Retinal Cell Biology BRAG2a, a Guanine Nucleotide Exchange Factor for Arf6, Is a Component of the -Associated Glycoprotein Complex at the Photoreceptor Terminal

Hiroyuki Sakagami,1 Osamu Katsumata,1 Yoshinobu Hara,1 Toshikuni Sasaoka,2 and Masahiro Fukaya1 1Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan 2Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University, Niigata, Japan

Correspondence: Hiroyuki Sakagami, PURPOSE. Mutations in encoding the dystrophin-associated glycoprotein complex (DGC) Department of Anatomy, Kitasato can cause and disturb synaptic transmission in the photoreceptor ribbon University School of Medicine, 1-15-1 . However, the molecular composition and specific functions of the photoreceptor Kitasato, Sagamihara, Kanagawa 252- DGC remain unknown. Brefeldin A–resistant Arf-GEF 2 (BRAG2), also known as IQSEC1, is a 0374, Japan; guanine nucleotide exchange factor for ADP-ribosylation factor 6 (Arf6), a critical GTPase that [email protected]. regulates endosomal trafficking and remodeling. In the present study, we Submitted: February 24, 2017 characterized the expression of BRAG2a, an alternative splicing isoform of BRAG2, in the Accepted: June 19, 2017 adult mouse photoreceptor. Citation: Sakagami H, Katsumata O, METHODS. Immunofluorescence and immunoelectron microscopic analyses of adult mouse Hara Y, Sasaoka T, Fukaya M. BRAG2a, a guanine nucleotide exchange factor were performed using a novel anti-BRAG2a antibody. Pull-down, immunoprecipitation, for Arf6, is a component of the and in situ proximity ligation assays were performed to examine the interaction between dystrophin-associated glycoprotein BRAG2a and the DGC in vivo. complex at the photoreceptor termi- RESULTS. Immunofluorescence demonstrated punctate colocalization of BRAG2a with b- nal. Invest Ophthalmol Vis Sci. in the outer plexiform layer. Immunoelectron microscopy revealed the 2017;58:3795–3803. DOI:10.1167/ iovs.17-21746 localization of BRAG2a at the plasma membrane of lateral walls and processes of photoreceptor terminals within the synaptic cavity. Pull-down and immunoprecipitation assays using retinal lysates demonstrated the complex formation between BRAG2a with the DGC. In situ proximity ligation assays further detected a close spatial relationship between BRAG2a and b-dystroglycan in the outer plexiform layer.

CONCLUSIONS. The present study provided evidence that BRAG2a is a novel component of the photoreceptor DGC, suggesting functional involvement of the BRAG2a–Arf6 pathway downstream of the DGC. Keywords: dystrophin, dystroglycan, , small GTPase

utations in genes encoding components of the dystrophin- Dp71) with tissue-specific expression patterns. In photorecep- M associated glycoprotein complex (DGC) can cause various tors, dystrophin Dp427 and Dp260, , and b- forms of muscular dystrophy such as Duchenne muscular dystrobrevin were immunohistochemically shown to localize in dystrophy (DMD) and Becker muscular dystrophy (BMD). In a specific microdomain of the plasma membrane of photore- addition to muscle-related symptoms, DMD and BMD patients ceptor presynaptic terminals.7–10 Recently, pikachurin was show altered retinal responses to light/dark stimuli without an identified as a novel physiologic ligand for a-dystroglycan and apparent loss of visual acuity.1–4 The DGC in skeletal muscles, shown to regulate photoreceptor synaptic transmission and which is composed of dystrophin, a- and b-dystroglycans, structure with the photoreceptor DGC.11,12 However, the exact sarcoglycans, sarcospan, syntrophins, and dystrobrevins, is molecular composition of the DGC differs depending on tissue proposed to function not only as a molecular bridge between and cell types,13 and we still have a poor understanding of the the intracellular cytoskeleton and the but molecular components and functions of the DGC in nonmuscle also as a molecular scaffold that recruits signaling molecules tissues. downstream of mechanical stimuli.5,6 Dystroglycan and dystro- The ADP-ribosylation factors (Arfs) are critical small GTPases phin constitute the core components of the DGC. Dystroglycan that regulate vesicle biogenesis and transport between various itself is composed of a and b subunits cleaved from a single subcellular compartments.14,15 Arfs are divided into three precursor protein: a-dystroglycan is an extensively glycosylated classes based on structural similarity: class I (Arf1, Arf2, and extracellular protein that functions as a receptor for extracel- Arf3), class II (Arf4 and Arf5), and class III (Arf6). Compared lular matrix components, whereas b-dystroglycan is a trans- with the other Arfs, Arf6 is the most divergent in terms of its membrane protein that is associated with a-dystroglycan molecular structure and subcellular localization. Furthermore, extracellularly and with dystrophin intracellularly. Dystrophin in contrast to the primary function of other Arfs in the Golgi exists in various isoforms (Dp427, Dp260, Dp140, Dp116, and complex, Arf6 regulates endosomal trafficking between endo-

