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• W2894580867 abstract "•Orphan receptors GPR158 and GPR179 are membrane partners for HSPGs•Pikachurin is the endogenous HSPG interacting with GPR179 in photoreceptor synapses•Loss of Pikachurin disrupts post-synaptic properties via GPR179•Pikachurin-GPR179 complex assembly is controlled by pre-synaptic dystroglycan complex Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify unique physiological properties of individual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft—members of the heparan sulfate proteoglycan (HSPG) family—associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the pre-synaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals. Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify unique physiological properties of individual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft—members of the heparan sulfate proteoglycan (HSPG) family—associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the pre-synaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals. Precise synaptic connectivity is one of the defining properties of the CNS. The ability of neurons to form synapses with an extremely defined spatial and temporal resolution is essential to establish functional neuronal circuits, but the molecular mechanisms involved in neuronal wiring specificity are still poorly understood. To establish proper connections, a network of transsynaptic interactions among membrane receptors, secreted ligands, and synaptic cell adhesion molecules coordinates pre- and post-synaptic assembly (Chia et al., 2013Chia P.H. Li P. Shen K. Cell biology in neuroscience: cellular and molecular mechanisms underlying presynapse formation.J. Cell Biol. 2013; 203: 11-22Crossref PubMed Scopus (60) Google Scholar, Sanes and Yamagata, 2009Sanes J.R. Yamagata M. Many paths to synaptic specificity.Annu. Rev. Cell Dev. Biol. 2009; 25: 161-195Crossref PubMed Scopus (229) Google Scholar, Siddiqui and Craig, 2011Siddiqui T.J. Craig A.M. Synaptic organizing complexes.Curr. Opin. Neurobiol. 2011; 21: 132-143Crossref PubMed Scopus (201) Google Scholar). Beyond a structural role, several components of the extracellular matrix (ECM) have been shown to play an active role in the formation and maintenance of correct synaptic connectivity (de Wit et al., 2013de Wit J. O’Sullivan M.L. Savas J.N. Condomitti G. Caccese M.C. Vennekens K.M. Yates 3rd, J.R. Ghosh A. Unbiased discovery of glypican as a receptor for LRRTM4 in regulating excitatory synapse development.Neuron. 2013; 79: 696-711Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Dityatev et al., 2010Dityatev A. Schachner M. Sonderegger P. The dual role of the extracellular matrix in synaptic plasticity and homeostasis.Nat. Rev. Neurosci. 2010; 11: 735-746Crossref PubMed Scopus (346) Google Scholar, Nitkin et al., 1987Nitkin R.M. Smith M.A. Magill C. Fallon J.R. Yao Y.M. Wallace B.G. McMahan U.J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ.J. Cell Biol. 1987; 105: 2471-2478Crossref PubMed Scopus (367) Google Scholar). Members of the G protein-coupled receptor (GPCR) family are among the most common resident proteins present at synapses. A wide variety of extracellular domains allows this large receptor family to sense a range of changes in the extracellular environment, including detection of all known neurotransmitters (Rosenbaum et al., 2009Rosenbaum D.M. Rasmussen S.G. Kobilka B.K. The structure and function of G-protein-coupled receptors.Nature. 2009; 459: 356-363Crossref PubMed Scopus (1634) Google Scholar). Traditionally, GPCRs have been considered powerful modulators of neurotransmission that shape properties of neuronal circuits (Bargmann and Marder, 2013Bargmann C.I. Marder E. From the connectome to brain function.Nat. Methods. 2013; 10: 483-490Crossref PubMed Scopus (322) Google Scholar, Marder, 2012Marder E. Neuromodulation of neuronal circuits: back to the future.Neuron. 2012; 76: 1-11Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). However, emerging proteomic studies increasingly point to their involvement in transsynaptic macromolecular complexes and interactions with ECM components (Bolliger et al., 2011Bolliger M.F. Martinelli D.C. Südhof T.C. The cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins.Proc. Natl. Acad. Sci. USA. 2011; 108: 2534-2539Crossref PubMed Scopus (111) Google Scholar, Cao et al., 2015Cao Y. Sarria I. Fehlhaber K.E. Kamasawa N. Orlandi C. James K.N. Hazen J.L. Gardner M.R. Farzan M. Lee A. et al.Mechanism for selective synaptic wiring of rod photoreceptors into the retinal circuitry and its role in vision.Neuron. 