Retinitis Pigmentosa Gtpase Regulator (RPGR) Protein Isoforms in Mammalian Retina: Insights Into X-Linked Retinitis Pigmentosa and Associated Ciliopathies

Vision Research 48 (2008) 366–376 www.elsevier.com/locate/visres

Retinitis Pigmentosa GTPase Regulator (RPGR) protein isoforms in mammalian retina: Insights into X-linked Retinitis Pigmentosa and associated ciliopathies

Shirley He a, Sunil K. Parapuram a, Toby W. Hurd b, Babak Behnam a,1, Ben Margolis b, Anand Swaroop a,c, Hemant Khanna a,*

a Department of Ophthalmology and Visual Sciences, University of Michigan, W. K. Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105, USA b Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48105, USA c Department of Human Genetics, University of Michigan, Ann Arbor, MI 48105, USA

Received 3 July 2007; received in revised form 3 August 2007

Abstract

Mutations in the cilia-centrosomal protein Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of retinal degeneration. The RPGR gene undergoes complex alternative splicing and encodes multiple protein isoforms. To elucidate the function of major RPGR isoforms (RPGR1–19 and RPGRORF15), we have generated isoform-specific antibodies and examined their expression and localization in the retina. Using sucrose-gradient centrifugation, immunofluorescence and co-immunoprecipitation methods, we show that RPGR isoforms localize to distinct sub-cellular compartments in mammalian photoreceptors and associate with a number of cilia-centrosomal proteins. The RCC1-like domain of RPGR, which is present in all major RPGR isoforms, is sufficient to target it to the cilia and centrosomes in cultured cells. Our findings indicate that multiple isotypes of RPGR may perform overlapping yet somewhat distinct transport-related functions in photoreceptors. Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Cilia; Centrosomes; Photoreceptor; Ciliary transport; Rod outer segments

1. Introduction (Breuer et al., 2002; Fishman, 1978; Jay, 1982). To date, six RP loci have been mapped on the X-chromosome: X-linked Retinitis Pigmentosa (XLRP) is a clinically RP2, RP3, RP6, RP23, RP24 and RP34 (Fujita et al., and genetically heterogeneous disorder, characterized by 1996; Gieser et al., 1998; Mears et al., 2000; Meindl early-onset visual symptoms, with night-blindness gener- et al., 1996; Melamud et al., 2006; Schwahn et al., 1998; ally in the ﬁrst decade and rapid progression towards blind- Shu et al., 2007; Wright et al., 1991)(http://www.sph.uth. ness by age 40 (Bird, 1975; Fishman, Farber, & Derlacki, tmc.edu/Retnet). The genes for RP3 and RP2 disease are 1988). Some XLRP patients also exhibit abnormal sperm Retinitis Pigmentosa GTPase Regulator (RPGR)and phenotype or hearing defects (Hunter, Fishman, & Kretzer, RP2, respectively (Meindl et al., 1996; Roepman et al., 1988; Iannaccone et al., 2003; Zito et al., 2003). XLRP 1996; Schwahn et al., 1998). accounts for 10–20% of inherited retinal dystrophies Mutations in RPGR account for a majority of XLRP and over 25% of simplex RP (Breuer et al., 2002; Sharon et al., 2000; Shu et al., 2007; Vervoort & Wright, 2002). Some of * Corresponding author. Fax: +1 734 936 7661. the patients with RPGR mutations exhibit a syndromic phe- E-mail address: [email protected] (H. Khanna). 1 Present address: Biomolecular Science Center, Burnett College of notype, including respiratory tract infections, hearing loss, Biomedical Sciences, University of Central Florida, Orlando, FL, 32816, and primary cilia dyskinesia (Iannaccone et al., 2003; Koene- USA. koop et al., 2003; Moore et al., 2006; van Dorp, Wright,

