Cellular/Molecular

5684 • The Journal of Neuroscience, June 23, 2004 • 24(25):5684 –5693 Cellular/Molecular A Retinal-Specific Regulator of G-Protein Signaling Interacts with G␣o and Accelerates an Expressed Metabotropic Glutamate Receptor 6 Cascade Anuradha Dhingra,1 Eva Faurobert,2 Nathan Dascal,3 Peter Sterling,1 and Noga Vardi1 Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, 2Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Propre de Recherche, 06560 Valbonne, France, and 3Department of Physiology and Pharmacology, Tel Aviv University, Tel Aviv 69978, Israel 1 Go is the most abundant G-protein in the brain, but its regulators are essentially unknown. In retina, G␣o1 is obligatory in mediating the metabotropic glutamate receptor 6 (mGluR6)-initiated ON response. To identify the interactors of Go , we conducted a yeast two-hybrid screen with constituitively active G␣o as a bait. The screen frequently identified a regulator of G-protein signaling (RGS), Ret-RGS1, the interaction of which we confirmed by coimmunoprecipitation with G␣o in transfected cells and in retina. Ret-RGS1 localized to the dendritic tips of ON bipolar neurons, along with mGluR6 and G␣o1. When Ret-RGS1 was coexpressed in Xenopus oocytes with mGluR6, G␣o1 , and a GIRK (G-protein-gated inwardly rectifying K ⫹) channel, it accelerated the deactivation of the channel response to glutamate in a concentration-dependent manner. Because light onset suppresses glutamate release from photoreceptors onto the ON bipolar dendrites, Ret-RGS1 should accelerate the rising phase of the light response of the ON bipolar cell. This would tend to match its kinetics to that of the OFF bipolar that arises directly from ligand-gated channels. Key words: ON bipolar neuron; retina; G␣o ; Ret-RGS1; GTPase-activating protein; GIRK channel Introduction A photoreceptor signals dimming and brightening by increasing and decreasing its release of glutamate. This fluctuating output is then transmitted to two classes of second-order neuron, one that depolarizes to glutamate (OFF bipolar cell) and another that hyperpolarizes (ON bipolar cell). In mammals the ON bipolar cells account for 75% of all bipolar cells and are responsible for both night and day vision (Sterling et al., 1988; Cohen and Sterling, 1990; Martin and Grünert, 1992; Strettoi and Masland, 1995). Thus it is important to understand the molecular basis for response of ON cells to light and glutamate. It is now established that the ON response is mediated by turning off a G-protein cascade that is active in darkness when glutamate binds to the metabotropic glutamate receptor 6 (mGluR6) (Nomura et al., 1994; Vardi and Morigiwa, 1997; Vardi et al., 2000). The heterotrimeric G-protein has been identified as Go1 (Nawy, 1999; DhReceived Feb. 11, 2004; revised April 16, 2004; accepted May 7, 2004. This work was supported by National Eye Institute (NEI) Grant EY 11105 (N.V.), Retina France (E.F.), NEI Grant EY 00128 (P.S.), National Institutes of Health (GM068493), and the Binational Israel–U.S. Science Foundation (2001122) (N.D.). We thank our colleagues for kindly providing materials: Ching-Hwa Sung (bovine retinal library), Donna Woulfe, (G␣o1 in pAS2–1), Dave Manning (polyclonal antibody against G␣o ), Yoshiaki Nakajima and Shigemoto Nakanishi (mGluR6), and Rick H. Cote (antibodies against PDE␥). We thank Sheryll Barker for providing the sequence information of mouse Ret-RGS1 clone. We also thank Erfie Bi for initial consultation on using the yeast two-hybrid system and Atif Qasim, Tehilla Bar-Yehuda, Marie Fina, Jian Li, and Rachel Barzilai for excellent technical assistance. Correspondence should be addressed to Anuradha Dhingra, Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104-6058. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.0492-04.2004 Copyright © 2004 Society for Neuroscience 0270-6474/04/245684-10$15.00/0 ingra et al., 2000, 2002), which during activation somehow closes a nonspecific cation channel (Nawy and Jahr, 1990; Shiells and Falk, 1990, 1992; de la Villa et al., 1995; Euler et al., 1996). Key questions are how Go affects the channel and how the cascade is regulated. Go is the most abundant G-protein in the brain (Sternweis and Robishaw, 1984; Huff et al., 1985; Li et al., 2000). It is involved in many signal transduction cascades, ranging from regulating several ion channels (voltage-gated Ca 2⫹, K ⫹, and possibly a cGMPdependent channel) to regulating multiple aspects of behaviors in Caenorhabditis elegans (Kleuss et al., 1991; Mendel et al., 1995; Berghard and Buck, 1996; Kojima et al., 1997; Valenzuela et al., 1997; Gomez and Nasi, 2000; Greif et al., 2000). Except for certain Ca 2⫹ and K ⫹ channels, however, where the ␤␥ subunits of Go directly bind the channel (Dolphin, 1998; Leaney and Tinker, 2000), the mechanisms of the transduction of Go are virtually unknown. The purpose of this study was to elucidate the mGluR6 cascade by finding interactors of G␣o1 in retina. Using active G␣o1 as bait in a yeast-two hybrid system, we identified a regulator of G-protein signaling (RGS), Ret-RGS1, which is a retina-specific splice variant of RGS20 (Faurobert and Hurley, 1997; Barker et al., 2001). After confirming the interaction by coimmunoprecipitation, we localized Ret-RGS1 to ON bipolar dendrites, in which mGluR6 and G␣o1 are coexpressed. Expressing Ret-RGS1 with mGluR6 and G␣o1 in Xenopus oocytes, we found that Ret-RGS1 accelerates the termination of the cascade. Ret-RGS1 is also expressed by photoreceptor terminals, where it may serve a different function. Dhingra et al. • Localization and Function of Ret-RGS1 in Retina Materials and Methods Yeast two-hybrid screen Yeast two-hybrid screen was performed using cDNA encoding constitutively active G␣o1 Q205L in pAS2–1 vector (Clontech, Cambridge, UK). The bovine retinal cDNA library (in pACT2–1) was generously provided by Dr. Ching-Hwa Sung (Cornell Medical School, Ithaca, NY). The presence of G␣o1 protein in yeast transformed with G␣o1 plasmid was checked by Western blots. Approximately 2 ⫻ 10 6 yeast clones cotransformed with G␣o1 and library plasmid were screened by Matchmaker GAL4 Two-Hybrid system (BD Biosciences, Palo Alto, CA). The colonies growing on SD/-His/-Leu/-Trp medium (BD Biosciences) were further assayed for LacZ. To eliminate false positives, in the initial screen, positive clones were subjected to cycloheximide counterselection to obtain colonies that had lost the bait plasmid and retained the library plasmid. These clones were then remated with clones carrying G␣o1 (in pACT2– 1), pACT2–1 vector alone, or pLAMC (negative control). The positive clones from remating were then grown, and DNA was transformed into Escherichia coli for sequencing. The insert was sequenced using primers in the pACT2–1 vector; these included rod and cone phosphodiesterase ␥ (PDE␥) and Ret-RGS1. J. Neurosci., June 23, 2004 • 24(25):5684 –5693 • 5685 West Grove PA), rinsed, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Digital images were acquired using a confocal microscope (Leica, Exton, PA). For immunoelectron microscopy, 100 ␮m radial Vibratome sections were immunostained as above except that Triton X-100 (0.1%) was added only to the preincubation medium (omitting Triton X-100 gave no staining). Sections were incubated with secondary antibody conjugated to HRP (anti-rabbit Fab fragments conjugated to HRP), developed using diaminobenzidine