2015; 87: 1248-1260Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, Kakegawa et al., 2015Kakegawa W. Mitakidis N. Miura E. Abe M. Matsuda K. Takeo Y.H. Kohda K. Motohashi J. Takahashi A. Nagao S. et al.Anterograde C1ql1 signaling is required in order to determine and maintain a single-winner climbing fiber in the mouse cerebellum.Neuron. 2015; 85: 316-329Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, Lanoue et al., 2013Lanoue V. Usardi A. Sigoillot S.M. Talleur M. Iyer K. Mariani J. Isope P. Vodjdani G. Heintz N. Selimi F. The adhesion-GPCR BAI3, a gene linked to psychiatric disorders, regulates dendrite morphogenesis in neurons.Mol. Psychiatry. 2013; 18: 943-950Crossref PubMed Scopus (57) Google Scholar, Luo et al., 2011Luo R. Jeong S.J. Jin Z. Strokes N. Li S. Piao X. G protein-coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical development and lamination.Proc. Natl. Acad. Sci. USA. 2011; 108: 12925-12930Crossref PubMed Scopus (195) Google Scholar, O’Sullivan et al., 2012O’Sullivan M.L. de Wit J. Savas J.N. Comoletti D. Otto-Hitt S. Yates 3rd, J.R. Ghosh A. FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.Neuron. 2012; 73: 903-910Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Such effects were primarily shown for the subfamily of “adhesion” receptors, and the scope of this involvement and extent of conservation across the GPCR superfamily are yet to be explored. Functional roles and signal transduction mechanisms of a large portion of the GPCR family remain poorly understood, with many receptors still “orphan” of endogenous ligands. Nonetheless, genomic studies in humans and the use of knockout animal models suggest a crucial role for the largely unexplored biology of orphan receptors in fundamental neuronal processes (Ahmad et al., 2015Ahmad R. Wojciech S. Jockers R. Hunting for the function of orphan GPCRs - beyond the search for the endogenous ligand.Br. J. Pharmacol. 2015; 172: 3212-3228Crossref PubMed Scopus (28) Google Scholar, Kroeze et al., 2015Kroeze W.K. Sassano M.F. Huang X.P. Lansu K. McCorvy J.D. Giguère P.M. Sciaky N. Roth B.L. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome.Nat. Struct. Mol. Biol. 2015; 22: 362-369Crossref PubMed Scopus (340) Google Scholar). Our progress in de-orphanizing these receptors and understanding their physiology has been slow, likely because of their unusual biology, which may deviate from the traditional role of GPCRs as mediators of neurotransmitter signaling. One of the classical models for studying synaptic organization whereby traditional and orphan GPCRs cooperate is offered by the first visual synapse of vertebrate photoreceptors. In the dark, photoreceptors tonically release the neurotransmitter glutamate, which is sensed by the mGluR6 receptor on the post-synaptic neuron: the ON-bipolar cell (ON-BC). The mGluR6 initiates a prototypic GPCR cascade that activates the G protein Gαo to keep the effector channel TRPM1 inhibited (Koike et al., 2010Koike C. Obara T. Uriu Y. Numata T. Sanuki R. Miyata K. Koyasu T. Ueno S. Funabiki K. Tani A. et al.TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade.Proc. Natl. Acad. Sci. USA. 2010; 107: 332-337Crossref PubMed Scopus (223) Google Scholar, Morgans et al., 2009Morgans C.W. Zhang J. Jeffrey B.G. Nelson S.M. Burke N.S. Duvoisin R.M. Brown R.L. TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 19174-19178Crossref PubMed Scopus (224) Google Scholar, Shen et al., 2009Shen Y. Heimel J.A. Kamermans M. Peachey N.S. Gregg R.G. Nawy S. A transient receptor potential-like channel mediates synaptic transmission in rod bipolar cells.J. Neurosci. 