Carothers, & Bleeker-Wagemakers, 1992; Zito et al., 2003). the phenotype of Rpgr-ko mouse (Hong, Pawlyk, Adami- RT-PCR studies have demonstrated complex alternative an, & Li, 2004; Hong, Pawlyk, Adamian, Sandberg, & splicing patterns of the RPGR gene, with over 20 different Li, 2005). Two spontaneous canine mutants of variant mRNAs (Ferreira, 2005; Hong & Li, 2002; Kirschner RPGRORF15—X-linked Progressive Retinal Atrophy et al., 1999; Neidhardt et al., 2007; Yan et al., 1998). All pro- (XLPRA) 1 and XLPRA2—harbor different frameshift tein isoforms are predicted to include an amino-terminal mutations and exhibit slow or rapid photoreceptor degen- domain (RCC1-like domain; RLD; encoded by exons 2– eration, respectively (Zhang et al., 2002). Different ORF15 11) homologous to Regulator of Chromosome Condensa- mutations can therefore result in distinct phenotypes, prob- tion 1 (RCC1), which is a guanine nucleotide exchange factor ably depending upon the degree of alternative splicing and for Ran-GTPase involved in nucleo-cytoplasmic transport loss-of-function of RPGR. (Meindl et al., 1996; Renault, Nassar, Wittinghofer, Roth, RPGR was first localized to the connecting cilium of & Vetter, 1999). However, no GTPase binding or activity mammalian photoreceptors and to the transition zone of has yet been associated with RPGR isoforms. motile cilia (Hong et al., 2003). Further studies reported Two widely-expressed isoforms of RPGR are: species-specific localization of RPGR to the connecting cil- RPGREx1–19 (derived from exons 1–19, encoding a protein ium (mouse) or photoreceptor outer segments (human and of 815 amino acids), which is detected in all cell types bovine) (Mavlyutov, Zhao, & Ferreira, 2002). By immuno- examined; and RPGRORF15 (exons 1—part of intron 15), gold microscopy, we demonstrated RPGRORF15 isoforms which has been associated with primary cilia (Khanna predominantly in the cilia and basal bodies of human et al., 2005; Kirschner et al., 1999; Shu et al., 2005; Ver- and mouse photoreceptors; however, additional staining voort et al., 2000; Yan et al., 1998). Interestingly, muta- in the outer and inner segments is detectable (Khanna tions in exons 1–14 account for less than 25% of XLRP et al., 2005). RPGR is also expressed in motile cilia of (Buraczynska et al., 1997; Fujita et al., 1997; Sharon mouse trachea, human and monkey cochlea, and localizes et al., 2000). An additional 50–60% of XLRP patients to centrosomes and primary cilia of renal epithelial cells reveal mutations in the terminal exon ORF15 of the (Hong et al., 2003; Iannaccone et al., 2003; Khanna RPGRORF15 isoform (Shu et al., 2007; Vervoort et al., et al., 2005; Shu et al., 2005). 2000), which includes a C-terminal acidic domain rich in A clearer understanding of sub-cellular localization and Glu-Gly repeats (EEEGEGE repeat in mouse, EEEGEGE- physiological relevance of RPGR isoforms is critical for GE repeat in human) (Vervoort et al., 2000) and undergoes elucidating the mechanism of retinal disease caused by additional alternative splicing due to the presence of pur- RPGR defects. Here, we show that RPGR1–19 and ine-rich exonic splicing enhancers (Hong & Li, 2002). RPGRORF15 isoforms are present as isotypes, which local- RPGR interacts directly with PDE6-d, RPGR-interact- ize to distinct subcellular compartments in the retinal pho- ing protein 1 (RPGRIP1), Structural Maintenance of Chro- toreceptors. We also demonstrate that, like RPGRORF15, mosomes (SMC) 1, SMC3, and nucleophosmin (Boylan & RPGR1–19 also associates with microtubule-based protein Wright, 2000; Hong, Yue, Adamian, & Li, 2001; Khanna assemblies and ciliary components in the retina. We pro- et al., 2005; Linari, Hanzal-Bayer, & Becker, 1999; Roep- pose that distinct RPGR isoforms mediate overlapping man et al., 2000; Shu et al., 2005). RPGR can be immuno- functions in regulating photoreceptor protein transport precipitated from retinal extracts with selected ciliary and by associating with discrete macromolecular complexes. microtubule-associated proteins, including motor proteins and intraflagellar transport polypeptide IFT88 (Khanna 2. Materials and methods et al., 2005). RPGR also interacts with nephrocystin (NPHP) family of ciliary disease proteins, NPHP5 and All animal experiments were performed according to the protocols CEP290/NPHP6; mutations in these are associated with approved by the University Committee on Use and Care of Animals (UCUCA). Senior-Loken Syndrome (NPHP5), Joubert Syndrome, Leber congenital amaurosis, and Meckel Syndrome 2.1. Antibodies and constructs (CEP290/NPHP6) (Baala et al., 2007; Brancati et al., 2007; Chang et al., 2006; den Hollander et al., 2006; Perra- We generated and characterized several isoform-specific antibodies ult et al., 2007; Sayer et al., 2006; Valente et al., 2006). All against RPGR (Fig. 1a). The ORF15CP and ORF15C2 antibodies were patients with NPHP5 or NPHP6/CEP290 mutations reveal raised against carboxyl-terminal epitopes, 1100HKTYQKKSVTNTQGN 1117 1097 1107 a retinal disease phenotype. These observations point to a GKE and YGKHKTQKKS , respectively, of the human RP GR-ORF15 protein. The CT-15 antibody was generated against the peptide key role of RPGR in photoreceptor ciliary transport. sequence 1131KNGPSGSKKFWNNILPHYLELK1152 of human RP A Rpgr-knockout (Rpgr-ko) mouse, generated by delet- GRORF15. Antibody common to both RPGR1–19 and RPGRORF15 isoforms, ing exons 4–6, exhibits a relatively slow retinal degenera- GR-P1 was raised against the peptide sequence 18GKSKFAENNPG tion with apparent mis-localization of cone opsins (Hong KFWFK33 encoded by exon 1 of human RPGR. The RPGR-E19 antibody 982 995 et al., 2000). Expression of truncated RPGRORF15-isoforms was against amino acid sequence NLQDSTTPNMEGKS , encoded by RPGR exon 19 (Invitrogen). Characterization of the ORF15CP, GR-P1, can be detected in the retina of this mutant mouse (Khanna C2 ORF15 CT-15 and ORF15 antibodies has been described (Khanna et al., 2005; et al., 2005). Notably, a truncated version of RPGR Otto et al., 2005; Shu et al., 2005; Yan et al., 1998). Antibodies against acet- can act as a dominant gain of function mutant or rescue ylated a-tubulin, SMC1, SMC3, c-tubulin, and p50-dynamitin were pro- 368 S. He et al. / Vision Research 48 (2008) 366–376