and hydrogen per- RT-PCR Retinal RNA was prepared using High Pure RNA Isolation kit (Roche, Mannheim, Germany). The reverse transcription was performed on 1 ␮g of total RNA with oligo dT primers by using Moloney murine leukemia virus reverse transcriptase (BD Biosciences). The PCR reaction was performed using degenerate primers for mouse cone and rod PDE␥ (forward: cagttcaagagcaagccncccaag; reverse: actgngcnagctc(g/a)tgcagctc). Thirty-five cycles (94°C for 1 min, 54°C for 1 min, and 72°C for 2 min) were performed on a programmable thermocycler (PerkinElmer Life Sciences, Boston, MA). Colony hybridization The yeast clones to be screened were grown on a master plate in a grid pattern and lifted onto nylon membrane followed by denaturation and neutralization by following the Matchmaker two-hybrid system supplier’s protocol (BD Biosciences). Digoxigenin-labeled cone and rod PDE␥ probes were made using PCR Dig Probe Synthesis kit (Roche). Hybridization was performed at 42°C in Dig Easy Hyb buffer with gentle agitation overnight followed by two 15 min washes (moderate stringency) in 0.5⫻ SSC, 0.1% SDS at 68°C. Immunological detection was performed with anti-Dig antibody (Roche). Primary antibodies The antibody against Ret-RGS1 was raised against the bovine N1 peptide (see Fig. 1 A) and has been characterized (Faurobert et al., 1999; this study). Anti-G␣o was a mouse monoclonal antibody (mAb) raised against the bovine protein (mAb 3073; Chemicon, Temecula, CA). AntiG␣o1 was a rabbit polyclonal raised against the specific peptide EYPGSNTYED (gift from D. Manning, University of Pennsylvania, Philadelphia, PA). The specificity of the monoclonal and polyclonal anti-G␣o has been established (Vardi et al., 1993; Dhingra et al., 2002). Anti-protein kinase C (PKC)␣ (clone MC5; Amersham Biosciences, Little Chalfont, UK), anti-calbindin (clone CB-955; Sigma, St. Louis, MO), and antisynaptophysin (mAb 5258; Chemicon) were mouse monoclonal antibodies. Polyclonal anti-Na/K ATPase was a gift from K. Geering (Institute of Pharmacology and Toxicology, Lausanne, Switzerland). Immunocytochemistry Bovine eyes were purchased from a local slaughterhouse and placed on ice until further treatment. An eye was hemisected along the ora serata, everted, and immersed in 2% or 4% buffered paraformaldehyde (0.1 M phosphate buffer, pH, 7.4) for 1 hr. After a rinse, the eye was cryoprotected in 30% sucrose in phosphate buffer overnight at 4°C; the retina was detached, embedded in a 2:1 mixture of 20% buffered sucrose and tissue freezing medium, and cryosectioned radially at 10 –15 ␮m thickness. Sections were preincubated in 0.1 M phosphate buffer containing 10% normal goat serum, 5% sucrose, and 0.3% Triton X-100, incubated in primary antibody for 48 –72 hr at 4°C, rinsed, incubated in a secondary antibody conjugated to a fluorescent marker (Jackson ImmunoResearch, Figure 1. G␣o interacts with Ret-RGS1. A, Alignment of amino acid residues of mouse (Mo), bovine (Bo), and human (Hu) Ret-RGS1 protein N termini (corresponding to the N1 bovine peptide used to generate the antibody against Ret-RGS1). B, HEK 293 cells transfected with bovine Ret-RGS1 expression plasmid (top) and mouse Ret-RGS1 EST clone (bottom), immunostained with N1 antibody (red) and counterstained with the nuclear dye SYTO 13 (green). This antibody recognized the bovine protein (top) but not the mouse protein (bottom). C, A Western blot stained with N1 antibody shows two bands of 45 and 40 kDa in bovine retina and a 45 kDa band in HEK cells transfected with Ret-RGS1 expression plasmid. These bands were eliminated by preabsorbing the antibody with the N1 peptide (preab). D, Coimmunoprecipitation of G␣o1 and Ret-RGS in HEK cells transfected with G␣o1 and Ret-RGS1 expression vectors. G␣o was immunoprecipitated in the presence or absence of AlF4 ⫺ (as indicated). The immunoprecipitates were subjected to Western blotting with G␣o (top) or Ret-RGS1 (bottom) antibodies in parallel. 1, Homogenate; 2 and 3, immunoprecipitation (IP) with anti-G␣o ; 4 and 5, mock IP without any antibody. In bottom blot, an empty lane next to lane 2 was cut out for alignment. For both D and E, 0.3% of total sample was applied for the homogenate lane, and 5% was applied for the rest of the lanes. E, Coimmunoprecipitation of G␣o and Ret-RGS1 from bovine retina. IP was done with anti-G␣o in the presence of AlF4 ⫺. h, Homogenate. 5686 • J. Neurosci., June 23, 2004 • 24(25):5684 –5693 Dhingra et al. • Localization and Function of Ret-RGS1 in Retina oxide, and intensified using the goldsubstitution silver intensification method (Johnson and Vardi, 1998). The sections were then treated with osmium tetroxide (2%; 60 min), stained with 1% uranyl acetate in 70% ethanol, dehydrated in graded ethanol series (70 –100%), cleared in propylene oxide, and embedded in Epon 812. Ultrathin sections were mounted on Formvar-coated slot grids and stained with uranyl acetate. Cell culture and transfection Human embryonic kidney (HEK) 293 cells were cultured in minimal essential medium supplemented with Penstrep (Invitrogen, Carlsbad, CA) and 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were transiently transfected with G␣o1 (in pDP; provided by D. Manning, University of Pennsylvania) and Ret-RGS1 (in pRC) expression plasmids using Fugene transfection reagent (Invitrogen). Cells were harvested 48 hr later. Coimmunoprecipitation Transfected cells or frozen bovine retinas were collected in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM MgCl2, 100 ␮M GDP, 30 mM PMSF) with or without AlF4 ⫺ (30 ␮M AlCl3 and 10 mM NaF). The cells were homogenized at low speed and centrifuged at 8000 ⫻ g in an Eppendorf centriFigure 2. Ret-RGS1 is localized to ON bipolar somas and dendrites. A, Radial section of bovine retina stained for Ret-RGS1. fuge for 5 min. The supernatant was precleared Staining is present in all retinal layers, most strongly in inner segments and then OPL. Staining in IPL and ganglion cell somas was by adding 20 ␮l of Protein G agarose beads (Insomewhat variable between tissues. IS, Inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear vitrogen), centrifuging, and collecting the sulayer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer. (Abbreviations apply to all Figures.) B, Double pernatant. The precleared supernatant was instaining for Ret-RGS1 (red) and PKC (green). Rod bipolar cells marked with anti-PKC (arrows) were also stained for Ret-RGS1. cubated with mouse anti-G␣o and Protein G Certain PKC-negative somas were Ret-RGS1 positive (arrowhead). C, Double staining for Ret-RGS1 (red) and G␣o (green). All ON agarose beads on a rotator at 4°C for ⬃12 hr. bipolar cells marked by G␣o express Ret-RGS1 (arrows). D, Electron micrograph of a rod synaptic complex. The invaginating rod The beads with protein complexes were then bipolar dendritic tip (RB) is stained for Ret-RGS1. r, Ribbon. E, Electron micrograph of a cone synaptic complex. The central element pulled down by centrifuging (10,000 ⫻ g), of the postsynaptic triad (CB) was stained for Ret-RGS1. H, Horizontal cell process. washed thoroughly in lysis buffer, resuspended in Lammelli buffer, boiled, and spin filtered. further homogenized, dissolved in Tris-HCl (10 mM, pH 6.8), and diThe proteins were run on 10% SDS-PAGE gel and transferred to a nitroluted in Tris-HCl (pH 6.8), KI, and KCl to a final concentration of 100 cellulose membrane using semiwet transfer