2009; 29: 6088-6093Crossref PubMed Scopus (168) Google Scholar). Suppression of the glutamate release by light leads to TRPM1 opening and requires rapid inactivation of Gαo. This is achieved by the action of the GTPase activating protein (GAP) complex, which involves coordinated action of several proteins, including catalytic subunits RGS7 and RGS11 (Martemyanov and Sampath, 2017Martemyanov K.A. Sampath A.P. The transduction cascade in retinal ON-bipolar cells: signal processing and disease.Annu. Rev. Vis. Sci. 2017; 3: 25-51Crossref PubMed Scopus (33) Google Scholar, Vardi and Dhingra, 2014Vardi N. Dhingra A. Mechanistic basis for G protein function in ON bipolar cells.in: Martemyanov K. Sampath A.P. G Protein Signaling Mechanisms in the Retina. Springer, New York2014: 81-98Crossref Scopus (3) Google Scholar). The abundance and subcellular localization of the GAP complex have a major impact on the synaptic transmission of light signal from photoreceptors to ON-BC and tuning the circuits for daylight and dim vision (Cao et al., 2009Cao Y. Masuho I. Okawa H. Xie K. Asami J. Kammermeier P.J. Maddox D.M. Furukawa T. Inoue T. Sampath A.P. Martemyanov K.A. Retina-specific GTPase accelerator RGS11/G beta 5S/R9AP is a constitutive heterotrimer selectively targeted to mGluR6 in ON-bipolar neurons.J. Neurosci. 2009; 29: 9301-9313Crossref PubMed Scopus (69) Google Scholar, Sarria et al., 2015Sarria I. Pahlberg J. Cao Y. Kolesnikov A.V. Kefalov V.J. Sampath A.P. Martemyanov K.A. Sensitivity and kinetics of signal transmission at the first visual synapse differentially impact visually-guided behavior.eLife. 2015; 4: e06358Crossref PubMed Scopus (11) Google Scholar). A critical role in this process belongs to the orphan receptor GPR179, which has been identified as a component of the GAP complex serving in a non-traditional capacity as membrane anchor for RGS proteins at the ON-BC post-synaptic site (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar). Knockout of GPR179 prevents post-synaptic accumulation of RGS proteins and severely compromises synaptic communication with photoreceptors (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, Peachey et al., 2012Peachey N.S. Ray T.A. Florijn R. Rowe L.B. Sjoerdsma T. Contreras-Alcantara S. Baba K. Tosini G. Pozdeyev N. Iuvone P.M. et al.GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 331-339Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), indicating that it is required for achieving temporal resolution needed for a rapid transduction of visual stimuli. However, the mechanisms of synaptic targeting of GPR179 and its integration into the synaptic architecture remain unknown. Although GPR179 is largely retina specific, its close homolog GPR158 is enriched in the brain, where it likewise plays a role in organizing RGS complexes (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, Orlandi et al., 2015Orlandi C. Xie K. Masuho I. Fajardo-Serrano A. Lujan R. Martemyanov K.A. Orphan receptor GPR158 is an allosteric modulator of RGS7 catalytic activity with an essential role in dictating its expression and localization in the brain.J. Biol. Chem. 2015; 290: 13622-13639Crossref PubMed Scopus (39) Google Scholar). Both proteins feature large extracellular segments, suggesting that they may be involved in the interactions with the ECM (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, Patel et al., 2013Patel N. Itakura T. Gonzalez Jr., J.M. Schwartz S.G. Fini M.E. GPR158, an orphan member of G protein-coupled receptor Family C: glucocorticoid-stimulated expression and novel nuclear role.PLoS ONE. 2013; 8: e57843Crossref PubMed Scopus (31) Google Scholar). In fact, ECM plays an essential role in the organization of the first visual synapse. One of the most prominent examples is provided by the complex of α/β-dystroglycan-dystrophins (DGCs) with the ECM protein Pikachurin, which is required for proper development and neurotransmission at the synapse (Omori et al., 2012Omori Y. Araki F. Chaya T. Kajimura N. Irie S. Terada K. Muranishi Y. Tsujii T. Ueno S. Koyasu T. et al.Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.J. Neurosci. 2012; 32: 6126-6137Crossref PubMed Scopus (68) Google Scholar, Sato et al., 2008Sato S. Omori Y. Katoh K. Kondo M. Kanagawa M. Miyata K. Funabiki K. Koyasu T. Kajimura N. Miyoshi T. et al.Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.Nat. Neurosci. 