Fig. 1. (a) Schematic representation of the major RPGR protein isoforms (constitutive RPGR1–19 and RPGRORF15) with distinct predicted domains. RPGR antibodies against peptides from different parts of the protein are represented by arrows. P-loop: ATP-GTP binding region; RLD: RCC1-like domain; Glu/Gly: Glutamic acid and Glycine rich domain; Prenyl: C-terminal isoprenylation site; C2: C-terminal basic domain. (b) COS-7 cells were transiently transfected with pcDNA4A-Ex16–19. The cell extracts were analyzed by SDS–PAGE and IB using anti-Xpress or RPGR-E19 antibodies. (c) Immunoblots of mouse retinal extract using pre-immune or RPGR-E19 anti-serum. Specific immunoreactive bands are indicated. cured from Chemicon (Temeculla, CA); anti-p150Glued and anti-KIF3A diluted with Buffer A, followed by centrifugation for 1 h on a cushion were obtained from BD Transduction Labs (San Jose, CA) and Sigma, of Buffer A with 50% (w/w) sucrose and collected at the interface. The respectively. Anti-RP1 antibody was a generous gift of Dr. Eric A. Pierce; fraction was extracted for 1 h by adding an equal volume of Buffer Anti-IFT88 (Polaris) was provided by Dr. Bradley Yoder and anti- A/1 mM DTT/2% Triton X-100/protease inhibitors. The detergent soluble RPGRIP1 and ORF15C2 antibodies were a gift of Dr. Alan Wright. To gen- and insoluble materials were separated by centrifugation for 1 h at erate the FLAG-RLD construct, PCR was performed to amplify the region 13,000g, and the supernatant was saved as soluble fraction. Insoluble of human RPGR encoding residues 34–372 of RPGR. This amplicon was cilia-enriched fractions were suspended in Buffer A/1 mM DTT/ 2%Triton ligated in-frame into the HindIII and BamH1 sites of p3xFLAG-CMV-10 X-100/protease inhibitors and sonicated. (Sigma). 2.3. Retinal dissociation, cell culture, and immunolocalization 2.2. Retinal fractionation Mouse retina was fixed in Histochoice (Amersco, Inc.) according to Frozen bovine retinas were thawed in 5 volumes (w/v) of a buffer con- manufacturer’s instructions. The sections were blocked in 0.5% Triton CP taining 100 mM Tris–HCl buffer (pH 7.4), 0.25 M sucrose, 10 mM MgCl2, X-100% and 5% bovine serum albumin in PBS and labeled with ORF15 , 5 mM dithiothreitol and Complete protease inhibitor cocktail (Roche), RPGR-E19, or acetylated a-tubulin antibodies followed by fluorescent- and homogenized using a Dounce homogenizer. Homogenates were frac- labeled secondary antibodies. tionated by differential centrifugation. The membrane/nuclei (800g, For retinal dissociation, adult mouse retina was cut into small pieces 10 min) and microsomal (100,000g, 60 min) fractions were washed with and incubated for 10 min in phosphate-buffered saline (PBS) containing and suspended in the buffer described above. The supernatant obtained papain activated with L-cysteine. The pieces of retina were then gently trit- after centrifugation at 100,000g for 60 min was used as the cytosol urated with a pipette and a sample checked under microscope for identi- fraction. fying single cells. Serum (10%)-containing DMEM was added to stop Cilia-enriched fractions were prepared according to (Fleischman, the reaction. Dissociated photoreceptors were put on slides and fixed with 1982), with minor modifications. Briefly, frozen bovine retinas were 4% paraformaldehyde. After 30 min, slides were washed with PBS and thawed in Buffer A (10 mM Pipes, 5 mM MgCl2, pH.7.0) containing used for immunocytochemistry. 50% sucrose (w/w) with 0.1 mM of PMSF and protease inhibitors and HeLa and MDCK II cells were propagated in DMEM plus 10%FBS, swirled on ice for 30 min. RIS-ROS were separated from the remainder supplemented with Penicillin, Streptomycin and L-glutamine. For cell syn- of the retina by centrifuge at 900g for 10 min, followed by centrifugation chronization, HeLa cells were growth-arrested by incubating with serum- for 1 h at 13,000g. Crude RIS-ROS were collected from the top of the free DMEM for 48 h and fixed for immunocytochemistry. MDCK II cells sucrose, injected at the bottom of a linear sucrose gradient (25–45%) in stably expressing FLAG-RLD were selected in media containing 600 lg/ Buffer A, and centrifuged for 1 h at 13,000g. Purified RIS-ROS were col- ml G418 (Invitrogen). For immunstaing cilia, MDCKII and FLAG- lected from the top half of the gradient. The purified RIS-ROS were RLD MDCKII cells were seeded onto transwell filters (Corning) and S. He et al. / Vision Research 48 (2008) 366–376 369 grown for 7 days post-confluence. Cells were fixed in 4% PFA/PBS followed by permeabilization in 0.1% TritonX100. Cells were blocked and immuno-stained in 2% goat serum/PBS with indicated antibodies. Alexa-labeled secondary antibodies were obtained from Invitrogen. Cells were mounted using ProLong Gold reagent (Invitrogen).

