Title: The Glutamic Acid-rich Protein-2 (GARP2) Is a High Affinity Rod Photoreceptor Phosphodiesterase (PDE6)-binding Protein That Modulates Its Catalytic Properties
Abstract: The glutamic acid-rich protein-2 (GARP2) is a splice variant of the β-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor “dark noise” and allowing these sensory cells to operate at the single photon detection limit. The glutamic acid-rich protein-2 (GARP2) is a splice variant of the β-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor “dark noise” and allowing these sensory cells to operate at the single photon detection limit. The visual transduction pathway in vertebrate photoreceptors is remarkable in many respects, including single photon detection capability (in rod photoreceptors), photoresponse kinetics on the millisecond time scale, and the ability to adapt to background illumination levels ranging from very dim illuminance levels (scotopic vision in rods) to bright sunlight (photopic vision in cones) (1Rodieck R.W. The First Steps in Seeing. Sinauer Associates, Sunderland, MA1998Google Scholar). The very first steps in vision occur in the photoreceptor outer segment when photo-isomerized rhodopsin activates the heterotrimeric G-protein transducin, which proceeds to bind to and displace the inhibitory γ-subunit (Pγ) 2The abbreviations used are: Pγ, inhibitory 10-kDa γ-subunit of PDE6; GARP, glutamic acid-rich protein; PDE6, photoreceptor phosphodiesterase; PrBP/δ, 17-kDa prenyl-binding protein; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ROS, rod outer segment; HIC, hydrophobic interaction chromatography; HPLC, high pressure liquid chromatography. of the photoreceptor phosphodiesterase (PDE6). Activated PDE6 rapidly lowers the cGMP concentration, resulting in closure of cGMP-gated channels in the plasma membrane and cell hyperpolarization (2Burns M.E. Baylor D.A. Annu. Rev. Neurosci. 2001; 24: 779-805Crossref PubMed Scopus (334) Google Scholar, 3Arshavsky V.Y. Lamb T.D. Pugh Jr., E.N. Annu. Rev. Physiol. 2002; 64: 153-187Crossref PubMed Scopus (506) Google Scholar, 4Zhang X. Cote R.H. Front. Biosci. 2005; 10: 1191-1204Crossref PubMed Scopus (69) Google Scholar). Several feedback mechanisms operate to actively terminate the photoresponse and restore the dark-adapted state, of which regulation of the lifetime of activated transducin is considered rate-limiting (2Burns M.E. Baylor D.A. Annu. Rev. Neurosci. 2001; 24: 779-805Crossref PubMed Scopus (334) Google Scholar, 3Arshavsky V.Y. Lamb T.D. Pugh Jr., E.N. Annu. Rev. Physiol. 2002; 64: 153-187Crossref PubMed Scopus (506) Google Scholar). Rebinding of Pγ to the PDE6 catalytic subunits following transducin deactivation returns PDE6 to its nonactivated state and allows cGMP levels to return to their dark-adapted levels. Electrophysiological evidence supports the hypothesis that factors in addition to transducin deactivation are involved in regulating the life-time of light-activated PDE6 during light adaptation of rod photoreceptors (5Calvert P.D. Govardovskii V.I. Arshavsky V.Y. Makino C.L. J. Gen. Physiol. 2002; 119: 129-146Crossref PubMed Scopus (48) Google Scholar, 6Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Neurosci. 2003; 23: 6965-6971Crossref PubMed Google Scholar). Several potential feedback mechanisms for modulating activated PDE6 have been proposed (7Erickson M.A. Robinson P. Lisman J. Science. 1992; 257: 1255-1258Crossref PubMed Scopus (30) Google Scholar, 8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 9Hayashi F. Matsuura I. Kachi S. Maeda T. Yamamoto M. Fujii Y. Liu H. Yamazaki M. Usukura J. Yamazaki A. J. Biol. Chem. 2000; 275: 32958-32965Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) but have not been explored in sufficient detail to validate their relevance to the phototransduction pathway. The catalytic activity of PDE6 in its dark-adapted state also must be tightly controlled to prevent any spontaneous activation of PDE6 that would consume metabolic energy unnecessarily and impair the ability of rod cells to reliably detect very dim flashes of light. Physiological measurements of “dark noise” reveal a component that represents spontaneous activation of PDE6 and which is much greater in magnitude in cones than in rods (10Rieke F. Baylor D.A. Biophys. J. 1996; 71: 2553-2572Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 11Rieke F. Baylor D.A. Neuron. 