apparatus (Bio-Rad, HermM, 0.6 M, and 0.1 M, respectively. The lysate was further centrifuged (20 cules, CA). Blots were then incubated sequentially in the following: 10% min; 25,000 ⫻ g) to yield a pellet containing lysed synaptosomal memnonfat dry milk in PBS containing 0.1% Tween 20 (PBST) for 1 hr at 4°C, branes and a supernatant containing synaptic vesicles. primary antibodies (against G␣o1 or Ret-RGS1) in PBST at 4°C overPurification of synaptic vesicles. The supernatant was centrifuged (2 hr; night, PBST, secondary antibodies (anti-rabbit Fab fragments linked to 65,000 ⫻ g), the resulting supernatant, referred as synaptosomal cytosol, HRP; Protos, Burlingame, CA), and detected by SuperSignal West Femto was removed, and the pellet containing the crude synaptic vesicles was Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL). washed in 10 mM HEPES, pH 7.4, and centrifuged (15 min; 400,000 ⫻ g). The pellet was homogenized in 40 mM sucrose and passed through a 23 Fractionation of bovine retinas and purification of synaptic gauge needle. The suspension was then layered on top of a linear continvesicles and plasma membrane on sucrose gradients uous sucrose gradient, which was centrifuged in a SW41 Beckman rotor Retinas for this purpose were obtained from François Le Madec (of the (5 hr; 65,000 ⫻ g). Fractions were collected from the bottom of the tube, slaughterhouse of Puget-Théniers, France). The following procedures concentrated by speed-vac, and precipitated with methanol/chloroform. were modified from the standard protocol for purification of synaptic Purification of synaptic plasma membranes. Synaptic plasma memvesicles from mammalian brain (Huttner et al., 1983). branes were prepared as described in Herms et al. (1999). In brief, the Preparation of crude synaptosomes. Freshly isolated bovine retinas were lysed synaptosomal membranes were resuspended in 1.2 M sucrose and disrupted in ice-cold homogenization buffer (320 mM sucrose, 1 mM loaded on discontinuous sucrose gradient containing 5 ml of 0.8 M suMgCl2, 4 mM HEPES–NaOH buffer, pH 7.3) supplemented with procrose and 1 ml of 0.3 M sucrose. After centrifugation at 65,000 ⫻ g for 2.5 tease inhibitors (Complete; Roche Diagnostics) and 50 ␮g/ml DNase I hr, the synaptic plasma membranes band between 0.8 and 1.2 M sucrose (Roche Diagnostics) by shear force with a Polytron (Kinematica, Littau, was collected, diluted in ice-cold water, and centrifuged (30 min; Switzerland). The homogenate was centrifuged (10 min; 800 ⫻ g), and 48,000 ⫻ g). the supernatant (S1) was collected and centrifuged again (15 min; 9200 ⫻ g) to yield a pellet P2 and a supernatant S2. An aliquot of S2 was centrifuged again (30 min; 400,000 ⫻ g) to yield a “cytosol” supernatant. The Tests of solubilization of Ret-RGS1 from retina membranes A bovine retina was homogenized in hypotonic buffer containing 5 mM pellet (P2) was washed and centrifuged (15 min; 10,200 ⫻ g). The subTris-HCl, pH 7.5, and protease inhibitors (Complete; Roche Diagnossequent pellet was frozen and thawed in homogenization buffer to yield tics). The homogenate was centrifuged (15 min; 800 ⫻ g) at 4°C. The the synaptosomal fraction. supernatant was dispensed equally in six microcentrifuge tubes and cenFractionation of synaptosomes. The crude synaptosomal fraction was Dhingra et al. • Localization and Function of Ret-RGS1 in Retina J. Neurosci., June 23, 2004 • 24(25):5684 –5693 • 5687 pg) was injected 12–16 hr before the experiment. An oocyte was placed in a chamber (⬃50 ␮l), impaled with two sharp electrodes with tips that were filled with 1% agarose plus 3 M KCl (resistance ⬃0.3 M⍀), and immediately perfused with ND96 at 0.2– 0.4 ml/sec. The holding potential was set to ⫺80 mV. Basal and evoked currents were measured in a high potassium solution containing (in mM): 24.5 KCl, 73.5 NaCl, 1 CaCl2, 1 MgCl2, 5 mM HEPES, pH 7.4. An oocyte was tested only once to remove variability caused by desensitization. Data were acquired with Axotape and pCLAMP software (Axon Instruments, Foster City, CA); statistical analysis (one way ANOVA followed by twotailed Student’s t test) was done with Microsoft Excel. Data are presented as either the mean or normalized mean ⫾ SE. To normalize data pooled from two experiments or more, each data point within a batch was divided by control values of this batch, and then normalized values were combined and analyzed as above. Results Identifying interactors of constitutively active G␣o1 To identify interactors of G␣o1, we screened a bovine retinal cDNA library in a Figure 3. Ret-RGS1 is expressed in presynaptic terminals. Top row, Immunostaining for Ret-RGS1 (red) and horizontal cell GAL4-based yeast two-hybrid system with marker, calbindin (green). Horizontal cell processes are unstained for Ret-RGS1. Middle row, Immunostaining for Ret-RGS1 (red) a constitutively active ␣ subunit of Go1 and synaptophysin (green). Ret-RGS1 and synaptophysin colocalize in presynatic photoreceptor terminals (PT) (arrows). Note that (G␣o1 *). An initial screen of ⬃5 ⫻ 10 4 Ret-RGS1 staining forms clusters, which often outline the synaptophysin-positive terminals (arrowheads). Bottom row, Immu- clones gave 36 positives, in which ⬎90% nostaining for Ret-RGS1 (red) and PKC (green). Staining for Ret-RGS1 forms large puncta throughout the IPL (arrowheads). Rod encoded the ␥ subunit of photoreceptor bipolar axon terminals (rba) (identified by PKC) are also stained (arrows). PDE. A full screen of ⬃2 ⫻ 10 6 clones, after hybridizing out the photoreceptor trifuged at 25,000 ⫻ g for 20 min. The resulting membrane pellets were PDE␥-encoding clones, gave ⬃1000 positive clones, of which we resuspended at 2 mg/ml in six different buffers: hypotonic (5 mM Trissequenced ⬃300. These fell into several categories: “potential HCl, pH 7.5), hypertonic (Tris-HCl, pH 7.5, 1 M NH4Cl), sodium careffector”: rod PDE␥ (87 hits), cone PDE␥ (41); “regulator of bonate (100 mM Na2CO3, pH 11.6), urea (5 mM Tris-HCl, pH 7.5, 4 M G-protein signaling”: Ret-RGS1 (37), RAP1-GTPase-activating urea), detergent (5 mM Tris-HCl, pH 7.5, 50 mM n-octyl glucoside), and protein (GAP) (2), RGS10 (3), RGS16 (18), G␣-interacting prohydroxylamine (1 M NH2OH, pH 7.5). The membranes were homogenized tein (GAIP) (2); “guanine nucleotide exchange factor”-like proby extrusion through a 23 gauge needle, incubated on ice for 20 min, and centrifuged for 15 min in a TLA100.1 rotor at 400,000 ⫻ g. For hydroxylteins: Synembryn (3), activator of G-protein signaling 3 (8); and amine washing, the suspension was incubated for 18 hr at room tem“guanine nucleotide dissociation inhibitors”: Purkinje cell perature. Equivalent amounts of supernatant and pellet were anaprotein-2 (28). Several of these interactors were identified previlyzed by immunoblot on a 12% polyacrylamide gel. ously by yeast two-hybrid using various nonretinal libraries (Jordan et al., 1999; Luo and Denker, 1999; Natochin et al., 2001, Tall cDNA constructs and RNA et al., 2003); however, PDE␥, Ret-RGS1, RGS10, and RGS16 are The coding sequences of all cDNA used for oocyte injection were prepared or obtained as described previously (Sharon et al., 1997; Vorobiov identified here as new potential interactors. et al., 2000). All cDNAs were inserted into high-expression oocyte vectors The finding of PDE␥ might have been important because the containing 5⬘ and 3⬘ untranslated sequences of Xenopus ␤-globin: effector of Go in