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FIGURE 1. Characterization of the anti-BRAG2a antibody. (A) Schematic domain structures of BRAG2a (NM_001134384.1) and BRAG2b (NM_001134383.1). Bars indicate the antigen regions used to produce anti-BRAG2a and anti-panBRAG2 antibodies. Note the presence of a unique C- terminal region containing a proline-rich domain and PDZ-binding motif in BRAG2a. IQ, IQ-like motif; PH, pleckstrin homology domain, Sec7, Sec7 domain. (B) Immunoblot analysis. Total lysates of adult mouse brain, , and HeLa cells transfected or untransfected with FLAG-BRAG2a or BRAG2bDN121/GEP100 were subjected to immunoblotting with antibodies against BRAG2a, panBRAG2, and the FLAG epitope. The positions and sizes (kDa) of molecular weight markers are indicated on the right side. (C) Immunostaining. HeLa cells transfected with FLAG-BRAG1a, BRAG2a, BRAG2bDN121/GEP100, or BRAG3a were subjected to double immunofluorescence with antibodies against BRAG2a and FLAG epitope. Note the absence of the cross-reactivity of the anti-BRAG2a antibody with other BRAG members. Scale bars denote 10 lm.

somes and the plasma membrane. Like other small GTPases, including immunohistochemistry, pull-down, immunoprecipi- the interconversion of Arfs between GTP-bound active and tation, and in situ proximity ligation assay (PLA). Furthermore, GDP-bound inactive states is under tight control by two types we show the effect of dystrophin (Dp427, Dp260, and Dp140) of regulatory : guanine nucleotide exchange factors loss on the photoreceptor presynaptic localization of BRAG2a (GEFs) that activate Arfs by catalyzing the exchange of GDP for using dystrophin exon 52 knockout mice lacking dystrophin GTP and GTPase-activating proteins (GAPs) that inactivate Arfs isoforms, Dp427, Dp260, and Dp140, which are expressed in by triggering the hydrolysis of bound GTP to GDP. photoreceptors.21 Brefeldin A–resistant Arf-GEF 2 (BRAG2), originally named Arf-guanine exchange protein 100 (Arf-GEP100)16 and also known as IQSEC1, was the first identified member of the BRAG/ MATERIALS AND METHODS IQSEC family of Arf-GEFs, and it functions to activate Arf6 in most of cellular contexts.17,18 There are at least two alternative Animals splicing isoforms, BRAG2a and BRAG2b, which share a We purchased male C57BL/6N mice at postnatal week 10 from conserved domain structure among the BRAG/IQSEC family, CLEA Japan (Tokyo, Japan). Dystrophin exon 52 knockout consisting of an N-terminal calmodulin-binding IQ-like motif, a mice were kindly provided to us by Motoya Katsuki.10,21 central catalytic Sec7 domain, and a pleckstrin homology (PH) Animals were kept on a 12-hour day/night cycle with free domain, but differ by the alternative use of N-terminal and C- access to food and water. All experimental procedures were in terminal regions (Fig. 1A). Particularly, BRAG2a is unique in compliance with the ARVO Statement for the Use of Animals in terms of the presence of a long C-terminal region containing a Ophthalmic and Vision Research and approved by the Animal proline-rich domain and a type I postsynaptic density protein-95 Experimentation and Ethics Committee of the Kitasato (PDS-95)/Disc /Zonula occludens 1 (PDZ)-binding motif. University School of Medicine. In the retina, we recently demonstrated using immunohis- tochemical analyses with an anti-panBRAG2 antibody that Plasmids recognizes all BRAG2 isoforms that BRAG2 colocalizes with the DGC at photoreceptor presynaptic terminals in contrast to its For bacterial expression vectors, the C-terminal region (amino postsynaptic localization in retinal bipolar cells and hippocam- acids 1014–1089) of mouse BRAG2a (BRAG2a [1014–1089]) pal neurons.19,20 In the present study, we provide several lines and the 120-amino acid C-terminal intracellular region of b- of evidence for BRAG2a as a novel DGC component in dystroglycan (b-dystroglycan [C120aa]) were amplified by PCR photoreceptor presynapses with the use of independent assays and subcloned into pGEX4T-2 (GE Healthcare, Piscataway, NJ,