2.4. Co-immunoprecipitation (Co-IP) assay

The details of co-IP experiments have been described earlier (Khanna et al., 2005).

3. Results

3.1. Characterization of the isoform-speciﬁc RPGR antibodies

Our previous studies using GR-P1 (Khanna et al., 2005; Yan et al., 1998), CT-15, ORF15CP (Khanna et al., 2005) and ORF15C2 (Shu et al., 2005) have demonstrated the existence of multiple isotypes of RPGR1–19 and RPGRORF15 isoforms in mammalian retina. Whereas GR-P1 and HR1 antibodies detected immunoreactive bands at 90 and 250 kDa, CT-15, ORF15CP, and ORF15C2 detected multiple bands in the molecular mass range of 100–250 kDa (data not shown; Khanna et al., 2005; Otto et al., 2005; Shu et al., 2005). To specifically analyze the RPGR1–19 isoform, we generated the RPGR-E19 antibody against an epitope in exon 19. The RPGR-E19 antibody recognizes Xpress-tagged Rpgr exon 16–19-derived protein Fig. 2. Immunoblot analysis of human, bovine, and mouse retinal in transiently-transfected COS-7 cells (Fig. 1b), and homogenates using the ORF15CP and RPGR-E19 antibodies. Equal RPGREx1–19 isoforms in mouse retina (Fig. 1c). Additional amount of protein (100 lg) was analyzed by SDS–PAGE followed by immunoblotting using indicated antibodies. b-Tubulin was used to assess protein bands of higher molecular weight probably indicate loading control. Molecular mass markers are shown on the right and post-translational modifications or alternative isoforms. individual immunoreactive bands for different RPGR isoforms are Pre-immune serum did not detect a signal. depicted on the left. For subsequent studies, we selected ORF15CP and RPGR-E19 antibodies to differentiate between the two pri- ORF15 RPGR -1 and 2 may represent proteolytically pro- mary RPGR isoforms. Pre-incubation of the antibodies ORF15 cessed fragments of RPGR . with specific peptide but not non-specific peptide elimi- CP The RPGR-E19 antibody detected bands of 90 nated the immuno-reactive signal for ORF15 antibody 1–19 1–19 1–19 (RPGR -1), 110 (RPGR -2), 140 (RPGR -3), 150 in our immunoblots analyses (data not shown; Otto 1–19 1–19 (RPGR -4), and 160 kDa (RPGR -5), while the et al., 2005). 1–19 expected molecular mass of the RPGR isoform is 90 kDa (Yan et al., 1998). The expression of RPGR1–19-2 3.2. RPGR isoforms in different species was not consistently detectable in retinal homogenates; however, upon fractionation it is enriched in the cytosolic ORF15 We examined the expression of different RPGR compartment (see Fig. 3). Some of the bands observed with 1–19 and RPGR isoforms in human, bovine, and mouse ret- these antibodies may represent post-translationally modi- CP inas. The ORF15 antibody detected bands at 100, 120, fied isotypes or alternatively spliced isoforms. Notably, less ORF15 and 140 kDa (Fig. 2; labeled as isoforms RPGR -1, expression of both RPGR isoforms is observed in mouse as 2, and 3, respectively). Higher molecular weight bands of compared to human and bovine retinal extracts. 240–250 kDa (RPGRORF15-4 and 5) were also observed in the retinal homogenates. The expected apparent molecular weight of the full-length RPGRORF15 isoform is 3.3. Compartmentalization of RPGR isoforms in bovine 140 kDa (Vervoort et al., 2000); however, due to the retina highly acidic carboxyl-terminal region, it may migrate at an aberrant rate. The expression of RPGRORF15-3 isoform We next examined the sub-cellular distribution of is not consistently detected in these experiments, indicating RPGR1–19 and RPGRORF15 isoforms in bovine retina. that this isoform is either unstable, expressed at very low Sucrose-gradient fractionation, followed by immunoblot levels, or post-translationally modified. Isoforms analysis, revealed the presence of RPGRORF15-4 and 5 370 S. He et al. / Vision Research 48 (2008) 366–376

