Ciliary trafficking defects underlie the pathogenesis of severe human ciliopathies, including Joubert Syndrome (JBTS), Bardet-Biedl Syndrome, and some forms of retinitis pigmentosa (RP). Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are common causes of RP-associated photoreceptor degeneration worldwide. While previous work has suggested that the localization of RPGR to cilia is critical to its functions, the mechanism by which RPGR and its associated cargo are trafficked to the cilia is unclear. Using proteomic and biochemical approaches, we show that RPGR interacts with two JBTS-associated ciliary proteins: PDE6δ (delta subunit of phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E). We find that PDE6δ binds selectively to the C-terminus of RPGR and that this interaction is critical for RPGR’s localization to cilia. Furthermore, we show that INPP5E associates with the N-terminus of RPGR and trafficking of INPP5E to cilia is dependent upon the ciliary localization of RPGR. These results implicate prenylation of RPGR as a critical modification for its localization to cilia and, in turn suggest that trafficking of INPP5E to cilia depends upon the interaction of RPGR with PDE6δ. Finally, our results implicate INPP5E, a novel RPGR-interacting protein, in the pathogenesis of RPGR-associated ciliopathies.

Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgrko retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgrko PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgrko PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies.

Introduction Primary cilia are microtubule-based extensions of the plasma membrane in nearly all cell types. In vertebrate photoreceptors, the sensory cilium develops as outer segment (OS) that contains the photopigment Rhodopsin and other proteins necessary for phototransduction. The distinct composition of proteins and lipids in the OS membrane is maintained by the selective barrier located at the border between the basal body and the ciliary compartment, called the Transition Zone (TZ). Areas covered In this review, we will discuss the identification and function of two ciliary TZ proteins, RPGR (retinitis pigmentosa GTPase regulator) and CEP290. Mutations in these proteins account for a majority of retinopathies due to ciliary dysfunction. We will also discuss the potential of such information in designing therapeutic approaches to treat cilia-dependent photoreceptor degenerative diseases. Expert opinion RPGR and CEP290 perform overlapping yet distinct functions in regulating trafficking of cargo via the TZ of photoreceptors. While RPGR modulates the trafficking by acting as a GEF for the small GTPase RAB8A, CEP290 may be involved in maintaining the polarized distribution of proteins in the OS by modulating intracellular levels of selected proteins involved in inhibiting OS formation.

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