Reiners Jan, Märker Tina, Jürgens Karin, Reidel Boris, Wolfrum Uwe
Department of Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg University of Mainz, Mainz, Germany.
Mol Vis. 2005 May 12;11:347-55.
The human Usher syndrome (USH) is the most common form of deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal onset of retinitis pigmentosa. Five corresponding genes of the seven USH1 genes have been cloned over the years. Recent studies indicated that three USH1 proteins, namely myosin VIIa (USH1B), SANS (USH1G), and cadherin 23 (USH1D) interact with the USH1C gene product harmonin. In these protein-protein complexes harmonin acts as the scaffold protein binding these USH1 molecules via its PDZ domains. The aim of the present study was to analyze whether or not the fifth identified USH1 protein protocadherin 15 (Pcdh15) also binds to harmonin and where these putative protein complexes might be localized in mammalian rod and cone photoreceptor cells.
In vitro binding assays (GST pull-down, yeast two-hybrid assay) were applied. Antibodies against bacterial expressed USH1 proteins were generated. Affinity purified antibodies were used in immunoblot analyses of brain fractions and isolated retinas, in immunofluorescence studies, and in immunoelectron microscopic studies of rodent retinas.
We showed that Pcdh15 (USH1F) interacted with harmonin PDZ2. Immunocytochemistry revealed that Pcdh15 is expressed in photoreceptor cells of the mammalian retina, where it is colocalized with harmonin, myosin VIIa, and cadherin 23 at the synaptic terminal. Colocalization of Pcdh15 with harmonin was found at the base of the photoreceptor outer segment, where newly synthesized disk membranes are present.
Our data indicate that harmonin-Pcdh15 interactions probably play a role in disk morphogenesis. Furthermore, we provide evidence that a complex composed of all USH1 molecules may assemble at the photoreceptor synapse. This USH protein complex can contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. Defects in any of the USH1-complex partners may result in photoreceptor dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of USH1 patients.
人类尤塞氏综合征（USH）是最常见的致盲致聋疾病形式。I型尤塞氏综合征（USH1）是最严重的形式，其特征为先天性重度耳聋、持续性前庭功能障碍以及青春期前发病的色素性视网膜炎。多年来已克隆出7个USH1基因中的5个相应基因。最近的研究表明，三种USH1蛋白，即肌球蛋白VIIa（USH1B）、SANS（USH1G）和钙黏蛋白23（USH1D）与USH1C基因产物和声蛋白相互作用。在这些蛋白质 - 蛋白质复合物中，和声蛋白作为支架蛋白，通过其PDZ结构域结合这些USH1分子。本研究的目的是分析第五个已鉴定的USH1蛋白原钙黏蛋白15（Pcdh15）是否也与和声蛋白结合结合这些假定的蛋白质复合物可能在哺乳动物视杆和视锥光感受器细胞中的定位。
应用体外结合试验（GST下拉试验、酵母双杂交试验）。制备针对细菌表达的USH1蛋白的抗体。亲和纯化的抗体用于脑部分和分离视网膜的免疫印迹分析、免疫荧光研究以及啮齿动物视网膜的免疫电子显微镜研究。
我们表明Pcdh15（USH1F）与和声蛋白的PDZ2相互作用。免疫细胞化学显示Pcdh15在哺乳动物视网膜的光感受器细胞中表达，在那里它与和声蛋白、肌球蛋白VIIa和钙黏蛋白23在突触末端共定位。在光感受器外段基部发现Pcdh15与和声蛋白共定位，那里存在新合成的盘膜。
我们的数据表明，和声蛋白 - Pcdh15相互作用可能在盘膜形态发生中起作用。此外，我们提供证据表明，由所有USH1分子组成的复合物可能在光感受器突触处组装。这种USH蛋白复合物可有助于突触前和突触后区域的皮质细胞骨架基质，这被认为在突触连接的结构和功能组织中起基本作用。USH1复合物中任何一个伙伴的缺陷可能导致光感受器功能障碍，从而引起色素性视网膜炎，这是USH1患者视网膜的临床表型。