ON cells was initially suggested to be a phosphopGEM-HJ [Muscarinic 2 receptor (m2R), G-protein-gated inwardly recdiesterase (Nawy and Jahr, 1990, 1991; Shiells and Falk, 1990); tifying K ⫹ channel (GIRK)1, GIRK2, G␣o1, Ret-RGS1], or pBluescript however, immunostainings with three antibodies against PDE␥ SK(⫺) (mGluR6). Plasmids were linearized with NotI, and mRNA was were negative in bipolar cells, and our efforts to identify a nontranscribed in vitro using a cap structure at the 5⬘ end site (Yakubovich et photoreceptor isoform using degenerate primers amplified only al., 2000). The plasmid containing the Ret-RGS1 coding sequence was photoreceptor isoforms. Presumably G␣o interacts with PDE␥ subcloned from pRC/CMV into pGEM-HJ using PCR and inserting an EcoRI sequence at the 5⬘ end and a HindIII sequence at the 3⬘ end. because of the homology of Go to transducin (G␣t), which in Pertussis toxin (PTX)-insensitive G␣o1 mutants (C351I and C351V) phototransduction binds PDE␥. The next important interactor were prepared with the Quick Change Site Directed Mutagenesis kit was Ret-RGS1, which was cloned previously from cow and hu(Stratagene, La Jolla, CA). man (Faurobert and Hurley, 1997; Barker et al., 2001). The rest of this report concerns Ret-RGS1. Xenopus oocyte recordings Xenopus laevis oocytes were maintained and injected as described (Dascal and Lotan, 1992; Dascal, 2000). mRNA was injected 3 d before experiments, and oocytes were maintained at 20°C in ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH 7.5) supplemented with 2.5 mM Na-pyruvate and 50 ␮g/ml gentamicin. Pertussis toxin (200 G␣o interacts with Ret-RGS1 in HEK cells and retina To test the interaction between Go and ret-RGS1 and localize ret-RGS1, we used a polyclonal antibody raised against the first 5688 • J. Neurosci., June 23, 2004 • 24(25):5684 –5693 Dhingra et al. • Localization and Function of Ret-RGS1 in Retina 35 amino acids of bovine Ret-RGS1 (N1 peptide) (Faurobert et al., 1999), but first we had to establish its specificity. Alignment of the bovine against the corresponding mouse (sequence obtained from Sheryll Barker, University of Texas, Dallas, TX; unpublished data) and human RetRGS1 shows a poor match (Fig. 1 A). We then transfected HEK 293 cells with an expression plasmid coding for bovine RetRGS1 or mouse Ret-RGS1 EST clone (IMAGE clone 5363855). The antibody recognized only the bovine form (Fig. 1 B). Western blots of bovine retinal protein gave a prominent band at ⬃45 kDa, in agreement with its predicted molecular weight (Fig. 1C, left). It also gave a band at 40 kDa. Western blots of transfected HEK cells gave a predominant band of 45 kDa and on longer exposure a weak 40 kDa band. Both 45 and 40 kDa bands were preabsorbed with 50-fold excess of antigenic peptide (Fig. 1C, right), suggesting that the 40 kDa band is a proteolytic degradation product, or possibly another splice variant of this gene. Thus, the N1 antibody is specific for bovine Ret-RGS1 but not for Figure 4. Ret-RGS1 associates with plasma membrane and synaptic vesicles in presynaptic terminals. A–C, Electron micromouse. Consequently, we restricted all ex- graphs of rod terminals. A, Diffused stain appears throughout the terminal; B, C, clusters of gold particles (denoted within the periments with this antibody to bovine. dashed pink ellipses) appear ⬃100 –200 nm from plasma membrane (outlined in blue). D, Western analysis of subfractions from To test whether Ret-RGS1 and G␣o1 are bovine retina with Ret-RGS1 antibody. Fifty micrograms of total proteins of each fraction were loaded on a 12% acrylamide gel. true interactors, we cotransfected HEK Ret-RGS1 is present in plasma membrane fraction and synaptic vesicles. Hom, Homogenate; crude synapt, crude synaptosomes. 293 cells with expression plasmids coding E, Fractions of synaptic vesicles on a sucrose gradient analyzed by Western blotting. Ret-RGS1 is co-present with synaptophysin in for G␣o1 and bovine Ret-RGS1 and immu- medium weight fractions; Na/K ATPase ␣-subunit (96 kDa; bottom band), a marker of a plasma membrane, settles in the heavier noprecipitated G␣o with monoclonal fractions. The 120 kDa (top band) is an aggregate of the ␣-subunit, which is commonly seen after sample boiling. As for Ret-RGS1, anti-G␣o. Under these conditions, a barely its migration was not perturbed by sample boiling. detectable amount of Ret-RGS1 coimmunoprecipitated with G␣o1, but when AlF4 ⫺ [G␣-GDP-AlF4 ⫺ complex stabilizes the interaction between G␣ and RGS proteins (Berman et al., 1996)] was added to the protein mix, the signal was stronger (Fig. 1 D, bottom, lane 2 vs 3). The level of immunoprecipitated G␣o was similar under both conditions (top, lane 2 vs 3). In the absence of immunoprecipitating antibody, the two proteins were undetectable (lanes 4 and 5) (four experiments). This experiment shows that G␣o1 and Ret-RGS1 can interact in mammalian cells. Next, we examined whether G␣o and Ret-RGS1 interact in the native tissue. Immunoprecipitating G␣o from bovine retina in the presence of AlF4 ⫺ also precipitated Ret-RGS1 (Fig. 1 E) (n ⫽ 2). When the immunoprecipitating antibody was omitted, Ret-RGS1 was undetectable. FiFigure 5. Ret-RGS1 is strongly bound to the plasma membrane. Retinal membranes from 25,000 ⫻ g pellets were washed with different treatments: hypotonic (hypo), hypertonic (hynally, in the converse experiment, immunoprecipitating Retper), alkaline (Na2CO3 ), octyl-glucoside (OG), urea, and NH2OH. For most treatments, Ret-RGS RGS1 with its antibody also precipitated G␣o (n ⫽ 1). This was recovered only in the pellet fraction (P); a small amount of Ret-RGS1 in supernatant (S) was experiment shows that G␣o1 and Ret-RGS1 interact in the recovered only after octyl-glucoside treatment. This suggests that Ret-RGS1 is tightly associnative tissue. ated with the plasma membrane. Expression pattern of Ret-RGS1 Immunostaining for Ret-RGS was present in many retinal cells: photoreceptor inner segments and cell bodies, outer synaptic layer, bipolar and amacrine somas, and inner synaptic layer (Fig. 2 A). Omitting the primary antibody or preabsorbing it with the N1 peptide eliminated staining. This general pattern agreed with in situ hybridization (Faurobert and Hurley, 1997). This suggests that the stain was specific. ON bipolar cells express Ret-RGS1 in their dendritic tips If Ret-RGS1 interacts with G␣o1 during the light response, it must be present in ON cells. To test this, we costained bovine retina for Ret-RGS1 and PKC, a known marker for rod bipolar cells, which comprise more than half of all ON cells. In the bipolar soma layer, all PKC-positive somas were positive for Ret-RGS1; however, certain somas were negative for PKC but positive for Ret-RGS1 Dhingra et al. • Localization and Function of Ret-RGS1 in Retina J. Neurosci., June 23, 2004 • 24(25):5684 –5693 • 5689 Ret RGS1 is present in photoreceptor and rod bipolar terminals and localized to vesicles and plasma membrane Certain structures in the outer plexiform layer stained strongly for Ret-RGS1 but not for G␣o. Horizontal cells, identified by the anti-calbindin antibody (Chun and Wassle, 1993), stained weakly for Ret-RGS1 in the soma but not in the dendrites (Fig. 3, top row); however, photoreceptor terminals, identified by synaptophysin (Brandstätter et al., 1996), were strongly positive for Ret-RGS1 (Fig. 3, middle). In the inner plexiform layer, staining for Ret-RGS1 was