2000; 26: 181-186Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 12Holcman D. Korenbrot J.I. The J. Gen. Physiol. 2005; 125: 641-660Crossref PubMed Scopus (36) Google Scholar). Subtle differences in the highly homologous rod and cone isoforms of PDE6 might account for the different dark noise in rods and cones, although this is not evident from biochemical comparisons of purified rod and cone PDE6 (13Baehr W. Devlin M.J. Applebury M.L. J. Biol. Chem. 1979; 254: 11669-11677Abstract Full Text PDF PubMed Google Scholar, 14Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar, 15Mou H. Grazio H.J. Cook T.A. Beavo J.A. Cote R.H. J. Biol. Chem. 1999; 274: 18813-18820Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 16Huang D. Hinds T.R. Martinez S.E. Doneanu C. Beavo J.A. J. Biol. Chem. 2004; 279: 48143-48151Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). An alternative possibility is that a rod- or cone-specific PDE6-binding protein suppresses the spontaneous activation of PDE6 by enhancing the affinity of Pγ at the PDE6 catalytic site. One candidate protein that might serve to regulate PDE6 in both its nonactivated and activated states is the glutamic acid-rich protein-2 (GARP2), a protein that exists in rod outer segments but is absent in cones (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). GARP2 is a product of alternative splicing of the β-subunit of the rod cGMP-gated ion channel (CNGB1) and contains a unique 8-amino-acid C-terminal extension (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This 32-kDa protein is unusual in that it has a high content of proline and glutamate residues (17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 18Sugimoto Y. Yatsunami K. Tsujimoto M. Khorana H.G. Ichikawa A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3116-3119Crossref PubMed Scopus (45) Google Scholar, 19Korschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (211) Google Scholar). The functions served by GARP2 in rod outer segments are unknown. Potential binding partners for GARP2 include proteins involved in phototransduction and disk membrane structural integrity (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 20Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), but the physiological significance of these interactions is unclear. In one previous study, it was reported that the addition of GARP2 to preparations of PDE6 reversed its activation by transducin, whereas GARP2 had no effect on the nonactivated PDE6 holoenzyme or on the catalytic dimer of PDE6 lacking bound Pγ (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar). It was proposed that GARP2 down-regulation of PDE6 activation in the vicinity of the plasma membrane might conserve metabolic energy during daylight when rod function is saturated. In this paper, we have examined the interaction of GARP2 with PDE6 and characterized the effect of GARP2 on PDE6 function. We show that GARP2 binds PDE6 with high affinity and co-purifies with the enzyme through several stages of purification. We have been unable to confirm the previously reported inhibitory effect of GARP2 on transducin-activated PDE6 (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar). Instead, we observed that purified, native GARP2 has a strong effect in suppressing the basal activity of PDE6 in its nonactivated state. The implications of GARP2 modulation of basal PDE6 activity in dark-adapted rods are discussed. Materials—Bovine retinas were purchased from W. L. Lawson, Inc. Chromatography supplies were purchased from G. E. Healthcare and Pierce. Supplies for immunoblotting were purchased from Schleicher & Schuell, Pierce, and Bio-Rad. Chemicals were obtained from Sigma. The bovine recombinant GST-PrBP/δ fusion protein was a kind gift of Dr. Joe Beavo (University of Washington). Rabbit polyclonal anti-GARP2 antibody to the unique C-terminal sequence of GARP2 (17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) was obtained from Affinity Bioreagents (catalog number PA1-728). Chicken and rabbit polyclonal anti-GARP antibodies to bovine sequences common to GARP1, GARP2, and the rod β-subunit CNGB1 were kind gifts of Dr. Steven Pittler (University of Alabama at Birmingham) and Dr. Benjamin Kaupp (Institut für Biologische