Dong-Hyun Hong

The retinitis pigmentosa GTPase regulator (RPGR)- interacting protein: Subserving RPGR function and participating in disk morphogenesis

Retinitis pigmentosa is a photoreceptor degenerative disease leading to blindness in adulthood. Leber congenital amaurosis (LCA) describes a more severe condition with visual deficit in early childhood. Defects in the retinitis pigmentosa GTPase regulator (RPGR) and an RPGR-interacting protein (RPGRIP) are known causes of retinitis pigmentosa and LCA, respectively. Both proteins localize in the photoreceptor connecting cilium (CC), a thin bridge linking the cell body and the light-sensing outer segment. We show that RPGR is absent in the CC of photoreceptors lacking RPGRIP, but not vice versa. Mice lacking RPGRIP elaborate grossly oversized outer segment disks resembling a cytochalasin D-induced defect and have a more severe disease than mice lacking RPGR. Mice lacking both proteins are phenotypically indistinguishable from mice lacking RPGRIP alone. In vitro, RPGRIP forms homodimer and elongated filaments via interactions involving its coiled-coil and C-terminal domains. We conclude that RPGRIP is a stable polymer in the CC where it tethers RPGR and that RPGR depends on RPGRIP for subcellular localization and normal function. Our data suggest that RPGRIP is also required for disk morphogenesis, putatively by regulating actin cytoskeleton dynamics. The latter hypothesis may be consistent with a distant homology between the C-terminal domain of RPGRIP and an actin-fragmin kinase, predicted by fold recognition algorithms. A defect in RPGRIP encompasses loss of both functions, hence the more severe clinical manifestation as LCA.

Retinitis Pigmentosa GTPase Regulator (RPGR)-interacting Protein Is Stably Associated with the Photoreceptor Ciliary Axoneme and Anchors RPGR to the Connecting Cilium

Retinitis pigmentosa (RP) is a blinding retinal disease in which the photoreceptor cells degenerate. Mutations in the gene for retinitis pigmentosa GTPase regulator (RPGR) are a frequent cause of RP. The function of RPGR is not well understood, but it is thought to be a putative guanine nucleotide exchange factor for an unknown G protein. Ablation of the RPGR gene in mice suggested a role in maintaining the polarized distribution of opsin across the cilia. To investigate its function, we used a protein interaction screen to identify candidate proteins that may interact physiologically with RPGR. One such protein, designated RPGR-interacting protein (RPGRIP), is expressed specifically in rod and cone photoreceptors. It consists of an N-terminal region predicted to form coiled coil structures linked to a C-terminal tail that binds RPGR. In vivo, both proteins co-localize in the photoreceptor connecting cilia. RPGRIP is stably associated with the ciliary axoneme independent of RPGR and is resistant to extraction under conditions that partially solubilized other cytoskeletal components. When over-expressed in heterologous cell lines, RPGRIP appears in insoluble punctate and filamentous structures. These data suggest that RPGRIP is a structural component of the ciliary axoneme, and one of its functions is to anchor RPGR within the cilium. RPGRIP is the only protein known to localize specifically in the photoreceptor connecting cilium. As such, it is a candidate gene for human photoreceptor disease. The tissue-specific expression of RPGRIP explains why mutations in the ubiquitously expressed RPGR confer a photoreceptor-specific phenotype.

Misexpression of the Constitutive Rpgrex1-19 Variant Leads to Severe Photoreceptor Degeneration

PURPOSE Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are a frequent cause of X-linked retinitis pigmentosa. The RPGR transcript undergoes complex alternative splicing to express both constitutive (Rpgr(ex1-19)) and Rpgr(ORF15) variants. Both variants localize to photoreceptor connecting cilia and are believed to play roles in ciliary function. This study examined variability in isoform expression and tested whether the constitutive variant could substitute for Rpgr function in photoreceptors. METHODS Rpgr(ex1-19) and Rpgr(ORF15) expression during retinal development were compared using immunoblot analysis and immunohistochemistry, and ciliary affinity in adult photoreceptors was assessed by protein fractionation. Transgenic mice expressing either the full-length Rpgr(ex1-19) or Rpgr(ORF15) variant were studied using light and electron microscopy and immunofluorescence imaging. The results were compared with those of wild-type and Rpgr(-/-) mice. RESULTS Rpgr expression undergoes dynamic temporal regulation during retinal development, and variants exhibit variability for ciliary localization in adult photoreceptors. Transgenic expression of both variants grossly exceeded endogenous Rpgr expression in photoreceptors. Although both variants exhibited normal ciliary localization, overexpression of the Rpgr(ex1-19) variant resulted in atypical accumulation of Rpgr in photoreceptor outer segments, abnormal photoreceptor morphology, and severe retinal degeneration. CONCLUSIONS The Rpgr isoform ratio in the adult retina is critical to photoreceptor integrity. The utilization of distinct Rpgr variants at different stages of photoreceptor maturation suggests independent roles in photoreceptor function. Finally, misexpression of Rpgr(ex1-19) causes retinal degeneration that is considerably more severe than that caused by Rpgr knockout but photoreceptors tolerate overexpression of Rpgr(ORF15) without evidence of degeneration.