Fig. 3. Bovine retinas were subjected to sucrose gradient centrifugation followed by immunoblotting using the ORF15CP and RPGR-E19 antibodies. Equal amount of protein (50 lg) was analyzed by SDS–PAGE and immunoblotting. Purity of fractions was determined by enrichment of marker proteins (data not shown). Protein amount control was not used as different fractions may have enrichment of distinct proteins, including b-tubulin. Different RPGR isoforms are identified on the left and the molecular mass markers (in kDa) are denoted on the right. isoforms in the nuclear and ciliary compartments and of 3.5. Cell-cycle dependent localization of RPGR to RPGRORF15-2 in the cytosolic fraction. RPGRORF15-4 centrosomes and RPGRORF15-5 were detected in considerably lower amounts in microsomes and excluded from the rod outer We previously demonstrated the localization of RPGR segment (ROS) fraction (Fig. 3). to centrosomes in cultured cells (Shu et al., 2005). Given Analysis of retinal fractions shows that the RPGR1–19-5 the importance of centrosomes in regulating cell division isoform is distributed in all fractions except soluble ROS and in recruiting components of microtubule-nucleation and ciliary compartments. RPGR1–19-3 does not seem to machinery (Badano, Teslovich, & Katsanis, 2005; Doxsey, be enriched in any of the fractions tested, whereas McCollum, & Theurkauf, 2005), we performed a more RPGR1–19-1 is enriched in the cytosolic and microsomal detailed analysis of the localization of RPGR during differ- fractions. We also observe the enrichment of a 250 kDa ent stages of cell division using synchronized He La cells. RPGR-E19 immuno-reactive band (RPGR1–19-5a) in the Immuocytochemical studies demonstrated that endoge- ciliary fraction (Fig. 3). nous RPGRORF15 exhibits cell cycle-dependent localization in the nucleus and centrosomes (Fig. 5A). During pro- 3.4. Localization of RPGR1–19 and RPGRORF15 in mouse phase, RPGR localization is predominant in the nucleus, retina whereas in other stages, it co-localizes with c-tubulin at the centrosomes (spindle poles). We also detected RPGR To validate the fractionation data, we performed immu- at the midbody in telophase (cytokinesis; Fig. 5B), a feature nohistochemistry of mouse retinal sections using the shared by several other core centrosomal proteins (Tsvet- RPGR-E19 and ORF15CP antibodies. As shown in kov, Xu, Li, & Stern, 2003). In addition to centrosomes Fig. 4A and B, RPGRORF15 and RPGR1–19 localize to and midbody, staining of other cellular structures is also the inner segments of photoreceptors, with punctate stain- detected for RPGRORF15. ing in the outer and inner plexiform layers of the retina. Labeling at the junction between the inner and outer seg- 3.6. RPGR-RLD is sufficient for basal body/ciliary ment indicates ciliary localization. We confirmed this find- localization of RPGR ing by co-staining with acetylated a-tubulin, a ciliary marker. Consistent with this, staining of dissociated photo- Given that disease-associated mutations in the RPGR- receptors of mouse retina also detected inner segment/con- RLD are predicted to affect its function and that all RPGR necting cilium staining for the RPGRORF15 and RPGR1–19 isoforms contain this domain (Shu et al., 2007), we tested isoforms (Fig. 4C; data not shown). the possibility that the RLD is sufficient for localization S. He et al. / Vision Research 48 (2008) 366–376 371 to the functionally relevant compartments, basal bodies localization of RPGR and may partially explain the pre- and cilia. Hence, we generated MDCKII cells stably dominant localization of RPGRORF15 and RPGR1–19 to expressing FLAG-tagged RPGR-RLD. Remarkably, the the connecting cilium of photoreceptors. However, evidence RLD localizes to the basal bodies/centrioles in polarized for the requirement of RLD in the localization of RPGR to non-ciliated MDCK cells (Fig. 5C; a–d), whereas after cil- basal bodies and cilia merits further investigation. iogenesis it traffics to the primary cilia (Fig. 5C; e–h). These results suggest that the RLD can assist in basal body/ciliary 3.7. RPGR1–19. interacting proteins in the retina

