RPGR-ORF15, Which Is Mutated in Retinitis Pigmentosa, Associates with SMC1, SMC3, and Microtubule Transport Proteins

Abstract

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued , and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia. X-linked retinitis pigmentosa (XLRP)4 (MIM 312610) is a relatively severe and genetically heterogeneous inherited retinal degeneration.RP3 is the major subtype of XLRP accounting for over 70% of affected families (1, 2). The RP3 gene, called retinitis pigmentosa GTPase regulator (RPGR), encodes several distinct alternatively spliced transcripts that are widely expressed (3-5). Mutations in the constitutive RPGR protein of 815 amino acids are detected in ϳ20% of XLRP (6). Subsequent studies revealed an unusual exon, ORF15 (immediately following exon 15) encoding a Gly-and Glu-rich carboxyl-terminal domain of 567 amino acids; mutations in ORF15 accounted for an additional 50% of XLRP patients and 25% of RP males with no family history (7-9). Several distinct RPGR isoforms that include complete or part of ORF15 (RPGR-ORF15) are detected preferentially, but not exclusively, in the retina (7, 10) and localized to the connecting cilium and/or outer segments of photoreceptors (11-13).The amino-terminal region of RPGR (termed RCC1-like domain, RLD) shows homology to RCC1, a guanine nucleotide exchange factor for Ran, a GTPase involved in nucleocytoplasmic transport (14). Hence, RPGR was predicted to be a guanine nucleotide exchange factor for a small GTP-binding protein. However, no such activity or interaction has yet been demonstrated. Yeast two-hybrid analysis using RPGR-RLD had previously identified two proteins, RPGRIP1 (RPGR-interacting prot...

Intravitreal injection of ciliary neurotrophic factor (CNTF) causes peripheral remodeling and does not prevent photoreceptor loss in canine RPGR mutant retina

“…Previous work has shown that CNTF delivered through intravitreal injections (Pearce-Kelling, unpublished study), or by means of an ECT device (Tao et al, 2002), rescues photoreceptors in the rcd1 dog, an early and rapidly progressing large animal model of RP caused by a stop mutation in PDE6B. The purpose of the present study was to determine whether intravitreal injections of CNTF could provide a similar neuroprotective effect in XLPRA2, an early onset model of X-linked RP caused by a frameshift mutation in RPGR exon ORF15 (Zhang et al, 2002;Ferreira, 2005;Khanna et al, 2005). Recently, we reported the morphologic retinal changes, and the kinetics of photoreceptor cell death, that occur during the course of this disease .…”

Hypomorphic CEP290/NPHP6 mutations result in anosmia caused by the selective loss of G proteins in cilia of olfactory sensory neurons

“…Selective entry through the nuclear pore requires cargo binding to a GTPbinding protein and guanine-nucleotide exchange involving a GEF to release cargo into the nucleus (27). We showed that RPGR ORF15 , a putative GEF (28), is a cilia/centrosomal protein that may regulate selective transport of cargo proteins from basal bodies distally along the ciliary axoneme in the photoreceptor connecting cilium (14,29). We now show that RPGR ORF15 is in complex with CEP290 in OSNs of both WT and rd16 mice, suggesting a possible role in regulating protein entry into the olfactory cilium.…”

“…Genetic mutations in ciliary, basal body, and centrosomal proteins lead to pleiotropic human diseases, including polycystic kidney disease, Bardet-Biedl syndrome, Senior-Loken syndrome, Meckel syndrome, and retinitis pigmentosa (RP) (2,7,(13)(14)(15). Nephrocystins are a family of ciliary proteins that are likely involved in cargo sorting during transport from the basal body to the ciliary axoneme (16)(17)(18)(19)(20).…”

Accumulation of the Raf-1 Kinase Inhibitory Protein (Rkip) Is Associated with Cep290-mediated Photoreceptor Degeneration in Ciliopathies

“…35 S signal was analyzed with STORM 840 (GE Healthcare) and GST pulldown input was analyzed by Coomassie Brilliant Blue staining. Immunoprecipitations were performed, as described (39). The precipitated proteins were resolved by two-dimensional gel electrophoresis and stained with SYPRO Ruby (Invitrogen).…”

“…GST Pulldown, Immunoprecipitations (IP), and Tandem Mass Spectrometry Analysis-GST pulldown analysis was carried out as described (39). Briefly, in vitro translation was performed using the [ 35 S]methionine in vitro translated reaction mix (PROMEGA TNT Quick kit).…”