punctate throughout the layer. Examination of sections costained for PKC and Ret-RGS1 proved that some of these were located within rod bipolar terminals (Fig. 3, bottom). Although Ret-RGS1 and synaptophysin were both present in photoreceptor terminals, their fine expression patterns differed. Aggregates of Ret-RGS1 stain appeared to outline the synaptophysin stain (Fig. 3, middle row, arrowheads). Electron microscopy confirmed that Ret-RGS1 was present in photoreceptor terminals (Fig. 4 A) and showed that it often clustered at the plasma membrane (Fig. 4 B, C). Similar results were obtained by fractionation experiments. After centrifugation of bovine retina, RetRGS1 was absent from the cytosol fraction (corresponding to 400,000 ⫻ g soluble proteins) but present in the crude synaptosomal fraction. Subsequent fractionation of these synaptosomes in the presence of potassium iodide, an actin depolymerizing agent, revealed that most of the Ret-RGS1 protein was associated with the plasma membrane and the rest with synaptic vesicles (Fig. 4 D). When synaptic vesicles were fractionated on a sucrose gradient, Ret-RGS1 colocalized with synaptophysin (Fig. 4 E). Plasma membrane, marked by Na/K ATPase, settled lower in the gradient. This shows that the fractions that were strongly positive for synaptophysin and Ret-RGS1 were not contaminated by plasma membrane. Figure 6. Ret-RGS1 modulates acetylcholine-evoked response. A, Experimental protocol (current responses to 10 ␮M acetylcholine). Bars above the records denote application of high K ⫹ and acetylcholine solutions. Before and after application of high K ⫹, oocytes were perfused with ND96 solution. Basal and evoked currents are defined on the left. Gray and black records correspond to records without (⫺) or with (⫹) 1 ng Ret-RGS RNA; current scale: 4 ␮A for ⫺ and 8 ␮A for ⫹. Oocytes were injected with RNA for (in nanograms) m2R (0.24), G␣o1 (1), and GIRK1/2 channels (0.8 each). For all RNAs in this and the following figures, the amounts are given in nanograms. B, C, Same experiment as in A showing mean and SEM for one experiment. Ret-RGS1 (1) and RGS4 (1) reduced deactivation half-time (t50% deact) ( B) and increased AChevoked current ( C) significantly. Above each bar is the number of oocytes. For all figures, a single asterisk denotes a significant difference from control ( p ⬍ 0.05) and double asterisks denote a highly significant difference ( p ⬍ 0.001). D, Ret-RGS1 increased the evoked current when interacting with the wild-type G␣o [normalized average of 3 experiments; oocytes were injected with RNA (in nanograms) for m2R (0.2) or mGluR6 (1), G␣o (0 or 1), GIRK1/2 channels (0.8 each), and Ret-RGS1 (0 or 1)]. (Fig. 2 B). To identify these cells, we costained sections for G␣o (expressed in all ON cells) (Vardi, 1998) and Ret-RGS1. In the inner nuclear layer, all somas positive for G␣o also stained for Ret-RGS1 (Fig. 2C). A further requirement for Ret-RGS1 to regulate G␣o is that it be present in the dendritic tips of the ON cell, where mGluR6 detects glutamate and activates G␣o. Electron micrographs of tissue stained for Ret-RGS1 confirmed its presence in dendritic tips that invaginate the rod and the cone synaptic terminals. These invaginating dendrites are known to arise from ON cells (Fig. 2 D, E). No stain was found in the tips of OFF dendrites located at the base of cone terminals. We conclude that all ON cells (but no OFF cells) coexpress Ret-RGS1 in exactly the site where it could regulate the mGluR6 cascade. Ret-RGS1 is strongly bound to membranes The demonstration by immunostaining that Ret-RGS1 associates with membrane seemed consistent with its amino acid sequence. This contains a putative transmembrane domain plus a cysteine string that might target the protein to membrane. To further investigate this association, we treated a crude fraction of bovine retinal membrane with buffers of various ionic strengths (as described for RGS9) (Cowan et al., 1998). These mild treatments (hypotonic and hypertonic washes), that would remove weakly bound proteins, did not solubilize Ret-RGS1, nor did more stringent treatments (sodium carbonate buffer at pH 11.6 denaturation with urea). Treatments that release proteins linked to membranes by palmitoylation (hydroxylamine) also did not release Ret-RGS1. Only when the membranes themselves were solubilized (octyl-glucoside) was a small amount of Ret-RGS1 recovered in the supernatant (Fig. 5). The low solubility of Ret-RGS1 in detergent could be explained either by its precipitation during solubilization and its subsequent partitioning in the 400,000 ⫻ g pellet or by its association with the actin cytoskeleton; however, the colocalization of Ret-RGS1 with plasma membranes and synaptic vesicles in conditions that disrupt the actin cytoskeleton (Fig. 4 D) strongly supports the hypothesis that Ret-RGS1 genuinely associates with membranes. One cannot exclude, however, an additional binding to cytoskeletal elements. Ret-RGS1 accelerates termination of the mGluR6 cascade So far we had established that Ret-RGS1 localizes to dendritic tips of ON bipolar cells at the same site as mGluR6 and its obligatory transducer, Go1. Next we asked whether Ret-RGS1 can modulate this cascade and how. To test this, we coexpressed (in Xenopus 5690 • J. Neurosci., June 23, 2004 • 24(25):5684 –5693 Dhingra et al. • Localization and Function of Ret-RGS1 in Retina oocytes) Ret-RGS1, mGluR6, and a G␣o1 that was engineered to be insensitive to PTX. The endogenous G␣i/o was then blocked with PTX. To read out the kinetics of Go1, we also expressed the inwardly rectifying potassium channel, GIRK. This channel opens during binding G␤␥, which occurs when an agonist-bound receptor activates the trimeric G-protein to release G␤␥. The kinetics of activation and especially deactivation of the agonist-induced response depend crucially on GTPase activity of the G␣ that contributes G␤␥, and this activity is typically regulated by RGS. The general protocol was as follows. An oocyte was voltage clamped to ⫺80 mV, and then its perfusion solution (ND96) was switched from low K ⫹ (2 mM) to high K ⫹ (24 mM). This caused an inward cur- Figure 7. Ret-RGS1 accelerates deactivation of mGluR6 response in a concentration-dependent manner. Pertussis toxin (200 rent representing basal GIRK activity. ng) was injected ⬃16 hr before the recordings. A, PTX completely diminished responses carried by endogenous G␣i/o protein. B, Current evoked by 1 mM glutamate versus quantity of injected mGluR6 RNA. Responses first grow linearly and then saturate ⬃1 When the basal current reached plateau, ng mRNA. Oocytes were injected with RNAs (in nanograms) for mGluR6 (as indicated), pertussis toxin-insensitive G␣o1 C351V (1) agonists were added to the perfusate. This and GIRK1/2 channels (0.8 each). C, Ret-RGS1 (0.25 ng) accelerated deactivation. Currents were normalized to the same amplitude evoked a large inward current that turned to show effect of time course. D, E, Responses to glutamate with increasing amounts of Ret-RGS1 (data pooled from 2 experioff when the agonist was removed (Fig. ments). The parameters quantified are denoted above the bar graphs. Act, activation. Cells were injected with mRNAs for mGluR6 6 A). (2), pertussis toxin-insensitive G␣o1 C351V (1), GIRK1/2 channels (0.8 each), and the indicated amount of Ret-RGS1. For activation Because RGS proteins had been tested and deactivation