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USA). For mammalian expression vectors, the coding regions of software (Carl Zeiss), and statistical analyses were performed BRAG2a and BRAG2b lacking the N-terminal 121 amino acids using Student’s t-test. (BRAG2bDN121/GEP100), which corresponded to the human 16 counterpart of GEP100, were amplified by PCR and Immunoblot Analysis subcloned into pCAGGS-FLAG.22,23 A mammalian expression vector for a BRAG2a mutant (P-PRD-A), in which proline Total lysates from the adult mouse brain and retina were residues that conform to the consensus WW domain-binding prepared as described previously.20 HeLa cells were transfected motif were replaced by alanine residues in the C-terminal with pCAGGS-FLAG-BRAG2a or pCAGGS-FLAG- proline-rich domain, was prepared as described elsewhere BRAG2bDN121/GEP100 using Lipofectamine 2000 (Invitro- (Fukaya M, unpublished observations, 2017). gen) and harvested with loading buffer. The lysates (10 lg) were subjected to immunoblot analyses with anti-BRAG2a, anti- 20 Antibodies panBRAG2, or anti-FLAG antibodies as described previously. Experiments were repeated at least three times. A glutathione S-transferase (GST) fusion protein of BRAG2a (1014–1089) was used as an antigen to immunize a rabbit. The Pull-Down serum was affinity-purified with the antigen. A guinea pig polyclonal anti-panBRAG2 antibody was characterized previ- HeLa cells were transfected with pCAGGS-FLAG vectors ously.20 Antibodies used in the present study were as follows: encoding BRAG2a, BRAG2bDN121/GEP100, or BRAG2a (P- mouse anti-b-dystroglycan IgG2a (clone 43DAG1/8D5; Novo- PRD-A) using Lipofectamine 2000. Twenty-four hours after castra Laboratories, Newcastle upon Tyne, UK); mouse anti- transfection, the cells were solubilized with lysis buffer PSD-95 IgG1 (clone 16; BD Transduction Laboratories, San consisting of 0.05 M Tris-HCl (pH 7.5), 0.15 M NaCl, 1% Triton Jose, CA, USA); rabbit anti-dystrophin IgG (ab15277; Abcam, X-100, and a cocktail of protease inhibitors (Complete Mini; Cambridge, MA, USA); mouse anti-Bassoon IgG2a (clone Roche, Mannheim, Germany). The retinal lysate was obtained SAP7F407; Stressgen, Victoria, BC, Canada); rabbit anti-RIBEYE by the solubilization of retinas from six to eight adult C57BL/ IgG24; and mouse anti-FLAG IgG1 (clone M2; Sigma-Aldrich, St. 6N mice in the lysis buffer by brief sonication and incubation Louis, MO, USA). for 30 minutes at 48C. The lysates (500 lg) were incubated with 20 lg GST-dystrophin (2937–3685),26 GST-b-dystroglycan Immunohistochemistry (C120aa), or GST alone, which had been immobilized on glutathione-Sepharose 4B, for 1 hour at 48C. After the beads Immunohistochemical procedures was described previous- were washed with the lysis buffer, proteins bound to the beads ly.20,24,25 Briefly, eyeballs, from which the cornea and lens were dissociated by boiling with loading buffer. Samples were had been removed, were immersed in 4% paraformaldehyde subjected to immunoblotting with anti-FLAG or anti-panBRAG2 for 10 minutes and cryoprotected in 30% (wt/vol) sucrose. antibodies. Experiments were repeated at least three times. Cryostat sections (20 lm) were treated with pepsin (1 g/L; DAKO, Carpinteria, CA, USA) in 0.2 N HCl for 5 minutes at Immunoprecipitation 378C. For immunofluorescence staining, sections were incu- bated with anti-BRAG2a IgG and antibodies against panBRAG2 The rabbit anti-dystrophin IgG and normal rabbit IgG were or b-dystroglycan overnight at room temperature. Immunore- conjugated with protein G-coupled magnetic beads (Dyna- actions were visualized with species-specific secondary anti- beads Protein G, Life Technologies AS, Oslo, Norway) in bodies conjugated with Alexa Fluor 488 or 594 (Invitrogen, phosphate-buffered saline (PBS; pH 7.4) containing 0.1% Triton Carlsbad, CA, USA). Sections were counterstained with 40,6- X-100 overnight at 48C. The complexes were cross-linked by diamidino-2-phenylindole dihydrochloride (DAPI) and analyzed incubation with 1 mM dithiobis(succinimidyl propionate) using a confocal laser scanning microscope (LSM 710; Carl (DSP; ThermoFisher Scientific, San Jose, CA, USA) in PBS for Zeiss, Jena, Germany). For immunoelectron microscopy, after 2 hours at 48C. After the cross-linking reaction was quenched incubation with primary antibodies, sections were incubated by the addition of Tris-HCl (pH 7.5) for 15 minutes at room with nanogold-conjugated secondary antibodies (1:100; temperature, beads were washed with PBS containing 0.1% Nanoprobes, Yaphank, NY, USA) and subjected to silver bovine serum albumin and stored at 48C. enhancement of gold particles (HQ kit; Nanoprobes). Ultrathin Retinas from six to eight adult C57BL/6N mice were sections (70 nm) were analyzed using a transmission electron solubilized in buffer consisting of 0.05 M Tris-HCl (pH 7.5), microscope (H-7650; Hitachi, Tokyo, Japan). Data were 0.15 M NaCl, 0.5% digitonin, 0.05% Nonidet P-40, and a confirmed using retinas from five mice for immunofluores- cocktail of protease inhibitors (Complete Mini; Roche) by brief cence and three for immunoelectron microscopy. sonication and subsequent incubation for 30 minutes at 48C. For quantification of immunofluorescent signals, coronal The retinal lysates (500 lg of 1 mg/mL) were incubated with sections of the retinas from three mice for each genotype were the cross-linked beads (5 lg IgG) overnight at 48C. The beads cut using a cryostat at the distance between 400 and 1000 lm were then washed three times with a buffer consisting of 0.05 from the posterior pole of the retina and mounted onto the M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.1% digitonin, and 0.05% same one glass slide. In our preliminary experiments, we Nonidet P-40 and boiled with loading buffer. Samples were confirmed that the immunoreactive intensities of puncta for subjected to immunoblotting with antibodies against pan- BRAG2a in the outer plexiform layer (OPL) on retinal sections BRAG2 and b-dystroglycan. Experiments were repeated three taken within this range were constant without significant times. regional differences. Immunofluorescent images of retinas immunostained with anti-BRAG2a or RIBEYE antibodies were In Situ PLA acquired from five areas randomly selected in the OPL from each mouse using a confocal laser scanning microscope (LSM In situ PLA was carried out in accordance with the 710; Carl Zeiss) using a 633 oil-immersion objective lens and a manufacturer’s instructions (Olink Bioscience, Uppsala, Swe- digital zoom of 3.5 within the linear range below the saturation den). Briefly, cryostat sections of retinas from three mice were of detection signals. The intensities of immunoreactive puncta treated with pepsin for 5 minutes at 378C, blocked with 5% (100–200 puncta per mice) were measured using ZEN imaging normal donkey serum, and incubated overnight at 48C with

