Abstract
Abstract: :
Purpose: Mutations in the X–linked gene RPGR give rise to RP, dominant cone dystrophy, recessive atrophic macular degeneration, and RP in combination with impaired hearing and sinorespiratory infections. The latter suggests a ciliary function in non–ocular tissue, but evidence about protein function on the molecular level remains scarce. The RPGR interacting protein 1 (RPGRIP1) was found to be involved in LCA and recessive CRD. We have set out to identify novel partners of the RPGR/RPGRIP1 protein network that can link this retina degeneration–associated complex with known pathways of photoreceptor function. Identified interactors will be candidate genes for inherited retina degeneration, as they are "guilty by association". Methods: We have created expression constructs of the domains that contain two protein kinase C conserved region 2 (C2) motifs, from bovine and human RPGRIP1 and their homologue KIAA1005. We performed a saturated yeast two–hybrid screen of both a randomly primed as well as an oligo dT primed bovine retina cDNA library, using the bovine C2 homologous domain as a bait. Putative interactors are being confirmed using GST pull–downs, co–IPs and immunohistochemistry. Results: The strongest interactor that was identified and confirmed by GST pull–down analysis links the RPGR/RPGRIP1 protein complex to focal adhesions, interaction sites of intracellular signalling molecules and cytoskeletal proteins. This interacting protein has previously been described to be part of a protein complex in primary cilia, and to be involved in a syndrome with a variable non–ocular phenotype and retinitis pigmentosa. Interaction with RPGRIP1 as well as with its only known homologue, KIAA1005, was confirmed. Additional putative interactors of the C2 homologous domain that were identified need to be confirmed. Preliminary data suggest the recruitment to the complex of proteins involved in membrane and cytoskeleton binding. These data fit very well in the hypothesis that RPGRIP1 has a function in vesicular transport, and the observation that at least a subset of RPGR and RPGRIP1 localize to the connecting cilium. Surprisingly, we also have identified a weaker, but frequent interactor of RPGRIP1 that suggests an interaction in the nucleus. This novel protein might be the first direct link with the apoptosis machinery and light sensitivity. Conclusions: We have identified novel members of the RPGR/RPGRIP1 protein complex that provide valuable links to functional pathways of the retina in health and disease.
Keywords: proteins encoded by disease genes • retinal degenerations: cell biology • photoreceptors