Prenylated retinal ciliopathy protein RPGR interacts with PDE6B and regulates ciliary localization of Joubert syndrome-associated protein INPP5E

Kollu Rao, Wei Zhang, Linjing Li, Manisha Anand, Hemant Khanna | Human Molecular Genetics | Vol. 25 (20) | 4533 - 4545 | 2016  October 15 | doi.org/10.1093/hmg/ddw281

Abstract

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.

Introduction

Cilia are microtubule-based membranous extensions of the apical plasma membrane (1) that serve as critical signalling nodes of diverse developmental processes, including sensory perception (2,3). As such, ciliary dysfunction due to mutations in ciliary proteins or trafficking of ciliary cargo is associated with severe human disorders, called ciliopathies. These include Meckel-Gruber Syndrome, Joubert Syndrome (JBTS), Bardet-Biedl Syndrome, and Usher Syndrome. Although these disorders differ in their age of onset and severity, they share common phenotypic features, including retinal degeneration due to defects in photoreceptor development and maintenance (4–6).

Photoreceptors are polarized sensory neurons with distinct inner segments involved in protein synthesis and trafficking, and photosensory outer segments (OS) (7,8). A photoreceptor OS extends from the apical region of the inner segment in the form of a microtubule based axoneme and contains stacks of membranous discs loaded with proteins involved in light detection. The region between the apical inner segment and the base of the OS is termed the connecting cilium, which is analogous to the transition zone (TZ) of primary cilia (9). The TZ is involved in regulating the composition of the OS. Commensurate with this role, proteins localized to the TZ are involved in regulating the composition of the photoreceptor cilia (6).

RPGR, is a ciliary TZ protein and has been suggested to regulate ciliary trafficking in photoreceptors (10). Mutations in RPGR result in severe photoreceptor degenerative diseases (11–15), the most prevalent of which is X-linked RP, a disorder characterized by progressive vision loss, usually by the third decade of life (16–24). RPGR mutations also account for 15–20% of simplex RP in males, making it a common cause of RP (17).

There are two major isoforms of RPGR: RPGRconst, a 19 exon constitutive transcript encoding a protein of 815 amino acids, and RPGRORF15, encoded by 15 exons (1152 amino acids) (21,22,25). The RPGRORF15 isoform shares exons 1-15 with the RPGRconst isoform; however, it continues into and terminates in intron 15. The terminal exon of this isoform is also termed ORF15. Although RPGRORF15 contains fewer exons, it is longer than RPGRconst because the length of exon ORF15 is greater than the combined length of exons 16-19 of RPGRconst. A schematic representation of these isoforms is depicted in Supplementary Material, Figure 1A and B.

Figure 1

Localization of RPGR domains to cilia. (A) hTERT-RPE1 cells were transiently transfected with constructs encoding the indicated GFP-fused RPGR proteins. After cilia formation, cells were fixed and stained with anti-GFP (green) and anti-ARL13B (red; cilia marker). Arrows in Merge point to ciliary staining (yellow, if co-localization; red, if no co-localization with ARL13B). Inset depicts enlarged view of ciliary staining of the cells. (B) Quantification of GFP-positive cilia is represented in the lower panel. One-way ANOVA analysis was performed to calculate the statistical significance of the differences in GFP-positive cilia. ***: P < 0.0001. AU: arbitrary units. Number (n) of cells analysed in this experiment: >200.

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