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anti-BRAG2a IgG or b-dystroglycan IgG alone or with the panBRAG2 or anti–b-dystroglycan antibody. In the retina, the combination of anti-BRAG2a IgG and anti–b-dystroglycan IgG anti-BRAG2a antibody strongly labeled the OPL and slightly or anti-Bassoon IgG. Sections were then incubated with labeled the inner plexiform layer (Fig. 2A). In the OPL, the anti- species-specific secondary antibodies conjugated with oligo- BRAG2a antibody perfectly traced puncta labeled by anti- nucleotides, anti-rabbit PLUS, and anti-mouse MINUS PLA panBRAG2 IgG (Figs. 2A–2C) or anti–b-dystroglycan IgG (Figs. probes (Sigma-Aldrich) for 1 hour at 378C and subjected to a 2E–2G). The detection of BRAG2a-immunoreactive puncta in rolling circle amplification reaction with the amplification- the OPL depended on the pepsin pretreatment of retinal polymerase solution (Duolink In situ Detection Reagent Red; sections with the optimal incubation time set at 5 minutes Sigma-Aldrich) for 100 minutes at 378C. Fluorescent signals (Supplementary Fig. S2), suggesting that the BRAG2a epitopes were analyzed using a confocal laser scanning microscope may be masked in the protein complex, as suggested by the (LSM 710; Carl Zeiss). Experiments were repeated twice. requirement for pepsin treatment in the immunodetection of various postsynaptic density proteins such as PSD-95.28 In the forebrain, BRAG2 was shown to form a protein complex with RESULTS PSD-95 in the postsynaptic density fraction.29 Thus, we also examined the colocalization of BRAG2a with PSD-95. In the Expression of BRAG2a in the Mouse Retina OPL, the anti–PSD-95 antibody labeled the plasma membrane of the photoreceptor terminals as described previously,30 BRAG2 exists in at least two alternative splicing isoforms: whereas BRAG2a-immunoreactive puncta were positioned BRAG2a and BRAG2b (Fig. 1A). In the present study, we within the PSD-95–labeled contour of photoreceptor terminals produced an antibody specific for BRAG2a using a BRAG2a- without any apparent overlapping (Figs. 2H–2J). In the control specific C-terminal region (amino acids 1014–1089) as an experiment, pre-absorption of the anti-BRAG2a antibody with antigen (Fig. 1A). In the immunoblot analysis, the anti- the antigen completely abolished the immunolabeling de- BRAG2a antibody recognized FLAG-BRAG2a but not FLAG- scribed above (Fig. 2D). BRAG2bDN121/GEP100 in the lysates of transfected HeLa To further examine the ultrastructural localization of cells and detected two major immunoreactive bands of 160 BRAG2a at photoreceptor ribbon , we performed and 140 kDa in the lysates of mouse brain and retina (Fig. 1B). pre-embedding immunoelectron microscopy using the silver Meanwhile, an anti-panBRAG2 antibody, which had previous- 20 enhancement method. With a subcellular distribution similar ly been raised against the common amino-terminal region to those of panBRAG2 and b-dystroglycan, BRAG2a-immunore- (Fig. 1A), recognized both FLAG-BRAG2a and FLAG- active metal particles accumulated along the plasma membrane BRAG2bDN121/GEP100 and detected three immunoreactive of the lateral walls and processes of photoreceptor terminals bands of 160, 140, and 110 kDa in the mouse brain and retina within the synaptic cavity (Fig. 3). Taken together, these 20 lysates, as described previously (Fig. 1B). The sizes of the findings suggest that BRAG2, especially BRAG2a, is localized at upper two panBRAG2-immunoreactive bands were consistent the specific plasma membrane domain of photoreceptor with those detected by the