Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas, USA.
Invest Ophthalmol Vis Sci. 2012 Mar 21;53(3):1519-29. doi: 10.1167/iovs.11-8845. Print 2012 Mar.
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are a frequent cause of X-linked retinitis pigmentosa. The RPGR transcript undergoes complex alternative splicing to express both constitutive (Rpgr(ex1-19)) and Rpgr(ORF15) variants. Because functional studies of Rpgr suggest a role in intracellular protein trafficking through the connecting cilia, the goal of this study was to identify potential binding partners for Rpgr(ORF15) and to identify the domains on whirlin necessary for Rpgr binding.
The C-terminus of mouse Rpgr(ORF15) was used as bait in a yeast two-hybrid system. Whirlin expression was analyzed using RT-PCR and Western blot analysis. Protein-protein interactions were confirmed using in vitro binding assays and coimmunoprecipitation. Subcellular colocalization was analyzed using immunohistochemistry on retinal cryosections.
Yeast two-hybrid analysis identified whirlin, a PDZ-scaffold protein, as a putative binding partner for Rpgr(ORF15). The RPGR(ORF15)-whirlin interaction was confirmed using in vitro binding assays and coimmunoprecipitation from retinal tissue, and both proteins were shown to colocalize in the photoreceptor connecting cilia in vivo. Results from RT-PCR, Western blot analysis, and immunocytochemistry demonstrated that whirlin expressed multiple isoforms in photoreceptors with variable subcellular localization.
Whirlin expression has been reported in photoreceptors and cochlear hair cells, and mutations in whirlin cause Usher syndrome (USH2D) and nonsyndromic congenital deafness (DFNB31). Because mutations in the 5' end of whirlin are associated with the syndromic phenotype associated with USH2D, the identification of novel N-terminal isoforms in the retina and a novel RPGR(ORF15)-whirlin interaction provide a potential mechanism for the retinal phenotype observed in USH2D.
视网膜炎色素变性 GTP 酶调节因子（RPGR）基因突变是 X 连锁视网膜炎色素变性的常见原因。RPGR 转录本通过复杂的选择性剪接表达组成型（Rpgr(ex1-19)）和 Rpgr(ORF15)变体。由于 Rpgr 的功能研究表明其在连接纤毛的细胞内蛋白运输中起作用，本研究的目的是鉴定 Rpgr(ORF15)的潜在结合伴侣，并确定 whirlin 上与 Rpgr 结合所必需的结构域。
使用小鼠 Rpgr(ORF15)的 C 末端作为酵母双杂交系统中的诱饵。使用 RT-PCR 和 Western blot 分析分析 whirlin 的表达。使用体外结合测定和共免疫沉淀证实蛋白-蛋白相互作用。使用视网膜冷冻切片的免疫组织化学分析亚细胞共定位。
酵母双杂交分析鉴定出 whirlin，一种 PDZ 支架蛋白，是 Rpgr(ORF15)的潜在结合伴侣。通过体外结合测定和从视网膜组织中进行的共免疫沉淀证实了 RPGR(ORF15)-whirlin 相互作用，并且两种蛋白质均在体内显示出在光感受器连接纤毛中共定位。来自 RT-PCR、Western blot 分析和免疫细胞化学的结果表明，whirlin 在光感受器中表达具有可变亚细胞定位的多种同工型。
已经报道 whirlin 在光感受器和耳蜗毛细胞中表达，并且 whirlin 突变导致 Usher 综合征（USH2D）和非综合征性先天性耳聋（DFNB31）。由于 whirlin 的 5'端突变与与 USH2D 相关的综合征表型相关，因此在视网膜中鉴定出新型 N 端同工型和新型 RPGR(ORF15)-whirlin 相互作用为 USH2D 中观察到的视网膜表型提供了潜在的机制。
