗性，表明在大鼠RPE的原代培养物和表达高水平转染ezrin的克隆RPE-J细胞系中的细胞骨架缔合。RPE细胞裂解物从大鼠视网膜的各种出生后的年龄显示EBP 50和SAP 97的水平增加与α v整联蛋白，蛋白质表达在恒定的成人水平从出生。GST pull-down和免疫沉淀实验表明EBP 50和SAP 97与ezrin之间存在直接相互作用。结论EBP 50定位于RPE细胞的顶面微绒毛，而SAP 97定位于基底面，可能通过与ezrin的直接相互作用。
PURPOSE Identification of binding partners for ezrin, an actin-binding protein crucial for morphogenesis of apical microvilli and basolateral infoldings in RPE cells. METHODS Rat eyes, rat primary RPE, the rat RPE-J cell line, and a clonal line of RPE-J cells transfected with human ezrin cDNA were analyzed by immunofluorescence microscopy and immunoblot. Immunofluorescence localization of two ezrin-binding proteins was performed in cryosections of rat eyes of various ages and in monolayers extracted with the detergent Triton X-100 and fixed in paraformaldehyde. The interaction of both proteins with ezrin and gluthathione-S-transferase (GST)-ezrin fusion proteins was analyzed by SDS-PAGE and immunoblot. RESULTS Immunofluorescence microscopy of adult rat eyes detected a polarized distribution of ERM (ezrin, radixin, and moesin)-binding phosphoprotein of 50 kDa (EBP50) at the apical microvilli and synaptic-associated protein of 97 kDa (SAP97) at the basolateral surface of RPE cells, which overlapped with ezrin. These two PDZ (postsynaptic density protein [PSD-95]/disc large [DLG]-A/ZO-1) domain proteins had a similar polarized distribution and high resistance to detergent extractability, indicative of cytoskeletal association, both in primary cultures of rat RPE and in a clonal RPE-J cell line expressing high levels of transfected ezrin. RPE cell lysates from rat retinas of various postnatal ages revealed increasing levels of EBP50 and SAP97 compared with alphav integrin, a protein expressed at constant adult levels from birth. GST pull-down and immunoprecipitation experiments demonstrated a direct interaction between EBP50 and SAP97 and ezrin. CONCLUSIONS The data indicate that EBP50 localizes at the apical microvilli, whereas SAP97 localizes at the basolateral surface of RPE cells, probably through a direct interaction with ezrin.