RPGRORF15 is shown to be a component of the multiprotein complexes involved in regulating the intraphotore- ceptor protein transport (Khanna et al., 2005). To investigate functional overlap between the RPGRORF15 and RPGR1–19 isoforms, we looked at the interaction of RPGR1–19 with other proteins involved in intracellular traﬃcking. Immunoprecipitation (IP) of RPGR1–19 from bovine retinal extract followed by immunoblotting revealed the presence of RPGRIP1, CEP290/NPHP6, SMC1, c- tubulin, kinesin motor subunits (KIF3A and KAP3), and dynein-dynactin subunits (p150Glued, dynein intermediate chain (DIC), and p50-dynamitin) (Fig. 6a). Reverse IP experiments demonstrated that RPGRIP1, CEP290/ NPHP6, SMC1, p150Glued, p50-dynatimin, KIF3A, and KAP3 can pull-down RPGR1–19 but SMC3, c-tubulin, and DIC did not, likely due to relatively low abundance of RPGR1–19 in these multi-protein complexes (Fig. 6b). We did not detect association of RPGR1–19 with RP1, Nephrocystin-5 (NPHP5), and IFT88 (Polaris). IP with the pre-immune serum did not detect a signal. Notably, RPGRORF15 interacts with NPHP5 and IFT88 (Khanna et al., 2005). Taken together, these results indicate that different RPGR isoforms may perform overlapping yet distinct roles in regulating photoreceptor protein traﬃcking.

4. Discussion

A majority of mammalian genes produce multiple RNA and protein isoforms, generating extensive diversity of cellular functions (Kapranov, Willingham, & Gingeras, 2007). Complex splicing patterns result in a large degree of heter- ogeneity of RPGR expression. Therefore, it is imperative to dissect the different protein isoforms of RPGR in the retina. We show that RPGR1–19 and RPGRORF15 are detected b Fig. 4. Localization of RPGR isoforms in mouse retina. Immunohisto- chemical analysis of cryosections of adult wild-type mouse retina was performed using ORF15CP (A) or RPGR-E19 (B) antibodies (green). Enlarged image if the inner segment/connecting cilium indicates co- localization with acetylated (Acet.) a-tubulin (red) (Merge; yellow). ORF15CP-specific staining is also observed in the Inner segment and proximal outer segment (A). DAPI was used to stain the nuclei (blue). OS: Outer segments; CC: connecting cilium; IS: inner segments; ONL: Outer nuclear layer; OLM: outer limiting membrane; OPL: outer plexiform layer; INL: Inner nuclear layer; GCL: ganglion cell layer. (C) Immuno- cytochemistry using dissociated GFP-tagged rod photoreceptors from Nrl- GFP mouse retina (Akimoto et al., 2006). ORF15CP antibody (red) co- localizes partially with acetylated a-tubulin (blue) (Merge; pink) at the proximal connecting cilium (CC) and not in outer segment (OS). N: nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) 372 S. He et al. / Vision Research 48 (2008) 366–376