anti-BRAG2a antibody. Because the presynaptic terminals with the DGC. 140-kDa band detected by both anti-BRAG2a and panBRAG2 antibodies was consistent with FLAG-BRAG2a, it is likely that the 140- and 110-kDa bands correspond to BRAG2a and Interaction of BRAG2a with Dystrophin and b- BRAG2b, respectively. The nature of the remaining 160-kDa Dystroglycan species detected by the anti-BRAG2a remains unknown at BRAG3, another member of the BRAG/IQSEC family, was present, but it could be explained by post-translational previously shown to interact with a WW domain of modification of BRAG2a such as phosphorylation or the dystrophin and utrophin through a proline-rich domain.26 presence of additional BRAG2 isoforms. Indeed, a previous Because BRAG2a also contains a similar proline-rich domain in proteomic analysis isolated BRAG2 as a phosphoprotein in the the C-terminal region (Fig. 1A), we examined the ability of 27 postsynaptic density, and the National Center for Biotech- BRAG2a to interact with dystrophin. HeLa cells were tran- nology Information (NCBI) database predicted the presence of siently transfected with BRAG2a, BRAG2bDN121/GEP100, or at least 12 possible BRAG2 isoforms, 8 of which possessed the a BRAG2a mutant (P-PRD-A) in which proline residues that same C-terminal region as BRAG2a and could be recognized by conform to the consensus WW domain-binding motif were the anti-BRAG2a antibody (Supplementary Fig. S1). To further mutated to alanine residues and subjected to pull-down assays examine the specificity of the anti-BRAG2a antibody, HeLa cells with the GST fusion protein of the dystrophin C-terminal 749 were transiently transfected with FLAG-BRAG1a, BRAG2a, amino acids containing a WW domain (GST-dystrophin [2937– BRAG2bDN121/GEP100, or BRAG3a and subjected to double 3685]). GST-dystrophin (2937–3685) pulled down wild-type immunofluorescence with the anti-BRAG2a and anti-FLAG FLAG-BRAG2a but not BRAG2bDN121/GEP100 or BRAG2a (P- antibodies (Fig. 1C). The anti-BRAG2a antibody only labeled PRD-A) (Fig. 4A), suggesting a specific interaction of BRAG2a HeLa cells expressing BRAG2a but not other BRAG isoforms, with dystrophin through the proline-rich domain. We further whereas the anti-FLAG antibody labeled HeLa cells expressing examined the possibility of an interaction between BRAG2 all FLAG-tagged isoforms (Fig. 1C). These findings suggested and b-dystroglycan using pull-down assays with GST fusion the specificity of the anti-BRAG2a antibody without any cross- proteins of the b-dystroglycan C-terminal intracellular domain reactivity to other BRAG family members. (GST-b-dystroglycan [C120]). GST-b-dystroglycan (C120) pulled down BRAG2a and BRAG2a (P-PRD-A) (Fig. 4B). In Localization of BRAG2a at Photoreceptor Ribbon addition, a small amount of FLAG-BRAG2bDN121/GEP100 was Synapses also detectable in the precipitates pulled down with GST-b- dystroglycan (C120). On the other hand, in the control Our previous immunohistochemical analysis using the anti- experiment, GST alone did not pull down any BRAG2 iso- panBRAG2 antibody demonstrated that BRAG2 colocalizes forms. with the DGC at photoreceptor presynaptic terminals.20 To We also performed a pull-down assay using retinal lysates examine the expression of BRAG2a in the photoreceptor, we (Fig. 4C). GST-dystrophin (2937–3685) efficiently pulled down performed double immunofluorescence staining of the mouse two protein species detected by the anti-BRAG2a antibody, retina with the anti-BRAG2a antibody and either an anti- with the lower band corresponding to BRAG2a being much