half-time, data were simply averaged. For currents, data were normalized to responses at 0 Ret-RGS1 as in this system using a muscarinic receptor explained in Materials and Methods. Numbers of oocytes denoted in D apply also to E. (m2R) and the endogenous Gi/o (Doupnik et al., 1997; Saitoh et al., 1997), we tested this first. When m2R was activated by 10 ␮M acetylcholine followed by atropine wash (10 ␮M), both RGS4 and Ret-RGS1 reduced the deactivation halftime from ⬃10 to 3 sec. Interestingly, both RGS proteins at the expressed level (1 ng RNA) increased the evoked current, rather than reducing it (Fig. 6C). This also occurred when mGluR6 was substituted for m2R and activated by 1 mM glutamate (Fig. 6 D). Next we blocked the endogenous G␣i/o with PTX and tested Ret-RGS1 against the PTX-insensitive exogenous G␣o1 (Fig. 7A). Larger amounts of mGluR6 RNA gave larger glutamate-evoked currents (Fig. 7B). With 0.2–1 ng of mGluR6 RNA and 1 ng of Ret-RGS1 RNA, the evoked responses were diminished drastically, and deactivation half-time could not be measured. With 2 ng of mGluR6 RNA and 1 ng of Ret-RGS1 RNA, the responses were reduced to ⬃70% (n ⫽ 8), and the deactivation half-time fell from 18.6 ⫾ 1.3 to 10.2 ⫾ 2.4 sec (n ⫽ 10). Activation time was unaffected (data not shown). Then, using a constant amount Figure 8. Ret-RGS1 is more potent than RGS4 and RGS7. A, B, Current responses ( A) and of mGluR6 RNA (2 ng), we tested different amounts of Ret-RGS1 deactivation half-time ( B) in the absence or presence of RGS4, RGS7, or Ret-RGS1. Oocytes were (0.2–2 ng mRNA). Ret-RGS1 reduced deactivation half-time in a injected with RNAs (in nanograms) for mGluR6 (1), pertussis toxin-insensitive G␣o1 C351V (1), concentration-dependent manner up to approximately twofold GIRK1/2 channels (0.8 each), and Ret-RGS1 (1), RGS4 (1), or RGS7 (1). (Fig. 7C,D). This effect was obtained with two different PTXinsensitive mutants of G␣o1 (G␣o1 C351V and G␣o1 C351I; five experiments). Ret-RGS1 also mildly reduced activation time and RGS1, which is normally associated with this cascade, was the basal current (Fig. 7E). With respect to evoked current, lower most effective regulator. quantities of Ret-RGS1 (0.5–1 ng RNA) did not change the evoked current, but higher quantities (2 ng) decreased it. When Discussion the ratio of evoked to basal currents (a measure of signal-toWe show here that Ret-RGS1 is expressed by ON bipolar cells at noise) was calculated, certain amounts of Ret-RGS1 (0.5 ng) acthe tips of their dendrites, where the mGluR63 G␣o1 cascade tually increased this ratio. We also compared several RGS procloses cation channels in the dark. We are confident that immuteins (at 1 ng RNA) coexpressed with mGluR6 and G␣o1 and nostaining for Ret-RGS1 is specific because the antibody against found effects on evoked current and deactivation half-time as Ret-RGS1 (1) stains HEK cells transfected with bovine Ret-RGS1 follows: Ret-RGS1⬎RGS7⬎RGS4 (Fig. 8). It is possible that a expression plasmid, (2) gives a prominent band at the expected constant amount of RNA did not result in a constant amount or molecular weight in Western blots, (3) staining in Western blots optimal folding of each RGS protein. Nevertheless, this experiand retinal tissue is completely blocked by the immunogen, and ment suggests that, at least under the conditions tested, Ret- Dhingra et al. • Localization and Function of Ret-RGS1 in Retina (4) staining perfectly fits the pattern observed with in situ hybridization (Faurobert and Hurley, 1997). We show further that Ret-RGS1 interacts with G␣o1 in yeast, cotransfected HEK cells, and native retina. The two-hybrid assay that repeatedly identified Ret-RGS1 never identified the RGS9 of the photoreceptor, although ⬃70% of retinal messages originate from photoreceptors. This suggests a preference of G␣o1 for the particular RGS with which it colocalizes in situ. Finally, we show in an expression system that Ret-RGS1 can serve as a GAP for G␣o1 to accelerate response termination. In the expressed muscarinic system, Ret-RGS1 was as effective as RGS4 in accelerating the deactivation of the m2R response; however, in the mGluR6 system, Ret-RGS1 was more potent than RGS4. Furthermore, although Ret-RGS1 can be a GAP for transducin (G␣t; the phototransduction G-protein), the rate constant is ⬃50-fold slower than with the natural GAP of G␣t, RGS9 (Grunwald et al., 1986; Lerea et al., 1986,1989; Faurobert and Hurley, 1997; He et al., 1998; Chen et al., 2000; Lyubarsky et al., 2001). Thus, the naturally colocalized pairs, G␣t-RGS9 and G␣o1-Ret-RGS1, interact more strongly than their alternative combinations. Because an RGS curtails the activity of a G-protein (Berman et al., 1996; Ross and Wilkie, 2000; Chidiac and Roy, 2003), it should reduce the amplitude of agonist-evoked current. Indeed, we observed this in many experiments, but in several, the evoked current was either constant or increased with certain levels of Ret-RGS1. This apparent paradox has been reported for several other RGS proteins. One possibility is that Ret-RGS also reduced the basal current (see below). This might allow more channels to be activated by the agonist and partially explain the nonreduction of evoked current; however, this could not be the only reason, because the total current increased when the evoked current increased, indicating another effect. Note that the effect on the evoked current depends on the precise stoichiometry of receptor, G␣, and RGS. For example, under the conditions of Figure 8, where injected mGluR6 RNA was 1 ng, Ret-RGS1 (1 ng) dramatically reduced the evoked current, whereas under the conditions of Figure 7, D and E, where injected mGluR6 RNA was 2 ng, evoked current was larger and Ret-RGS1 hardly affected the evoked current. The increase in evoked current is thought to arise from a direct interaction of the RGS protein with the receptor or the channel (Doupnik et al., 1997; Saitoh et al., 1997; Jeong and Ikeda, 2001; Keren-Raifman et al., 2001). In our experiments, whether Ret-RGS1 increased or decreased the evoked current also depended on modification of the G-protein: Ret-RGS1 (1 ng RNA) increased evoked current with wild-type G␣o1 (3 of 3 experiments) but decreased the evoked current with PTXinsensitive G␣o (six of seven experiments). An additional effect of Ret-RGS was to reduce the basal current; this is expected because its GAP activity leaves more G␣-GDP, which binds G␤␥, and thus reduces the free G␤␥ available to open the GIRK channel. Because the basal current sets the noise, when Ret-RGS decreases the basal current and increases the evoked current, it increases the signal-to-noise ratio. J. Neurosci., June 23, 2004 • 24(25):5684 –5693 • 5691 2002); however, the effect of Ret-RGS1 and the dependence on its amount should be comparable. Ret-RGS1 is present in both the slow rod bipolar cell and the faster cone bipolar cell; we expect the cone bipolar to express more Ret-RGS1 relative to its G␣o1. This seems plausible because for photoreceptors, where the rod is slow and the cone is faster, the cone expresses more RGS9 than the rod (Cowan et al., 1998; Zhang et al., 2003). Our current assays, however, cannot quantify these ratios precisely enough to test this. Under certain conditions, Ret-RGS1 increases the agonist response. It is therefore tempting to speculate that in rod bipolar cells Ret-RGS1 should increase glutamate response. The rod bipolar