RPGRIP1 and the different products differentially localize to subcellular compartments (Lu & Ferreira, 2005; Lu, Guruju, Oswald, & Ferreira, 2005). Proteolysis of ciliary proteins, such as polycystin-1 and Gli transcription factors serves as a mechanism of transducing extracellular signals to cell interior (Haycraft et al., 2005; Low et al., 2006; Sin- gla & Reiter, 2006). We previously demonstrated that nuclear proteins SMC1 and SMC3 localize to the photoreceptor connecting cilium and interact with RPGR-RLD (Khanna et al., 2005). Given that RPGR isoforms can be detected in the nuclear fractions, we hypothesize that RPGR undergoes proteolytic processing or other post- translational modifications, resulting in association with SMC1/3 complex and transport to the nucleus as a macromolecular complex. As demonstrated earlier for RPGRIP1 (Lu & Ferreira, 2005), expression of multiple RPGR isoforms in the retina and their differential distribution merits the consideration of isoform ratios. It is plausible that some RPGR isoforms are enriched in specific species and cell-types, resulting in apparent differences in localization and functional compen- sation. Given that over-expression of a truncated RPGRORF15 isoform can result in a dominant gain-of- function effect (Hong et al., 2004) and that a splice-site mutation upstream exon 9a of RPGR, which is associated with RP, leads to high levels of exon 9a-containing tran- scripts (Neidhardt et al., 2007), abnormal enrichment of some isoforms over others may drastically change the RPGR isoform ratio and result in retinopathy. Microsomes contain fragmented endoplasmic reticulum and are enriched in cytoskeletal proteins, metabolic enzymes, and transmembrane moieties (Han, Eng, Zhou, Fig. 5. (A and B) Cell cycle dependent localization of RPGRORF15: & Aebersold, 2001). We had previously demonstrated that 1–19 Synchronized HeLa cells were fixed and analyzed by immunocytochem- RPGR is prenylated at the carboxyl-terminus, a modi- istry using ORF15CP (green) or c-tubulin (red) antibodies. RPGRORF15 is fication that assists in membrane association (Yan et al., detected in the nuclei at prophase and co-localizes with c-tubulin at the 1998). Although the RPGRORF15 isoform(s) do not contain centrosomes during cell division (A; arrows). Staining for other cellular CP the isoprenylation site (CTIL) encoded by exon 19 (Yan structures is also detected with the ORF15 antibody. DAPI was used to ORF15 stain the nuclei (blue). RPGRORF15 also localizes to the midbody at et al., 1998), RPGR may interact with other proteins telophase, depicted by co-localization with c-tubulin and b-tubulin (B). in order to localize to microsomes. (C) Non-ciliated (a–d) and ciliated (e–h) MDCK cells stably expressing The association of RPGR with midbody and centro- FLAG-tagged RPGR-RLD were stained with anti-FLAG (green) or anti- somes indicates its involvement in regulating cell division. polaris (IFT88; red) antibodies. Nuclei were stained with DAPI. Merge Midbody marks the intercellular bridge during the forma- image shows basal body (d) and ciliary (h) localization of RPGR-RLD prior to and after ciliogenesis, respectively. (For interpretation of the tion of cleavage furrow at telophase (Tsvetkov et al., references to color in this figure legend, the reader is referred to the web 2003). It is possible that RPGR is involved in the assembly version of this paper.) of proteins involved in the formation of the furrow by regulating microtubule-based membrane trafficking, a phe- as multiple immunoreactive bands in mammalian retina nomenon critical for normal cytokinesis (Tsvetkov et al., and localize to distinct subcellular compartments, likely 2003). Support of this hypothesis comes from a previous contributing to the reported disparate localization of report demonstrating the localization of another cilia-cen- RPGR. Notably, these isotypes associate with different sets trosomal disease protein BBS6 at midbody (Kim et al., of proteins involved in microtubule transport in the retina. 2005). The precise role of RPGR in the midbody and cen- Different isotypes generated by the RPGR1–19 and trosomes during cell division warrants additional studies. RPGRORF15 splice variants can occur due to post-transla- In postmitotic cells, such as photoreceptors, the mother tional modifications (such as phosphorylation, prenylation, centriole of the centrosomes migrates to the apical mem- or proteolytic processing) or additional splicing, as demon- brane of cells and nucleates the assembly of cilia (Badano strated with exon ORF15 (Hong & Li, 2002; Yan et al., et al., 2005). A predominant ciliary localization of both 1998). Proteolytic processing has been demonstrated for RPGRORF15 and RPGR1–19 isoform may be due to the S. He et al. / Vision Research 48 (2008) 366–376 373