Informationsverarbeitung, Jülich, Germany). Affinity-purified anti-peptide rabbit polyclonal antibodies directed to the PDE6 GAFb domain (termed NC) and to the C terminus of the Pγ-subunit of PDE6 (CT-9710) were produced in our laboratory. The ROS1 monoclonal antibody used for immunoprecipitations (21Hurwitz R.L. Bunt-Milam A.H. Beavo J.A. J. Biol. Chem. 1984; 259: 8612-8618Abstract Full Text PDF PubMed Google Scholar) was a gift of Dr. Richard Hurwitz (Baylor College of Medicine). ROS Membrane Isolation and Purification—ROS membranes from bovine retina were prepared as described previously (22Pentia D.C. Hosier S. Collupy R.A. Valeriani B.A. Cote R.H. Methods Mol. Biol. 2005; 307: 125-140PubMed Google Scholar). Briefly, ROSs were isolated from frozen bovine retinas on a discontinuous sucrose gradient. ROS membranes were homogenized in an isotonic buffer (10 mm Tris, pH 7.5, 60 mm KCl, 40 mm NaCl, 2 mm MgCl2, 1 mm dithiothreitol, 0.3 mm phenylmethylsulfonyl fluoride) using a glass, handheld homogenizer. The soluble proteins were separated from membranes by centrifugation. Native GARP2 Purification—GARP2 was isolated from ROS membranes and purified to homogeneity as follows. First, ROS membranes were homogenized in a hypotonic buffer (5 mm Tris, pH 7.5, 0.2 mm MgCl2, 1 mm dithiothreitol). The soluble proteins were separated from membranes by centrifugation at 100,000 × g for 45 min. The hypotonic extraction was repeated three times. The pooled hypotonic extract was then adjusted to 500 mm ammonium sulfate and applied to a 15-ml butyl-Sepharose column. The column was washed of unbound proteins using two column volumes of 500 mm ammonium sulfate in 5 mm Tris, pH 7.5, and bound proteins were eluted by a step gradient (400 mm ammonium sulfate, 150 mm ammonium sulfate, and no ammonium sulfate in a solution containing 5 mm Tris, pH 7.5, 1 mm dithiothreitol). The GARP2-containing fractions were pooled, adjusted to 500 mm ammonium sulfate, and rechromatographed on butyl-Sepharose. To concentrate and further purify GARP2 from other contaminating proteins, anion exchange chromatography on Mono Q was used exactly as described for PDE6 purification. In some instances, the GARP2-containing fractions from the first butyl-Sepharose column were chromatographed on a Mono Q column prior to a final purification using a reversed phase HPLC column (Vydac 214TP54) with a gradient of 0–100% acetonitrile containing 0.1% trifluoroacetic acid. Under these conditions, GARP2 eluted at 48% acetonitrile, Pγ was found at 43% acetonitrile, and PDE6 catalytic subunits were undetectable by immunoblot analysis. HPLC-purified GARP2 behaved identically to butyl-Sepharose-purified GARP2 in its effects on PDE6 catalysis. PDE6 Purification—Purified PDE6 was prepared as described elsewhere (22Pentia D.C. Hosier S. Collupy R.A. Valeriani B.A. Cote R.H. Methods Mol. Biol. 2005; 307: 125-140PubMed Google Scholar). Briefly, a hypotonic extract of purified ROS membranes was loaded onto a Mono Q column. The proteins were eluted using a linear gradient from 100 mm NaCl to 1 m NaCl in 5 mm Tris, pH 7.5. The PDE6 peak was collected, concentrated, and further purified on a Superdex 200 gel filtration column using the following buffer: 5 mm Tris, pH 7.5, 300 mm NaCl, 1 mm dithiothreitol, and 3 mm phenylmethylsulfonyl fluoride. The gel filtration column was calibrated using the following: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (43 kDa), ovalbumin (29 kDa), blue dextran for the void volume, and adenosine triphosphate for the included volume. Immunoprecipitation of PDE6 with the Monoclonal Antibody ROS1—The ROS1 antibody to PDE6 (21Hurwitz R.L. Bunt-Milam A.H. Beavo J.A. J. Biol. Chem. 1984; 259: 8612-8618Abstract Full Text PDF PubMed Google Scholar) coupled to Sulfolink beads (Pierce) was used for immunoprecipitation of PDE6 and its binding partners. Hypotonic extracts from bovine ROS or Mono Q-purified PDE6 (containing 5–10 pmol of PDE6) were incubated for 2 h at 4°C with 20 μl of prewashed ROS1 beads in a total volume of 100 μl. The samples were centrifuged to separate bound from unbound proteins, and the beads were washed extensively before the proteins were eluted in Laemmli sample buffer. As a control for nonspecific binding of GARP2 to the beads, purified GARP2 was also tested with the ROS1-Sulfolink beads. Portions of the starting