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FIGURE 2. Immunohistochemical localization of BRAG2a in the mouse retina. (A–C) Double immunofluorescence staining of the retina with antibodies against BRAG2a (A, E, H), and panBRAG2 (B), b-dystroglycan (F), or PSD-95 (I). Merged images (C, G, J) include nuclear staining with DAPI. (D) Control experiment in which the anti-BRAG2a antibody was preabsorbed with its antigen (10 lg/mL). BV, blood vessel; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars denote 10 lminD;1lm in G and J.

more efficiently pulled down than the upper band. On the upper band being most densely labeled. On the other hand, other hand, GST-b-dystroglycan (C120) pulled down two normal rabbit IgG immunoprecipitated only a small amount of species equally, although the efficiency of pull-down with the lower BRAG2 species but not the two upper species. The GST-b-dystroglycan (C120) was much lower than that with reversed immunoprecipitation with the anti-panBRAG2 or anti- GST-dystrophin (2937–3685). In the control, GST alone did not BRAG2a antibodies did not yield detectable immunoprecipi- pull down any BRAG2 species from the retinal lysate. tates for dystrophin or b-dystroglycan, because these antibod- To further confirm the in vivo interaction between BRAG2 ies for BRAG2 did not work well for the immunoprecipitation and the DGC in the retina, we performed immunoprecipitation from the retinal lysate under the present condition (data not from the retina with anti-dystrophin IgG (Fig. 4D). Anti- shown). dystrophin IgG precipitated b-dystroglycan and three BRAG2 Finally, to verify the in situ interaction in photoreceptor synapses, we performed in situ PLA of the mouse retina. This is species detected by the anti-panBRAG2 antibody, with the a sensitive method to visualize pairs of endogenous proteins that are localized in close proximity to form a protein complex in vivo.31 When retinal sections were subjected to in situ PLA with antibodies against BRAG2a and b-dystroglycan, discrete punctate fluorescent signals were detected in the OPL (Fig. 5A). In the control experiment, in situ PLA performed with the anti-BRAG2a or b-dystroglycan antibody alone, or the combi- nation of antibodies against BRAG2a and Bassoon, an irrelevant presynapticactivezoneprotein,didnotproduceany fluorescent signals in the OPL (Figs. 5B–5D), thus confirming the specificity of the present in situ PLA.

FIGURE 3. Ultrastructural localization of BRAG2a, panBRAG2, and b- dystroglycan in the photoreceptor presynaptic terminal. Cryosections Synaptic Localization of BRAG2 in Photoreceptors of the adult mouse retina were subjected to immunoelectron of Dystrophin Exon 52 Knockout Mice microscopy using the pre-embedding silver-enhanced immunogold method. Note the similar subcellular accumulation of silver particles The interaction of BRAG2a with the DGC led us to examine for BRAG2a, panBRAG2, and b-dystroglycan along the plasma whether the presynaptic localization of BRAG2a in the OPL membrane of the photoreceptor terminal within the synaptic cavity. depends on the expression of dystrophin. Photoreceptors Scale bars denote 500 nm. were shown to express three dystrophin isoforms, Dp426,

