MECHANISMS OF PHOTORECEPTOR CELL DEGENERATION

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Mutations in the gene encoding the disk rim specific protein, peripherin/rds, have been implicated in the pathogenesis of both autosomal dominant retinitis pigmentosa (ADRP) and various forms of macular dystrophy (MD). Peripherin/rds plays an important role in the morphogenesis and maintenance of the disk rim structure. This role is supported by interactions with other proteins. The primary goals of the proposed research are to determine the molecular basis of action and functional properties of peripherin/rds and these interacting proteins, and to investigate the molecular abnormalities that lead to photoreceptor degeneration in transgenic models of human retinal diseases. Specific Aims 1, 2 and 3 are to use the structural, electrophysiological and biochemical studies to evaluate transgenic retinas expressing different mutations in the peripherin/rds gene. These mutations include: (1) the ARG172TP mutation that causes MD in humans, (2) the CYS214Ser mutation to determine the role of intermolecular disulfide bonds in the assembly of functional peripherin/rds-rom-1 complexes. We will analyze the effects of these mutations on morphogenesis of the disc membrane and on regulation of complex formation by peripherin/rds and rom-1. We can evaluate the different roles played by peripherin/rds in rods versus cones by comparing the effects of the Arg172Trp and Cys214Ser mutations on the structure and function of rod and cone outer segments. Comparisons of the Cys214 and Cys150Ser transgenic mice will provide insight into the role of inter- and intramolecular disulfide bond formation in peripherin/rds function. In Specific im 4, we will use a yeast two-hybrid system to identify the sites of interaction involved in formation of multimeric complexes by peripherin/rds and rom-1. This system provides a powerful genetic mechanism for detecting protein-protein interactions. We will also use the yeast two-hybrid system to screen a retinal cDNA library for other proteins that are involved in the assembly of functional peripherin/rds-rom-1 complexes. We have identified regions of high homology between all known peripherin/rds and rom-1 that are located in the large intradiscal loop. We hypothesize that these regions mediate the interactions between peripherin/rds-rom-1 complexes and are required to hold the rod discs or cone lamellae in their flattened shape. These interactions may be mediated by direct association of the subunit complexes or indirectly through other proteins. Mutagenesis studies will be performed to evaluate these interactions. These studies will provide insight into the functional role of peripherin/rds in normal and diseased retinas.