material, bound proteins, and unbound proteins were subjected to SDS-PAGE followed by Western blotting for PDE6 (NC antibody) and GARP (chicken anti-GARP antibody). PrBP/δ Expression and Purification—Recombinant bovine PrBP/δ was expressed in the Escherichia coli strain BL21 (23Norton A.W. Hosier S. Terew J.M. Li N. Dhingra A. Vardi N. Baehr W. Cote R.H. J. Biol. Chem. 2005; 280: 1248-1256Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Protein expression was induced by the addition of 1 mm isopropyl-β-d-thiogalactopyranoside to log phase cultures. Bacterial cells were incubated for 1 h at 37 °C, lysed by sonication, and soluble proteins were recovered following centrifugation. GST-PrBP/δ was purified on a glutathione-agarose column. GST-PrBP/δ concentration was determined by a colorimetric protein assay (24Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Protein Quantification—The amount of rhodopsin in dark-adapted ROS membranes was determined by difference spectroscopy (25Bownds D. Gordon-Walker A. Gaide Huguenin A.C. Robinson W. J. Gen. Physiol. 1971; 58: 225-237Crossref PubMed Scopus (137) Google Scholar). The PDE6 concentration was routinely determined by measurements of trypsin-activated PDE6 maximum activity (Vmax) (26Cote R.H. Methods Enzymol. 2000; 315: 646-672Crossref PubMed Google Scholar) and knowledge of the turnover number (kcat = 5600 cGMP/s/PDE6; [PDE6] = Vmax/kcat) (27Mou H. Cote R.H. J. Biol. Chem. 2001; 276: 27527-27534Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Independent determinations of the ratio of rhodopsin to PDE6 in purified bovine ROS gave a value of 310 ± 20 rhodopsins per PDE6 (n = 5), very similar to the values for amphibian ROS of 270 (28Dumke C.L. Arshavsky V.Y. Calvert P.D. Bownds M.D. Pugh Jr., E.N. J. Gen. Physiol. 1994; 103: 1071-1098Crossref PubMed Scopus (50) Google Scholar) to 330 (29Cote R.H. Brunnock M.A. J. Biol. Chem. 1993; 268: 17190-17198Abstract Full Text PDF PubMed Google Scholar) rhodopsins per PDE. The amount of purified GARP2 was routinely estimated by immunoblot analysis. Samples of purified GARP2 and known amounts of ROS membranes were resolved on SDS-PAGE and immunoblotted. GARP2 was detected using a GARP2-specific antibody. The intensities of GARP2 immunoreactive bands were determined using Quantiscan (Biosoft) and then compared with GARP2 immunoreactivity in ROS membranes containing known amounts of rhodopsin and PDE6. SDS-PAGE and Western Blotting—SDS-PAGE was performed by the method of Laemmli (30Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207537) Google Scholar) in 10, 12, or 15% acrylamide gels. The immunoblotting procedure followed the protocols in Gallagher (31Gallagher S. Current Protocols in Protein Science Unit 10.10.in: Coligan J.E. Dunn B.M. Ploegh H.L. Speicher D.W. Wingfield P.T. John Wiley & Sons, Inc., New York1998Google Scholar). Note that GARP2 typically migrates at ∼60 kDa (roughly 2-fold higher than predicted based on its amino acid sequence) and shows size heterogeneity, in accord with previous observations (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 19Korschen H.G. Illing M. Seifert R. Sesti F. Williams A. Gotzes S. Colville C. Müller F. Dosé A. Godde M. Molday L. Kaupp U.B. Molday R.S. Neuron. 1995; 15: 627-636Abstract Full Text PDF PubMed Scopus (211) Google Scholar). This anomalous behavior was recently explained as being due to GARP2 existing in solution as a natively unfolded protein (32Batra-Safferling R. Abarca H.K. Korschen H.G. Tziatzios C. Stoldt M. Budyak I. Willbold D. Schwalbe H. Klein-Seetharaman J. Kaupp U.B. J. Biol. Chem. 2006; 281: 1449-1460Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). GARP2 Is a High Affinity PDE6-binding Protein—Because there is uncertainty about the proteins with which GARP2 interacts in rod photoreceptors (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 20Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), we first re-examined whether PDE6 interacts in a specific manner with GARP2. Following homogenization of purified ROS from bovine retina and removal of soluble proteins with isotonic washes, PDE6 and GARP2 both remained associated with ROS disk membranes (Fig. 1A). Release of PDE6 from ROS membranes by exposure to a hypotonic buffer also caused the release of almost all detectable GARP2 (Fig. 1A). Subsequent