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FIGURE 4. Pull-down and immunoprecipitation assays. (A–C) Pull-down assay. The lysates of HeLa cells (A, B) transfected with pCAGGS-FLAG- BRAG2a, BRAG2bDN121/GEP100, or BRAG2a (P-PRD-A), and mouse retinas (C) were subjected to pull-down assays with GST-dystrophin (2937– 3685) (A, C) or GST-b-dystroglycan (C120) (B, C). (D) Immunoprecipitation. Retinal lysates were subjected to immunoprecipitation with anti- dystrophin IgG or normal rabbit IgG conjugated to protein G magnetic beads. The lysate and precipitates were subjected to immunoblotting with antibodies against panBRAG2 and b-dystroglycan. The positions and sizes (kDa) of molecular weight markers are indicated on the right side.

32 Dp260, and Dp140, at the mRNA level. We examined the DISCUSSION expression of BRAG2a in the OPL of dystrophin exon 52 knockout mice, in which the expression of these three Recent evidence of the existence of multiple Arf regulators in isoforms were abolished.10,21 Although the density of immu- the photoreceptor terminal, including BRAG1, BRAG2, EFA6A, nofluorescent puncta was indistinguishable between wild and and ArfGAP3, suggests the functional importance of Arfs in the mutant retinas, the intensity of BRAG2a-immunoreactive photoreceptor ribbon synapse.20,33,34 We previously demon- puncta in the OPL was drastically decreased in mutant retinas strated by immunohistochemistry using the anti-panBRAG2 by approximately 50% compared with wild-type retinas (Fig. 6; antibody that BRAG2, a GEF specific for Arf6, colocalizes with control, 100 6 10.2%; mutant, 48.9 6 6.2%; P ¼ 0.00428). On the DGC at the photoreceptor presynaptic terminal.18 In the the other hand, we also examined the expression of RIBEYE, a present study, we produced a novel antibody that recognized component of synaptic ribbon in photoreceptor terminals, and the C-terminal region of BRAG2a and extended our previous found that there were no significant differences in the pattern finding by showing that BRAG2, especially BRAG2a, is a novel of distribution and the immunofluorescent intensity of puncta component of the DGC at the photoreceptor terminal using between wild-type and mutant retinas (Supplementary Fig. S3; independent assays. First, both immunofluorescence and control, 100 6 22.3%; mutant, 107.0 6 15.9%; P ¼ 0.68), immunoelectron microscopy demonstrated that BRAG2a ex- excluding the possibility that the loss of dystrophin isoforms hibited subcellular localization in the same subcompartment of could generally affect the expression of presynaptic compo- the photoreceptor terminal as b-dystroglycan. Second, pull- nents in photoreceptor terminals. Therefore, our results down assays showed both GST fusion proteins of dystrophin suggested that synaptic localization of BRAG2a in photorecep- and b-dystroglycan could interact with BRAG2a that was tors was partially dependent on dystrophin isoforms Dp426, artificially overexpressed in HeLa cell and endogenously Dp260, and Dp140. expressed in the mouse retina. Third, immunoprecipitation

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the b-dystroglycan intracellular domain can interact with both BRAG2a and BRAG2b. Thus, multiple interactions of BRAG2a with dystrophin and b-dystroglycan enable BRAG2a to build a more specific and stable complex with the DGC than BRAG2b. However, the present immunofluorescence showed that BRAG2a was significantly decreased but still present in the OPL of dystrophin exon 52 knockout mice lacking photore- ceptor dystrophin isoforms, although this finding remains to be further verified by independent quantitative assays such as immunoblot and Northern bot analyses. Because this mutant retina was previously shown to contain very little b-dystrogly- can in the OPL,10 it was suggested that dystrophin isoforms (Dp426, Dp260, and Dp140) and b-dystroglycan are not essential for the presynaptic localization of BRAG2a in the photoreceptor. Therefore, additional mechanisms are required to anchor BRAG2a to photoreceptor terminals. In the brain, BRAG2a is enriched at the postsynaptic density of excitatory synapses probably through the direct interaction of the C- terminal PDZ-binding motif of BRAG2a with PSD-95.29,42,43 However, the PSD-95 family members are unlikely to be responsible for the synaptic targeting of BRAG2a in the photoreceptor terminal, because PSD-95 and PSD-93 exhibit