2008; 11: 923-931Crossref PubMed Scopus (224) Google Scholar). Notably, ablation of Pikachurin in mice results in ultrastructural abnormalities of the photoreceptor synapse and deficits in synaptic transmission. However, it is unclear how the photoreceptor Pikachurin-DGC complex engages ON-BCs and what its post-synaptic molecular targets are. Here we identify ECM components heparan sulfate proteoglycans (HSPGs) as interaction partners of the orphan GPCRs, GPR179 and GPR158, and demonstrate an essential role of these interactions in synaptic targeting. Using the first visual synapse as a model, we provide evidence that the photoreceptor-released HSPG Pikachurin dictates the post-synaptic organization of the GAP complex by anchoring GPR179-RGS at the dendritic tips of ON-BCs. We further show that this function involves transsynaptic interaction with the DGC at the axonal terminals and that its disruption alters synaptic neurotransmission of photoreceptors in a process that involves RGS protein recruitment. Orphan receptors GPR158 and GPR179 contain large extracellular segments that feature an EGF-like Ca2+-binding domain and a leucine repeat sequence (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, Patel et al., 2013Patel N. Itakura T. Gonzalez Jr., J.M. Schwartz S.G. Fini M.E. GPR158, an orphan member of G protein-coupled receptor Family C: glucocorticoid-stimulated expression and novel nuclear role.PLoS ONE. 2013; 8: e57843Crossref PubMed Scopus (31) Google Scholar), suggesting their possible role in association with ECM proteins. To test this possibility, we conducted an unbiased proteomics search for their extracellular binding partners (Figure 1A) in HEK293 cells known to express a wide range of receptors, cell adhesion molecules, and matrix proteins (Geiger et al., 2012Geiger T. Wehner A. Schaab C. Cox J. Mann M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins.Mol. Cell. Proteomics. 2012; 11 (M111.014050)Crossref Scopus (577) Google Scholar, Lin et al., 2014Lin Y.C. Boone M. Meuris L. Lemmens I. Van Roy N. Soete A. Reumers J. Moisse M. Plaisance S. Drmanac R. et al.Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations.Nat. Commun. 2014; 5: 4767Crossref PubMed Scopus (282) Google Scholar, Thomas and Smart, 2005Thomas P. Smart T.G. HEK293 cell line: a vehicle for the expression of recombinant proteins.J. Pharmacol. Toxicol. Methods. 2005; 51: 187-200Crossref PubMed Scopus (467) Google Scholar). Given high sequence homology between GPR158 and GPR179 (Figure S1A), our initial experiments were conducted with the N-terminal ectodomain of GPR158 fused to a human IgG Fc fragment (ecto-GPR158-Fc) directing its secretion to the medium. Following transfection of HEK293 cells with the ecto-GPR158-Fc, secreted proteins were purified using protein G beads that captured Fc fragments. In parallel, the same experiment was conducted with Fc construct alone and used as a negative control to assess non-specific binding. Mass spectrometric identification of the proteins eluted from the beads identified 129 proteins specifically co-isolated with the ecto-GPR158-Fc but not with the Fc fragment (Figure 1A). A Gene Ontology analysis of the data revealed that about half of these proteins were classified as secreted, with the largest group (21.71%) constituting ECM components. Remarkably, the most abundant proteins in this group belonged to a family of proteoglycans post-translationally modified by HS, classified as HSPGs (Bishop et al., 2007Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1265) Google Scholar). In total, we found 12 different HSPGs specifically co-purified with GPR158 ectodomain (Figures 1B and S1B). To confirm the interactions, we studied binding of several representative HSPG members to full-length GPR158 by co-immunoprecipitation upon co-expression in HEK293 cells (Figure 1C). We found a robust pull-down of all tested HSPGs by GPR158. This interaction was specific, as no binding was detected upon omitting the bait protein from the transfection. Reciprocal experiments similarly revealed effective and specific pull-down of GPR158 when HSPGs were used as baits (Figure S1C). We next tested whether GPR179 could also bind to HSPGs given considerable sequence conservation between the extracellular domains of GPR158 and GPR179 (Figure S1A). These experiments were designed similarly, and the interaction was first studied upon co-transfection of epitope-tagged full-length GPR179 and candidate HSPGs into HEK293 cells. Again, we found that immunoprecipitation of GPR179 specifically pulled down all of the HSPGs tested (Figure 1D). The binding was further confirmed in the reverse direction where HSPGs were also able to pull down GPR179 (Figure S1D). The ubiquitous nature of GPR158/179 interactions with various HSPG members, which do not share extensive homology in their amino acid sequences, prompted us to evaluate the role of HS chains in binding. In these experiments, we used beads directly conjugated to heparin, a highly sulfated form of HS often used in affinity chromatography to isolate HS-binding