purification of PDE6 by anion-exchange chromatography on a Mono Q column resulted in co-elution of PDE6 and GARP2 at 400 mm NaCl. Gel filtration chromatography of the Mono Q-purified GARP2-PDE6 also failed to separate GARP2 from PDE6 (Fig. 1A). The results in Fig. 1A conclusively demonstrate that PDE6 and GARP2 are both associated with ROS membranes, are co-eluted by exposure to a hypotonic buffer, and co-purify by two different chromatographic procedures. To directly show that GARP2 is associated with PDE6, we immunoprecipitated PDE6 with the ROS1 antibody (21Hurwitz R.L. Bunt-Milam A.H. Beavo J.A. J. Biol. Chem. 1984; 259: 8612-8618Abstract Full Text PDF PubMed Google Scholar) coupled to Sulfolink beads. Fig. 1B shows that unpurified PDE6 obtained from hypotonic extraction of ROS membranes was pulled down in a complex with GARP2 and that very little GARP2 remained unbound under these conditions. Mono Q-purified PDE6 was also immunoprecipitated in tight association with GARP2, whereas purified GARP2 failed to bind to the ROS1 antibody in the absence of PDE6 (Fig. 1B). To estimate whether a significant amount of the PDE6 exists free of bound GARP2, we performed gel filtration chromatography on proteins solubilized from dark-adapted ROS membranes with a hypotonic buffer. A single peak of PDE6 hydrolytic activity (Fig. 1C) and immunoreactivity (Fig. 1D) was observed at an apparent molecular mass of 330 kDa. (The higher than predicted molecular mass for the PDE6-GARP2 complex by gel filtration chromatography may be due to its asymmetric shape (14Gillespie P.G. Beavo J.A. J. Biol. Chem. 1988; 263: 8133-8141Abstract Full Text PDF PubMed Google Scholar).) Qualitatively, the observed ratio of PDE6 and GARP2 immunoreactivity did not vary in the fractions containing PDE6, indicating that there is not a large fraction of the total PDE6 that exists free of bound GARP2. Further, only small amounts of GARP2 immunoreactivity could be detected at an apparent molecular mass of ∼30–60 kDa (Fig. 1D). This result indicated that PDE6 is tightly associated with GARP2 and that there is no evidence for a significant amount of unbound GARP2 or PDE6. To assess whether GARP2 binding to PDE6 might be an artifact of the initial hypotonic extraction of ROS membrane proteins, we also solubilized PDE6 and GARP2 from dark-adapted ROS membranes with 1% Triton X-100. After removing the ROS membranes by centrifugation and immunoprecipitation of the detergent-solubilized PDE6 with the ROS1 antibody, we detected both PDE6 and GARP2 in the immunoprecipitates; control samples with beads lacking the ROS1 antibody failed to pull down either protein (data not shown). Together, these results demonstrate that most of the GARP2 in ROS co-purifies with PDE6 through several stages of purification. The fact that GARP2 remained bound to PDE6 after repeated washing of the ROS1 immune complex (Fig. 1B) and that little unbound GARP2 was observed during gel filtration chromatography (Fig. 1D) demonstrates that GARP2 binds PDE6 with high affinity. GARP2 Content in Rod Photoreceptors—If GARP2 is to regulate PDE6 activity during phototransduction, it would need to be present in ROS in molar equivalence to PDE6. A previous study by Kaupp and colleagues (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar) has suggested that GARP2 is actually ∼3-fold more abundant than PDE6 (1 GARP2 per 100 rhodopsins). Another GARP2-interacting protein, peripherin, is believed to bind only 10% of the total GARP2 in ROS (20Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Our observation that practically all GARP2 in bovine ROS co-purified with PDE6 (Fig. 1) suggested that GARP2 was not significantly more abundant than PDE6 in ROS. To directly address the question of the GARP2 content in ROS, we purified native GARP2 from bovine ROS, as described in the next section, and compared the immunoreactivity of known amounts of GARP2 to that of intact ROS whose rhodopsin and PDE6 concentration were measured. The precision of our measurements were hampered by uncertainties in the concentration of purified GARP2 used for quantitative immunoblots, because the poor staining of GARP2 by Coomassie and other protein stains limited our ability to assess its purity on SDS-PAGE (see next section). Taking this into consideration, we estimate that there are 