FIGURE 5. In situ PLA. Cryosections of adult mouse retinas were a distinct subcellular localization from BRAG2a and the DGC in subjected to in situ PLA using the following combination of primary the photoreceptor terminal,30,44 and SAP102 is expressed in antibodies: (A) anti-BRAG2a and anti–b-dystroglycan antibodies; (B) horizontal cell processes but not in the photoreceptor anti-BRAG2a antibody alone; (C) anti–b-dystroglycan antibody alone; terminal.44 Among the DGC components in the skeletal and (D) anti-BRAG2a and anti-Bassoon antibodies. Note the occurrence muscle, syntrophin and neuronal nitric synthase are known of fluorescent signals in the OPL only with the combination of anti- to be PDZ domain-containing proteins. Particularly, the BRAG2a and anti–b-dystroglycan antibodies. denotes 5 lm. Scale bar syntrophin family consists of a1-, a2-, b1-, b2-, c1-, and c2 isoforms and functions as a scaffold for signaling proteins.5,45 from retinal lysates with the anti-dystrophin antibody demon- Although a1 syntrophin exhibits a distinct punctate distribu- strated the formation of an immunoprecipitable protein tion from the DGC in the OPL,46 information on the subcellular complex between BRAG2a and the DGC. Finally, in situ PLA localization of other syntrophin isoforms in the photoreceptor showed a close spatial relationship between BRAG2a and b- is not available at present. Therefore, further studies are dystroglycan in the OPL. Taken together, these findings necessary to elucidate the targeting mechanisms for the strongly supported the in vivo complex formation of BRAG2a synaptic localization of BRAG2 in photoreceptors and to and the DGC in the photoreceptor terminal. investigate the potential interaction between BRAG2a and Concerning the targeting mechanism for BRAG2a to the syntrophins. photoreceptor terminal, accumulating evidence for the exis- What is the functional role of BRAG2a in the DGC at the tence of multiple BRAG2-interacting proteins19,29,35–41 (Table) photoreceptor terminal? Although the functions of the DGC suggested that BRAG2a can be recruited to various subcellular in the photoreceptor are not yet fully understood, the recent locations by partner proteins, thereby regulating the precise discovery of pikachurin as a ligand for a-dystroglycan has spatiotemporal activation of Arf6. In the present study, we advanced our understanding of the functions of the DGC in demonstrated the dystrophin WW motif can interact specifi- the photoreceptor terminal.11 Pikachurin is a 1017-amino- cally with BRAG2a through its proline-rich domain, whereas acid extracellular matrix protein that is synthesized by

TABLE. Summary of BRAG2-Interacting Proteins

Interactors Functional Consequences of the Interaction References

a-Catenin Arf6 activation and possible regulation of cellular content and HGF-dependent internalization of E-cadherin 35 Synaptic PSD-95 Association with the PSD-95 complex at the postsynaptic density 29 complex EGFR/ErbB1 EGF-dependent Arf6 activation and enhancement of invasive and metastatic activities of breast cancer cells 36 AMPAR (GluA2, Arf6 activation and subsequent internalization of AMPARs during the hippocampal long-term depression 19 3 and 4short) Her2/ErbB2/Neu Arf6 activation and enhancement of invasive activity in lung adenocarinoma cells 37 VEGFR2 VEGF-dependent Arf6 activation and enhancement of angiogenic activity, cell permeability and VE-cadherin 38 endocytosis in human umbilical vein endothelial cells Clathrin Clathrin-dependent endocytosis of integrin (a5b1) and cell spreading mediated through Arf5 activation 39 Adaptor protein Clathrin-dependent endocytosis of integrin (a5b1) and cell spreading mediated through Arf5 activation 39 (AP)-2 (b-adaptin) MAP4K4 Arf6 phosphorylation and activation, and regulation of cell motility and turnover of focal adhesions through 40 b1-integrin endocytosis NMDAR (GluN2A) NMDA-dependent Arf6 activation and AMPAR internalization in hippocampal neurons 41 Calmodulin Calcium-dependent conformational change in BRAG2 and possible modulation of the interaction with GluN2 41 subunits

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FIGURE 6. Reduced clustering of BRAG2a at photoreceptor synapses in dystrophin exon 52 knockout mice. (A) Representative images of the OPL from wild-type and dystrophin exon 52 knockout mouse retinas immunostained with anti-BRAG2a IgG. Scale bar denotes 5 lm. (B) Quantification of the immunofluorescent intensity of puncta for BRAG2a in the OPL. Note the marked decrease in the intensity for BRAG2a in the OPL between wild-type and mutant OPL. The values represent the mean 6 SD with n ¼ 3 mice. Statistical analysis was performed using Student’s t-test (*P ¼ 0.00428).

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