cell carries the tiny single photon signal from rods to the AII amacrine cell via a high gain system (Ashmore and Falk, 1979; Falk, 1988; Shiells and Falk, 1994). During dark periods, glutamate is released continuously by the rod and hyperpolarizes the rod bipolar cell. If Ret-RGS1 increased the response to glutamate, it would further hyperpolarize the cell and so increase the driving force of the conducting ions. Additionally, a cell maintained at a more hyperpolarized state could give more graded levels of responses at mesopic light levels (twilight). Thus, Ret-RGS1 can increase the single photon response at scotopic luminances and the dynamic range of the rod bipolar cell at mesopic luminances. In short, the location, interaction, and apparent function of RetRGS1 are all suited to terminate the mGluR63 G␣o1 cascade and allow rapid opening of cation channels to light onset. One natural way to test these hypotheses would be to see whether a knock-out slows the ON response. Unfortunately, this experiment is not straightforward because Ret-RGS1 is also expressed in photoreceptor and ON bipolar axon terminals. Possible functions of Ret-RGS1 in synaptic terminals Ret-RGS1 is abundant in photoreceptor and rod bipolar synaptic terminals. There, Ret-RGS1 tightly associates with the plasma membrane and also with the synaptic vesicles. Ret-RGS1 shares the property of membrane association with several RGS proteins (RGS9, GAIP, and RGSZ1), but these attach by different mechanisms. Thus, RGS9 attaches via its DEP domain, and both RGSZ1 and GAIP attach via a highly palmitoylated cysteine string motif (De Vries et al., 1996, 1998; Wang et al., 1998; Martemyanov et al., 2003). For Ret-RGS1, the membrane association resists all treatments short of dissolving the membrane. This might be because Ret-RGS1 contains both a cysteine string domain and a putative transmembrane domain that might connect it to the membrane (Faurobert and Hurley, 1997). With respect to vesicular localization, GAIP is located on clathrin-coated vesicles near the plasma membrane and transGolgi vesicles (De Vries et al., 1998, 2000). This suggests a role in vesicular trafficking. This was also suggested for RGS-PX1, which serves G␣s and is localized in early endosomes where it contributes to vesicular trafficking (Zheng et al., 2001). By analogy, RetRGS1 in synaptic terminals might also serve in vesicle trafficking. References Implication of the function of Ret-RGS1 in ON bipolar cells Light-evoked current in ON bipolar cells is caused by a decrease in glutamate concentration. Therefore, Ret-RGS1 should accelerate the rising phase of the light response (rather than its falling phase). The precise time of activation and deactivation in the Xenopus oocyte is obviously different from the light responses of the ON bipolar cell because of huge differences in rates of solution exchange and other intrinsic oocyte factors (Zhang et al., Ashmore JF, Falk G (1979) Transmission of visual signals to bipolar cells near absolute threshold. Vision Res 19:419 – 423. Barker SA, Wang J, Sierra DA, Ross EM (2001) RGSZ1 and Ret RGS: two of several splice variants from the gene RGS20. Genomics 78:223–229. Berghard A, Buck LB (1996) Sensory transduction in vomeronasal neurons: evidence for G␣o, G␣i2, and adenylyl cyclase II as major components of a pheromone signaling cascade. J Neurosci 16:909 –918. Berman DM, Kozasa T, Gilman AG (1996) The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J Biol Chem 271:27209 –27212. 5692 • J. Neurosci., June 23, 2004 • 24(25):5684 –5693 Brandstätter JH, Löhrke S, Morgans CW, Wässle H (1996) Distributions of two homologous synaptic vesicle proteins, synaptoporin and synaptophysin, in the mammalian retina. J Comp Neurol 370:1–10. Chen C-K, Burns ME, He W, Wensel TG, Baylor DA, Simon MI (2000) Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9 –1. Nature 403:557–560. Chidiac P, Roy AA (2003) Activity, regulation, and intracellular localization of RGS proteins. Receptors Channels 9:135–147. Chun MH, Wassle H (1993) Some horizontal cells of the bovine retina receive input synapses in the inner plexiform layer. Cell Tissue Res 272:447– 457. Cohen E, Sterling P (1990) Convergence and divergence of cones onto bipolar cells in the central area of cat retina. Philos Trans R Soc Lond B Biol Sci 330:323–328. Cowan CW, Fariss RN, Sokal I, Palczewski K, Wensel TG (1998) High expression levels in cones of RGS9, the predominant GTPase. Proc Natl Acad Sci USA 95:5351–5356. Dascal N (2000) Voltage clamp recordings from Xenopus oocytes. In: Current protocols in neuroscience, pp 6.12.1– 6.12.20. New York: Wiley. Dascal N, Lotan I (1992) Expression of exogenous ion channels and neurotransmitter receptors in RNA injected Xenopus oocytes. In: Methods in molecular biology (Longstaff A, Revest P, eds), pp 205–225. Totowa, NJ: Humana. de la Villa P, Kurahashi T, Kaneko A (1995) L-Glutamate-induced responses and cGMP-activated channels in three subtypes of retinal bipolar cells dissociated from the cat. J Neurosci 15:3571–3582. De Vries L, Elenko E, Hubler L, Jones TL, Farquhar MG (1996) GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G alpha i subunits. Proc Natl Acad Sci USA 93:15203–15208. De Vries L, Elenko E, McCaffery JM, Fischer T, Hubler L, McQuistan T, Watson N, Farquhar MG (1998) RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles. Mol Biol Cell 9:1123–1134. De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG (2000) The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 40:235–271. Dhingra A, Lyubarsky A, Jiang M, Pugh Jr EN, Birnbaumer L, Sterling P, Vardi N (2000) The light response of ON bipolar neurons requires G␣o. J Neurosci 20:9053–9058. Dhingra A, Jiang M, Wang T-L, Lyubarsky A, Savchenko A, Bar-Yehuda T, Sterling P, Birnbaumer L, Vardi N (2002) Light response of retinal ON bipolar cells requires a specific splice variant of G␣o. J Neurosci 22:4878 – 4884. Dolphin AC (1998) Mechanisms of modulation of voltage-dependent calcium channels by G proteins. J Physiol (Lond) 506:3–11. Doupnik CA, Davidson N, Lester HA, Kofuji P (1997) RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K⫹ channels. Proc Natl Acad Sci USA 94:10461–10466. Euler T, Schneider H, Wässle H (1996) Glutamate responses of bipolar cells in a slice preparation of the rat retina. J Neurosci 16:2934 –2944. Falk G (1988) Signal transmission from rods to bipolar and horizontal cells: a synthesis. Prog Retin Res 8:255–279. Faurobert E, Hurley JB (1997) The core domain of a new retina specific RGS protein stimulates the GTPase. Proc Natl Acad Sci USA 94:2945–2950. Faurobert E, Scotti A, Hurley JB, Chabre M (1999) RET-RGS, a retinaspecific regulator of G-protein signaling, is located in synaptic regions of the rat retina. Neurosci Lett 269:41– 44. Gomez MP, Nasi E (2000) Light transduction in invertebrate hyperpolarizing photoreceptors: possible involvement of a Go-regulated guanylate cyclase. J Neurosci 20:5254 –5263. Greif GJ, Sodickson DL, Bean BP, Neer EJ, Mende U (2000) Altered regulation of potassium and calcium channels by GABA(B) and adenosine receptors in hippocampal neurons from mice lacking Galpha(o). J Neurophysiol 83:1010 –1018. Grunwald GB, Gierschik P, Nirenberg M, Spiegel A (1986) Detection of alpha-transducin in retinal rods but not cones. Science 231:856 – 859. He W, Cowan CW, Wensel TG (1998) RGS9, a GTPase accelerator for phototransduction Galpha(i)-coupled