MOLECULAR BASIS OF PROTEIN TRANSPORT IN PHOTORECEPTORS

描述(申请者的描述)：本研究的长期目标 应用是理解分子机制，通过这些机制 视紫红质羧基末端自然产生的突变， 视杆感光细胞中的感光蛋白可引起视网膜色素变性。 遗传性人类视网膜退行性疾病。这些突变被认为是 干扰视紫质向视杆感受器的正确运输 外部管段。我们发现分子马达的轻链Tctex-1 胞质动力蛋白，与视紫红质的羧基末端特异结合。 因此，细胞质动力蛋白可能在胞浆动力蛋白的转运中起关键作用。 视紫红质。此应用程序的具体目标旨在研究 Tctex-1在视紫红质转运中的生理作用(S)。第一个目标将是 开发阻断正常功能和/或表达的方法 Tctex-1在细胞培养和光感受器中均有表达。第二个目标将是 使用产生的试剂和技术来检查抑制的效果 Tctex-1在极化上皮细胞视紫红质转运中的功能/表达 细胞模型系统。通过使用细胞培养系统收集的信息 因为视紫红质贩运对于我们的第三个目标将是有价值的，这将是 使用转基因小鼠系统在体内阻断Tctex-1的功能/表达。这 实验将明确测试Tctex-1的作用，进而， 胞浆动力蛋白在引导视紫质在脑内的适当转运中的作用 光感受器。最后，我们的第四个目标将是描述潜在的 蛋白磷酸化在Tctex-1功能调节中的作用 多种体外培养和细胞培养技术。如果成功，这些 实验将大大加深我们对分子机制的理解。 指导和调节视紫红质和其他物质的正确运输 感光蛋白。这一认识将具有重要的临床意义。 相关性，因为提出了视紫红质错误定位和 一些RP病例中的感光细胞死亡。首先，蛋白质是 视紫红质运输所必需的可能代表新的候选基因 遗传性视网膜疾病。其次，我们可能会获得对 视紫红质错位导致细胞死亡的机制。是这样的 洞察力最终可能被证明对设计视觉保护很有用 RP患者的治疗方法。

DESCRIPTION (Applicant's Description): The long-term goals of this research application are to understand the molecular mechanisms by which naturally-occurring mutations in the carboxy-terminus of rhodopsin, the light-sensing protein in rod photoreceptors, cause retinitis pigmentosa (RP), a hereditary human retinal degenerative disease. These mutations are thought to interfere with the correct transport of rhodopsin to the rod photoreceptor outer segment. We have found that Tctex-1, a light chain of the molecular motor cytoplasmic dynein, binds specifically to the carboxy terminus of rhodopsin. Cytoplasmic dynein may therefore play a critical role in the transport of rhodopsin. The specific aims of this application are designed to study the physiologic role(s) of Tctex-1 in rhodopsin transport. The first aim will be to develop approaches to blocking the normal function and/or expression of Tctex-1, both in cell culture and in photoreceptors. The second aim will be to use the resulting reagents and techniques to examine the effects of suppressing Tctex-1 function/expression on rhodopsin transport in a polarized epithelial cell model system. The information gathered from using a cell culture system for rhodopsin trafficking will be valuable for our third aim, which will be to block Tctex-1 function/expression in vivo using a transgenic mouse system. This experiment will definitively test the role of Tctex-1, and by extension, cytoplasmic dynein, in directing the appropriate transport of rhodopsin in the photoreceptor. Finally, our fourth aim will be to characterize the potential role of protein phosphorylation in the regulation of Tctex-1 function using a variety of in vitro and cell culture techniques. If successful, these experiments will greatly increase our understanding of the molecular mechanisms that direct and regulate the correct transport of rhodopsin and possibly other photoreceptor proteins. This understanding will be of significant clinical relevance because of the proposed link between rhodopsin mistargeting and photoreceptor cell death in some cases of RP. First, proteins that are necessary for rhodopsin transport may represent novel candidate genes for hereditary retinal diseases. Second, we may gain novel insights into the mechanisms by which rhodopsin mislocalization leads to cell death. Such insights may eventually prove to be useful for designing sight-preserving therapies for individuals with RP.