MOLECULAR BASIS OF PROTEIN TRANSPORT IN PHOTORECEPTORS

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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.