1–2 GARP2 molecules per PDE6 holoenzyme in bovine ROS (data not shown). This value agrees well with two other reports (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 32Batra-Safferling R. Abarca H.K. Korschen H.G. Tziatzios C. Stoldt M. Budyak I. Willbold D. Schwalbe H. Klein-Seetharaman J. Kaupp U.B. J. Biol. Chem. 2006; 281: 1449-1460Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and indicates that GARP2 is present in rod photoreceptors in sufficient amounts to bind all of the PDE6. Purification of Native GARP2 Free of Contamination with PDE6—To study the effects of GARP2 on PDE6 catalytic activity, we needed to purify GARP2 free of PDE6 subunits. We discovered that the association of GARP2 with PDE6 could be disrupted using high concentrations of ammonium sulfate. This permitted separation of the two proteins by hydrophobic interaction chromatography (HIC). A hypotonic extract containing PDE6 and GARP2 was mixed with 500 mm ammonium sulfate, and the sample was applied to a butyl-Sepharose column. A decreasing, discontinuous ammonium sulfate gradient permitted the separation of PDE6 (at higher ammonium sulfate concentrations) from the GARP2 (which eluted only when ammonium sulfate was omitted from the buffer) (Fig. 2A). Examination of the PDE6 peak by immunoblot analysis revealed no detectable GARP2 (Fig. 2B, HIC-PDE). The disruption of GARP2 binding to PDE6 by ammonium sulfate suggested that hydrophobic domains in GARP2 (which contains 27% hydrophobic amino acids) may be important in promoting its binding to PDE6 catalytic subunits. The GARP2 peak eluting from the butyl-Sepharose column in the absence of ammonium sulfate still contained traces of the inhibitory Pγ-subunit immunoreactivity at ∼12 kDa as well as a faint band of GARP1 immunoreactivity at ∼130 kDa (Fig. 2B, HIC1). By exposing the partially purified GARP2 sample to 500 mm ammonium sulfate and running the sample on butyl-Sepharose again, most of the residual Pγ was removed from the GARP2 (Fig. 2B, HIC2). Alternatively, the GARP2-enriched fractions from the butyl-Sepharose could be completely separated from PDE6 subunits by reversed phase HPLC. On Coomassie-stained gels of purified GARP2, >50% of the total staining was observed at ∼60 kDa, corresponding to GARP2 (Fig. 2C). No detectable protein was observed at molecular masses corresponding to PDE6 catalytic or inhibitory subunits. The GARP2 purity was likely to be much higher, because this glutamate-rich protein binds Coomassie protein stain very poorly relative to other proteins. It is therefore unlikely that the effects of GARP2 on PDE6 activity reported in this paper could be ascribed to a contaminating protein in our purified GARP2. The 17-kDa Prenyl-binding Protein (PrBP/δ) Releases PDE6, but Not GARP2, from ROS Membranes—The 17-kDa prenyl-binding protein (PrBP/δ), originally described as the δ-subunit of PDE6 (33Gillespie P.G. Prusti R.K. Apel E.D. Beavo J.A. J. Biol. Chem. 1989; 264: 12187-12193Abstract Full Text PDF PubMed Google Scholar), is able to solubilize membrane-associated rod PDE6 in vitro (23Norton A.W. Hosier S. Terew J.M. Li N. Dhingra A. Vardi N. Baehr W. Cote R.H. J. Biol. Chem. 2005; 280: 1248-1256Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 34Florio S.K. Prusti R.K. Beavo J.A. J. Biol. Chem. 1996; 271: 1-12Abstract Full Text Full Text PDF Google Scholar) by binding to the hydrophobic prenyl groups attached to the C termini of the PDE6 catalytic subunits (35Cook T.A. Ghomashchi F. Gelb M.H. Florio S.K. Beavo J.A. Biochemistry. 2000; 39: 13516-13523Crossref PubMed Scopus (47) Google Scholar). Because hydrophobic interactions may stabilize GARP2-PDE6 interactions (see previous section), we wondered whether PrBP/δ binding to PDE6 would solubilize the enzyme as a complex with GARP2 or, alternatively, compete with GARP2 for binding to a hydrophobic region on PDE6. Fig. 3 shows an experiment in which increasing amounts of PrBP/δ were added to ROS membranes (containing bound PDE6 and GARP2) and the solubilization of PDE6 monitored by centrifugal separation of bound and soluble PDE6. Although PDE6 catalytic and Pγ-subunits were released from ROS membranes by PrBP/δ in a concentration-dependent manner, GARP2 remained completely associated with ROS membranes (Fig. 3). The ability of PrBP/δ to disrupt GARP2-PDE6 interactions was not