proteins (Ori et al., 2011Ori A. Wilkinson M.C. Fernig D.G. A systems biology approach for the investigation of the heparin/heparan sulfate interactome.J. Biol. Chem. 2011; 286: 19892-19904Crossref PubMed Scopus (169) Google Scholar). Indeed, we found that heparin beads were able to effectively pull down native GPR158 from a membrane-enriched brain lysate. The interaction was specific, as inclusion of excess free unbound heparin prevented GPR158 retention by the beads. We further examined the role of divalent cations in the interaction, given their role in regulating many extracellular interactions (Maurer and Hohenester, 1997Maurer P. Hohenester E. Structural and functional aspects of calcium binding in extracellular matrix proteins.Matrix Biol. 1997; 15: 569-580Crossref PubMed Scopus (54) Google Scholar) and the presence of the putative Ca2+-binding motif in GPR158/179 (Orlandi et al., 2012Orlandi C. Posokhova E. Masuho I. Ray T.A. Hasan N. Gregg R.G. Martemyanov K.A. GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes.J. Cell Biol. 2012; 197: 711-719Crossref PubMed Scopus (72) Google Scholar, Patel et al., 2013Patel N. Itakura T. Gonzalez Jr., J.M. Schwartz S.G. Fini M.E. GPR158, an orphan member of G protein-coupled receptor Family C: glucocorticoid-stimulated expression and novel nuclear role.PLoS ONE. 2013; 8: e57843Crossref PubMed Scopus (31) Google Scholar). We found that retention of GPR158 by heparin beads was insensitive to either addition of excess Ca2+/Mg2+ or their chelation by EDTA (Figure 1E). Native GPR179 exhibited similar behavior, suggesting that divalent cations may not be involved in modulating this interaction (Figure 1F). Together, these results establish HSPGs as extracellular binding partners of GPR158/179 orphan receptors and reveal sufficiency of HS side chains for the interaction. To explore physiological relevance of the GPR158/179-HSPG interactions in the context of native neuronal circuits, we turned our attention to one particular candidate HSPG identified in the screen: an Agrin-like protein, Pikachurin, one of the least characterized members of the family (Manabe et al., 2008Manabe R. Tsutsui K. Yamada T. Kimura M. Nakano I. Shimono C. Sanzen N. Furutani Y. Fukuda T. Oguri Y. et al.Transcriptome-based systematic identification of extracellular matrix proteins.Proc. Natl. Acad. Sci. USA. 2008; 105: 12849-12854Crossref PubMed Scopus (102) Google Scholar). Pikachurin is specifically expressed in the retina by both rod and cone photoreceptors and released in the synaptic cleft, where it has been identified as a ligand for the pre-synaptic DGC (Sato et al., 2008Sato S. Omori Y. Katoh K. Kondo M. Kanagawa M. Miyata K. Funabiki K. Koyasu T. Kajimura N. Miyoshi T. et al.Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.Nat. Neurosci. 2008; 11: 923-931Crossref PubMed Scopus (224) Google Scholar) (Figure 2A). Intriguingly, the function of photoreceptor synapses requires the presence of GPR179, expressed by the post-synaptic ON-BC and targeted to the dendritic tips in apposition to pre-synaptic release sites of both rods and cones (Audo et al., 2012Audo I. Bujakowska K. Orhan E. Poloschek C.M. Defoort-Dhellemmes S. Drumare I. Kohl S. Luu T.D. Lecompte O. Zrenner E. et al.Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 321-330Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, Peachey et al., 2012Peachey N.S. Ray T.A. Florijn R. Rowe L.B. Sjoerdsma T. Contreras-Alcantara S. Baba K. Tosini G. Pozdeyev N. Iuvone P.M. et al.GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 331-339Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In contrast, brain-enriched GPR158 is not detected in either photoreceptors or ON-BC (Sarin et al., 2018Sarin S. Zuniga-Sanchez E. Kurmangaliyev Y.Z. Cousins H. Patel M. Hernandez J. Zhang K.X. Samuel M.A. Morey M. Sanes J.R. et al.Role for Wnt signaling in retinal neuropil development: analysis via RNA-seq and in vivo somatic CRISPR mutagenesis.Neuron. 2018; 98: 109-126.e8Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Shekhar et al., 2016Shekhar K. Lapan S.W. Whitney I.E. Tran N.M. Macosko E.Z. Kowalczyk M. Adiconis X. Levin J.Z. Nemesh J. Goldman M. et al.Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics.Cell. 2016; 166: 1308-1323.e30Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar), making GPR179 at this synapse non-redundant. First, we confirmed the interaction between GPR179 and Pikachurin in transfected HEK293 cells, where we detected robust and specific co-immunoprecipitation of both proteins in both forward and reverse directions (Figure 2B). We further established that the binding is mediated by the ectodomain of GPR179 using an overlay approach. A live staining of Pikachurin upon transfection in HEK293 cells revealed its predominant extracellular localization within the ECM (Figure 2C). These Pikachurin-positive patches were stained by application of ecto-GPR179-Fc (Figure 2C). The interaction of the ecto-GPR179 and Pikachurin in the ECM was specific, as no staining was observed when Fc carrier alone was used or when Pikachurin was omitted from the transfection, indicating low expression of endogenous Pikachurin in HEK293 cells, which was nevertheless detectable by mass spectrometry in our proteomic screen. Because HS side chains were sufficient for interaction with GPR158/179 (Figure 1E), we next asked a converse question: whether HS modification of Pikachurin is the sole requirement for GPR179 binding. Enzymatic treatment with heparinase III resulted in a clear mobility shift of Pikachurin isolated from HEK293 medium, confirming its modification with HS side chains (Figure 2D). However, Pikachurin stripped from HS still effectively co-immunoprecipitated with GPR179, indicating the involvement of additional binding sites on Pikachurin for the interaction with GPR179 (Figure 2D). Like other secreted members of the HSPG family, Pikachurin contains several conserved structural domains (Sato et al., 2008Sato S. Omori Y. Katoh K. Kondo M. Kanagawa M. Miyata K. Funabiki K. Koyasu T. Kajimura N. Miyoshi T. et al.Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.Nat. Neurosci. 2008; 11: 923-931Crossref PubMed Scopus (224) Google Scholar) that can possibly be involved in HS-independent binding to GPR179. Multiple sequence alignment across 197 species revealed a high degree of amino acid conservation across the three laminin G and the two EGF-like domains in the C terminus of the protein, with the highest similarity (55.5%) in the third laminin G domain (Figure 2E). Accordingly, we generated several deletion constructs lacking various conserved domains of Pikachurin (Figure 2F). In these experiments, we used cell lysates in which HS modification does not occur to specifically analyze contribution of protein moiety to binding. Pull-down experiments with ecto-GPR179-Fc as a bait and the Fc carrier as negative control revealed that the C-terminal 251 amino acids (Pika-LG3) were both necessary and sufficient for the interaction with GPR179 (Figure 2G). Thus, we conclude that Pikachurin, when secreted in the ECM, specifically interacts with the ectodomain of GPR179 receptor via multiple sites including the HS side chains and the LG3 domain of the protein core. We next examined GPR179 and Pikachurin expression and interaction in vivo, in the mouse retina. First, we confirmed the ability of the ectodomain of GPR179 to interact with endogenous Pikachurin in a pull-down experiment with mouse retina lysates (Figure 3A). Western blot analysis revealed robust capture of retina-derived Pikachurin by the beads coated with ecto-GPR179-Fc protein but not by the control Fc beads, indicating that the binding was specific. Second, we examined immunohistochemical staining of retina cross-sections and found extensive colocalization of Pikachurin and GPR179 in the outer plexiform layer (OPL), where both proteins showed characteristic punctate staining at photoreceptor synapses (Figure 3B). Third, we tested their interaction in situ, using a proximity ligation assay (PLA). Using this approach, we found numerous positive signals generated by the antibodies against Pikachurin and GPR179 intersecting at the complex (Figure 3C). The staining pattern corresponded to characteristic synaptic puncta and was confined to the OPL. The puncta predominantly decorated dendritic ti" @default.
• W2894580867 created "2018-10-12" @default.
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• W2894580867 date "2018-10-01" @default.
• W2894580867 modified "2023-10-12" @default.
• W2894580867 title "Transsynaptic Binding of Orphan Receptor GPR179 to Dystroglycan-Pikachurin Complex Is Essential for the Synaptic Organization of Photoreceptors" @default.
• W2894580867 cites W127884157 @default.
• W2894580867 cites W1555249890 @default.
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