muscarinic m2 receptors in Xenopus oocytes: the role of receptor precoupling in RGS modulation. Neuron 20:95–102. Herms J, Tings T, Gall S, Madlung A, Giese A, Siebert H, Schurmann P, Windl Dhingra et al. • Localization and Function of Ret-RGS1 in Retina O, Brose N, Kretzschmar H (1999) Evidence of presynaptic location and function of the prion protein. J Neurosci 19:8866 – 8875. Huff RM, Axton JM, Neer EJ (1985) Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex. J Biol Chem 260:10864 –10871. Huttner WB, Schiebler W, Greengard P, de Camilli P (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J Cell Biol 96:1374 –1388. Jeong SW, Ikeda SR (2001) Differential regulation of G protein-gated inwardly rectifying K(⫹) channel kinetics by distinct domains of RGS8. J Physiol (Lond) 535:335–347. Johnson MA, Vardi N (1998) Regional differences in GABA and GAD immunoreactivity in rabbit horizontal cells. Vis Neurosci 15:743–753. Jordan JD, Carey KD, Stork PJ, Iyengar R (1999) Modulation of Rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein. J Biol Chem 274:21507–21510. Keren-Raifman T, Bera AK, Zveig D, Peleg S, Witherow DS, Slepak VZ, Dascal N (2001) Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K(⫹) channel and control regulation of RGS7 by G beta 5. FEBS Lett 492:20 –28. Kleuss C, Hescheler J, Ewel C, Rosenthal W, Schultz G, Wittig B (1991) Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature 353:43– 48. Kojima D, Terakita A, Ishikawa T, Tsukahara Y, Maeda A, Shichida Y (1997) A novel Go-mediated phototransduction cascade in scallop visual cells. J Biol Chem 272:22979 –22982. Leaney JL, Tinker A (2000) The role of members of the pertussis toxinsensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel. Proc Natl Acad Sci USA 97:5651–5656. Lerea CL, Somers DE, Hurley JB, Klock IB, Bunt-Milam AH (1986) Identification of specific transducin alpha subunits in retinal rod and cone photoreceptors. Science 234:77– 80. Lerea CL, Bunt-Milam AH, Hurley JB (1989) Alpha transducin is present in blue-, green-, and red-sensitive cone photoreceptors in the human retina. Neuron 3:367–376. Li Y, Mende U, Lewis C, Neer EJ (2000) Maintenance of cellular levels of G-proteins: different efficiencies of alpha s and alpha o synthesis in GH3 cells. Biochem J 318:1071–1077. Luo Y, Denker BM (1999) Interaction of heterotrimeric G protein G-alphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor. J Biol Chem 274:10685–10688. Lyubarsky AL, Chen C-K, Naarendorp F, Zhang X, Wensel T, Simon MI, Pugh Jr EN (2001) RGS9 –1 is required for normal inactivation of mouse cone phototransduction. Mol Vis 7:71–78. Martemyanov KA, Lishko PV, Calero N, Keresztes G, Sokolov M, Strissel KJ, Leskov IB, Hopp JA, Kolesnikov AV, Chen CK, Lem J, Heller S, Burns ME, Arshavsky VY (2003) The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo. J Neurosci 23:10175–10181. Martin PR, Grünert U (1992) Spatial density and immunoreactivity of bipolar cells in the Macaque monkey retina. J Comp Neurol 323:269 –287. Mendel JE, Korswagen HC, Liu KS, Hajdu-Cronin YM, Simon MI, Plasterk RH, Sternberg PW (1995) Participation of the protein Go in multiple aspects of behavior in C. elegans. Science 267:1652–1655. Natochin M, Gasimov KG, Artemyev NO (2001) Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs. Biochemistry 40:5322–5328. Nawy S (1999) The metabotropic receptor mGluR6 may signal through Go, but not phosphodiesterase, in retinal bipolar cells. J Neurosci 19:2938 –2944. Nawy S, Jahr CE (1990) Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells. Nature 346:269 –271. Nawy S, Jahr CE (1991) cGMP-gated conductance in retinal bipolar cells is suppressed by the photoreceptor transmitter. Neuron 7:677– 683. Nomura A, Shigemoto R, Nakamura Y, Okamoto N, Mizuno N, Nakanishi S (1994) Developmentally-regulated postsynaptic localization of a metabotropic glutamate-receptor in rat rod bipolar cells. Cell 77:361–369. Ross EM, Wilkie TM (2000) GTPase-activating proteins for heterotrimeric Dhingra et al. • Localization and Function of Ret-RGS1 in Retina G proteins: regulators of G protein signaling (RGS) and RGS like proteins. Annu Rev Biochem 69:795– 827. Saitoh O, Kubo Y, Miyatani Y, Asano T, Nakata H (1997) RGS8 accelerates G-protein-mediated modulation of K ⫹ currents. Nature 390:525–529. Sharon D, Vorobiov D, Dascal N (1997) Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K ⫹ channel, GIRK, in Xenopus oocytes. J Gen Physiol 109:477– 490. Shiells RA, Falk G (1990) Glutamate receptors of rod bipolar cells are linked to a cyclic GMP cascade via a G-protein. Proc R Soc Lond B Biol Sci 242:91–94. Shiells RA, Falk G (1992) Properties of the cGMP-activated channel of retinal on-bipolar cells. Proc R Soc Lond B Biol Sci 247:21–25. Shiells RA, Falk G (1994) Responses of rod bipolar cells isolated from dogfish retinal slices to concentration-jumps of glutamate. Vis Neurosci 11:1175–1183. Sterling P, Freed MA, Smith RG (1988) Architecture of the rod and cone circuits to the On-␤ ganglion cell. J Neurosci 8:623– 642. Sternweis PC, Robishaw JD (1984) Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem 259:13806 –13813. Strettoi E, Masland R (1995) The organization of the inner nuclear layer of the rabbit retina. J Neurosci 15:875– 888. Tall GG, Krumins AM, Gilman AG (2003) Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor. J Biol Chem 278:8356 – 8362. Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ, Fishman MC (1997) G alphao is necessary for muscarinic regulation of Ca 2⫹ channels in mouse heart. Proc Natl Acad Sci USA 94:1727–1732. Vardi N (1998) Alpha subunit of Go localizes in the dendritic tips of ON bipolar cells. J Comp Neurol 395:43–52. J. Neurosci., June 23, 2004 • 24(25):5684 –5693 • 5693 Vardi N, Morigiwa K (1997) ON cone bipolar cells in rat express the metabotropic receptor mGluR6. Vis Neurosci 14:789 –794. Vardi N, Matesic DF, Manning DR, Liebman PA, Sterling P (1993) Identification of a G-protein in depolarizing rod bipolar cells. Vis Neurosci 10:473– 478. Vardi N, Duvoisin RM, Wu G, Sterling P (2000) Localization of mGluR6 to dendrites of ON bipolar cells in primate retina. J Comp Neurol 423:402– 412. Vorobiov D, Bera AK, Keren-Raifman T, Barzilai R, Dascal N (2000) Coupling of the muscarinic m2 receptor to G protein-activated K(⫹)channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha(z) eliminates catalytic (collision) coupling. J Biol Chem 275:4166 – 4170. Wang J, Ducret A, Tu Y, Kozasa T, Aebersold R, Ross EM (1998) RGSZ1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Galphaz phosphorylation, and relationship to a Gz GTPaseactivating protein subfamily. J Biol Chem 273:26014 –26025. Yakubovich D, Pastushenko V, Bitler A, Dessauer CW, Dascal N (2000) Slow modal gating of single G protein-activated K ⫹ channels expressed in Xenopus oocytes. J Physiol (Lond) 524:737–755. Zhang Q, Pacheco MA, Doupnik CA (2002) Gating properties of GIRK channels activated by G alpha o– and G alpha i– coupled muscarinic m2 receptors in Xenopus oocytes: the role of receptor precoupling in RGS modulation. J Physiol (Lond) 545:355–373. Zhang X, Wensel TG, Kraft TW (2003) GTPase regulators and photoresponses in cones of the Eastern chipmunk. J Neurosci 23:1287–1297. Zheng B, Ma Y-C, Ostrom RS, Lavoie C, Gill GN, Insel PA, Huang X-Y, Farquhar MG (2001) RGS-PX1, a GAP for Galphas and sorting nexin in vesicular trafficking. Science 294:1939 –1942.