restricted to PDE6 associated with ROS membranes. If PDE6 and GARP2 were first released from ROS membranes by hypotonic extraction and then PrBP/δ was added, some of the GARP2 that normally co-migrated with PDE6 at ∼300 kDa during gel filtration chromatography was now observed eluting at an apparent molecular mass of ∼60 kDa (data not shown). The disruption of GARP2-PDE6 interactions by PrBP/δ suggests that one site of interaction may be at the hydrophobic isoprenyl groups at the C termini of PDE6 catalytic subunits. It is possible that this same binding interface may be disrupted during hydrophobic interaction chromatography. Purified, Native GARP2 Suppresses Basal PDE6 Catalytic Activity but Does Not Inhibit Activated PDE6—It has been previously reported (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar) that GARP2 potently inhibited PDE6 hydrolytic activity when purified PDE6 was activated by transducin in solution; in contrast, trypsin-activated PDE6 (lacking Pγ) or nonactivated enzyme (αβγγ) were not greatly affected by GARP2. However, Korschen et al. (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar) used a recombinant GARP2 fusion protein for these experiments, and unpublished results from the same group have called into question the ability of native GARP2 to inhibit transducin-activated PDE6 (36Kaupp U.B. Seifert R. Physiol. Rev. 2002; 82: 769-824Crossref PubMed Scopus (943) Google Scholar). Furthermore, transducin activates PDE6 more effectively when both proteins are associated with the disk membrane compared with activation in solution (37Fung B.K.K. Nash C. J. Biol. Chem. 1983; 258: 10503-10510Abstract Full Text PDF PubMed Google Scholar, 38Bruckert F. Catty P. Deterre P. Pfister C. Biochemistry. 1994; 33: 12625-12634Crossref PubMed Scopus (23) Google Scholar). Therefore, we chose to examine whether native, purified GARP2 exerted an effect on nonactivated or activated PDE6 under more physiological conditions in which PDE6 remains associated with the disk membrane. As seen in Fig. 4, the addition of purified, native GARP2 to nonactivated PDE6 attached to ROS membranes (which contain endogenous GARP2) inhibited the basal rate of PDE6 activity by 80%. The suppression of PDE6 activity by GARP2 was maximal when an amount of purified GARP2 was added equal to its endogenous level in ROS (as determined by quantitative immunoblot analysis). A similar result was obtained with purified PDE6 that had been extracted from ROS membranes and chromatographically purified (data not shown). In contrast, no significant effect of GARP2 on either transducin-activated PDE6 (attached to ROS membranes) or trypsin-activated PDE6 was seen (Fig. 4). Even following the addition of a 10-fold excess of purified GARP2 relative to its endogenous concentration in ROS membranes, the PDE6 activity of both transducin- and trypsin-activated PDE6 remained within 20% of its activity in the absence of GARP2. Although the results in Fig. 4 for trypsin-activated PDE6 are in accord with those of Korschen et al. (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar), we failed to observe the inhibitory effect of GARP2 on transducin-activated PDE6 that they reported. To more sensitively test whether GARP2 influences the ability of transducin to activate PDE6, we first supplemented light-exposed ROS membranes with either GARP2 or nothing and then added increasing amounts of GTPγS to persistently activate transducin in a concentration-dependent manner. Fig. 5 shows that, in the presence of GARP2 (at a concentration 2-fold greater than required to maximally suppress PDE6 basal activity), the ability of transducin to activate PDE6 was only slightly impaired; the small decrease in activation at any given GTPγS concentration was not statistically significant. Once sufficient GTPγS was added to activate all of the transducin present (the transducin:PDE6 ratio in bovine ROS being 30:1), adding more GTPγS had no further effect on PDE6 activation in the absence or presence of GARP2. This result shows that the activity-lowering effect of GARP2 on the nonactivated PDE6 holoenzyme is distinct from the molecular events by which the activated α-subunit of transducin binds to PDE6 and displaces its Pγ-subunit, thereby causing light activation of PDE6. Proposed Physiological Role for GARP2 Regulation of PDE6 in