Fig. 6. Co-immunoprecipitation (IP) of bovine retinal homogenates (200 lg) using RPGR-E19 (a) was performed followed by SDS–PAGE and immunoblotting using indicated antibodies. Input represents 20% of the protein used for IP. Lanes are: 1, Input; 2: IP with pre-immune serum; 3: IP with RPGR-E19. (b) Reverse IP using indicated antibodies was performed and proteins analyzed by SDS–PAGE and immunoblotting using the RPGR-E19 antibody. ability of the RCC1-like domain of RPGR to localize to and IFT88 in the retina. We and others have shown that the primary cilia. The photoreceptor connecting cilium RPGR isoforms localize to the proximal region of photore- serves as a conduit for bidirectional inter-segmental trans- ceptor connecting cilium, called the transition zone, and to port of protein complexes along the microtubule network basal bodies, which may act as a selection barrier for the (Besharse, Baker, Luby-Phelps, & Pazour, 2003; Young proteins being transported distally to the axoneme and & Droz, 1968). The cargo vesicles undergo polarized outer segments (Liu et al., 2007; Singla & Reiter, 2006). post-Golgi trafficking and dock at the basal bodies to be Moreover, RPGRORF15 and RPGR1–19 do not associate transported along the connecting cilium. This vectorial with RP1, which localizes to the distal axoneme of photo- transport depends upon the activity of small GTPases, receptors (Liu, Zuo, & Pierce, 2004). Taken together, we including Rab8 (Deretic et al., 1995; Moritz et al., 2001). predict that different RPGR isoforms facilitate the assem- Localization of RPGR1–19 isoform to the Golgi (Yan bly, selection, and transport of multiprotein cargo com- et al., 1998) and other compartments in the inner segment plexes by interacting with distinct ciliary–basal body– (present study) may indicate a possible involvement of centrosome (CBC) proteins, and that RPGR function is RPGR in post-Golgi sorting of cargo-containing vesicles necessary for maintaining efficient inter-segmental trans- for delivery to the base of the cilium. We previously port. Further studies will be necessary to understand the showed that the carboxyl-terminal amino acids CTIL are composition of the CBC macromolecular complexes in involved in the localization of RPGR1–19 to Golgi com- the retina and the connections made by distinct RPGR iso- partments in transfected cells (Yan et al., 1998). Given forms within these transport particles. the involvement of Golgi in processing membrane proteins We propose that diversity of RPGR isoforms is a critical destined to the cilium (Cai et al., 1999) and the localization determinant of its function. However, the potential of func- of IFT20 to Golgi (Follit, Tuft, Fogarty, & Pazour, 2006), tional redundancy between RPGR1–19 and RPGRORF15 we postulate that RPGR1–19 may be involved in sorting of isoforms in the retina requires additional investigations membrane versus soluble proteins for transport towards aiming at delineation of the role of individual RPGR iso- the connecting cilium. Owing to a lack of disease-associ- forms. It will also be necessary to differentiate between ated mutations in exons 16–19 of RPGR1–19 isoform, this hypomorphic, loss-, and gain-of-function RPGR alleles domain is either dispensable for RPGR function or muta- to delineate the pathogenesis of photoreceptor tions in this domain may lead to a lethal phenotype. degeneration. Protein trafficking via the connecting cilium is important for outer segment disc morphogenesis and renewal (Besh- Acknowledgments arse et al., 2003; Young & Droz, 1968). Though basic components associated with the transport have been discovered We thank Edwin Oh and other members of the Swaroop (Rosenbaum, 2002; Rosenbaum, Cole, & Diener, 1999), the Lab for stimulating discussions, and Sharyn Ferrara for mechanisms of cargo sorting and assembly of protein com- administrative assistance. This research was supported by plexes, and their regulation by signaling pathways have not grants from the NIH (EY007961), the Foundation Fight- been elucidated. 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