Rod Photoreceptors—In this study, we have shown that GARP2 is a high affinity PDE6 regulatory protein capable of suppressing the basal activity of nonactivated PDE6 but with negligible effects on transducin-activated PDE6. The exclusive localization of GARP2 to rod outer segments (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) suggests that this protein may regulate rod PDE6 in a way that helps distinguish the rod and cone phototransduction pathways. One feature that differentiates rods from cones is the amplitude of fluctuations in the dark current (dark noise) in the photoreceptor outer segment (10Rieke F. Baylor D.A. Biophys. J. 1996; 71: 2553-2572Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 11Rieke F. Baylor D.A. Neuron. 2000; 26: 181-186Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The low dark noise of rods permits reliable signaling at the single photon level (39Baylor D.A. Lamb T.D. Yau K.-W. J. Physiol. (Lond.). 1979; 288: 613-634Google Scholar), whereas cones require several photons to generate a detectable signal (40Schnapf J.L. Nunn B.J. Meister M. Baylor D.A. J. Physiol. (Lond.). 1990; 427: 681-713Crossref Scopus (370) Google Scholar). Because the rates of PDE6 activation/inactivation determine the characteristics of photoreceptor dark noise (12Holcman D. Korenbrot J.I. The J. Gen. Physiol. 2005; 125: 641-660Crossref PubMed Scopus (36) Google Scholar), GARP2 is an attractive candidate for regulating rod PDE6 to lower its spontaneous activation. This study suggests that the binding of GARP2 to nonactivated rod PDE6 will lower its catalytic activity, most likely by enhancing the affinity of Pγ for the active site of the enzyme. The observed localization of GARP2 to the rim of the disk membrane in ROS (8Korschen H.G. Beyermann M. Müller F. Heck M. Vantler M. Koch K.W. Kellner R. Wolfrum U. Bode C. Hofmann K.P. Kaupp U.B. Nature. 1999; 400: 761-766Crossref PubMed Scopus (118) Google Scholar, 17Colville C.A. Molday R.S. J. Biol. Chem. 1996; 271: 32968-32974Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) may serve as a mechanism to reduce spontaneous PDE6 activation and thereby minimize fluctuations in cGMP concentrations in the vicinity of the cGMP-gated ion channel. The lack of an effect of GARP2 on transducin-activated PDE6 is also consistent with the need for rod PDE6 to be rapidly and stoichiometrically activated upon binding of activated transducin. The single photon sensitivity of rod photoreceptors would likely be impaired if GARP2 were to reduce the efficiency of PDE6 activation by transducin. In summary, our results support a role for GARP2 in maintaining a very low spontaneous activation of PDE6 without interfering with the efficiency of the visual excitation pathway in rod photoreceptors in response to photic stimuli. Reports that GARP2 is associated with the disk rim protein peripherin (20Poetsch A. Molday L.L. Molday R.S. J. Biol. Chem. 2001; 276: 48009-48016Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 32Batra-Safferling R. Abarca H.K. Korschen H.G. Tziatzios C. Stoldt M. Budyak I. Willbold D. Schwalbe H. Klein-Seetharaman J. Kaupp U.B. J. Biol. Chem. 2006; 281: 1449-1460Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) leave open the intriguing possibility that GARP2 might preferentially regulate PDE6 in the vicinity of the cGMP-gated channel, where cGMP metabolic flux might be most stringently controlled. Summary—This work has demonstrated that GARP2 is a novel PDE6 interacting protein that is capable of regulating the basal activity of the PDE6 holoenzyme in rod outer segments. The high affinity with which it binds nonactivated PDE6 suppresses catalytic activity without adversely affecting the ability of transducin to activate PDE6. This novel regulatory mechanism may be of fundamental importance in establishing the high signal-to-noise ratio needed for single photon detection in rod photoreceptors. Future studies will be directed toward determining the molecular mechanism of GARP2 interaction with the catalytic and/or inhibitory subunits of dark-adapted and light-activated PDE6. We thank Dr. U. Benjamin Kaupp (Institut für Biologische Informationsverarbeitung Forschungszentrum, Jülich, Germany) for intellectual contributions and for sharing results prior to publication and Suzanne Matte for technical assistance.