- Email: [email protected]
[28] Heterologous expression and reconstitution of rhodopsin with rhodopsin kinase and arrestin
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
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[281 H e t e r o l o g o u s E x p r e s s i o n a n d R e c o n s t i t u t i o n o f Rhodopsin with Rhodopsin Kinase and Arrestin By
SHOJI O S A W A , D A Y A N I D H I R A M A N ,
and
ELLEN R. WEISS
Introduction Rhodopsin, the light receptor of the vertebrate rod cell. mediates a rapid but controlled response to light through its interactions with transducin (the rod cell G protein), rhodopsin kinase (also known as GRKI), and arre~.tin. The phosphorylation of rhodopsin by rhodopsin kinase and the subsequent binding of arrestin mediate desensitization, a rapid turnoff mechanism that limits the lifetime of photoactivated rhodopsin. Seven serine and threonine residues at the rhodopsin COOH terminus can serve as substrates in vitro for phosphorylation by rhodopsin kinase. Rhodopsin kinase belongs to a unique family of G-protein-coupled receptor kinases (GRKs) that phosphorylate only activated receptors. This may be explained by the observation that the cytoplasmic loops of light-activated rhodopsin stimulate rhodopsin kinase activity, promoting phosphorylation of the rhodopsin COOH terminus. 1 Although phosphorylation at multiple sites can support arrestin binding, 2 only one or two sites are thought to be necessary for arrestin binding in vivo.1 The interaction of arrestin with the phosphorylated COOH terminus of rhodopsin has been shown to induce a conformational change in arrestin that promotes its stable binding to a site presumed to include the cytoplasmic loops. 3 Therefore, multiple domains of rhodopsin participate in its interactions with both rhodopsin kinase and arrestin. Our laboratory is interested in identifying these domains as a step toward understanding the complex process of desensitization. This chapter describes procedures developed to express wild-type and site-directed mutants of rhodopsin in HEK293 cells and to measure the ability of these recombinant proteins to be phosphorylated by rhodopsin kinase and to bind arrestin. Expression of Bovine Rhodopsin in HEK293 Cells The cDNA for bovine rhodopsin, 4 obtained from Dr. Jeremy Nathans (Johns Hopkins University, Baltimore, MD), was truncated to remove part l K. Palczewski, Eur. J. Biochem. 2,48, 261 (1997). 2 L. Zhang, C. D. Sports, S. Osawa, and E. R. Weiss, J. BioL Chem. 272, 14762 (1997). 3 R. Sterne-Marr and J. L. Benovic, Vit. Hormones 51, 193 (1995). 4 j. N a t h a n s and D. S. Hogness, Cell 34, 807 (1983).
METHODS IN ENZYMOLOGY, VOL. 315
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PROTEINS THAT INTERACT WITH RHODOPSIN
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of the 5' and 3' noncoding domains using the restriction enzyme Sinai, creating a 1.3-kb fragment. Truncation of the cDNA significantly enhances the level of rhodopsin expression in cultured mammalian cells. After the addition of HindlII linkers, the cDNA is ligated into the vector pcDNAI/ Amp (Invitrogen, Carlsbad, CA). The construct is transfected into HEK293 cells using DEAE-dextran following procedures similar to those described previously. 5 HEK293 cells are plated at approximately 75% confluence in 10-cm dishes in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 10% fetal calf serum (complete medium) 1 day prior to transfection. On the day of transfection, the cells are rinsed once with phosphate-buffered saline (PBS; 4.3 mM Na2HPO4" 7H20, 1.4 mM KH2PO4, 2.7 mM KCI, 137 mM NaCI) and incubated with 4 ml of DMEM/F12 containing either 10% (v/v) NuSerum (Collaborative Biomedical Products) or 2.5% newborn calf serum. This prevents heavy protein precipitation during the transfection. Each 10-cm dish of cells is cotransfected with 4/zg of pcDNAI/Amprhodopsin and 2 /zg of pRSV-TAg (T antigen; a gift from Dr. Jeremy Nathans). Transfection with pRSV-TAg increases the level of rhodopsin expression in the HEK293 cell line. The solutions are prepared as described next. The transfection buffer is composed of 25 mM Tris-HC1, pH 7.4, 137 mM NaCI, 5 mM KCI, 1.4 mM Na2 HPO4, 10 mM CaCI2, and 5 mM MgC12. To prepare 100 ml of the transfection buffer, mix 10 ml of solution A (250 mM Tris-HC1, pH 7.4, 1.37 M NaC1, 50 mM KC1, 14 mM Na2 HPO4) and 1 ml of solution B (1 M CaCI2, 0.5 M MgClz) with 89 ml of distilled, deionized water and filter sterilize. The D N A needed for transfection is resuspended at a concentration of 0.2 mg/ml in the transfection buffer. A 10 mg/ml solution of DEAE-dextran (500,000 MW; Pharmacia, Piscataway, NJ) and a 10 mM solution of chloroquine are also prepared in the transfection buffer and filter sterilized. For each dish, 20/zl of pcDNAI/Amp-rhodopsin and 10/zl of pRSVTAg are mixed with 200/zl of the DEAE-dextran solution at room temperature. Chloroquine (40/zl) is also added to the DNA/DEAE-dextran solution and the entire mixture is added dropwise to the plate, swirling slowly to distribute evenly. The cells are incubated for 3 hr in a 5% (v/v) CO2 incubator at 37 °, then rinsed with 2 ml PBS and incubated in 4 ml 10% dimethyl sulfoxide (DMSO) in PBS for 2 min. Because prolonged exposure to DMSO is highly toxic to the cells, the DMSO solution is removed immediately and each dish is gently rinsed with 2-4 ml complete medium. Care must be taken during this step not to dislodge the cells, which are very loosely attached. The cells are refed with 10 ml complete medium 5 E. R. Weiss, S. Osawa, W. Shi, and C. D. Dickerson,
Biochemistry 33, 7587 (1994).
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
and incubated in a 5% before harvesting.
CO 2
413
incubator at 37 ° for approximately 3 days
Preparation of Plasma Membranes Plates of transfected cells are rinsed with 2 ml ice-cold PBS, scraped in 2 ml PBS, then transferred to a chilled 50-ml conical centrifuge tube. Typically, the cells from 5-10 plates are pooled in one centrifuge tube. The cells are pelleted by centrifugation at 400g (Sorvall, Newtown, CA; H1000B, 1,500 rpm) for 5 rain at 4°. The cell pellet is frozen at - 8 0 ° to improve cell disruption, thawed on ice, and resuspended in 18 ml of 0.25 M sucrose in buffer containing 0.1 M sodium phosphate, pH 6.5, 1 mM EDTA, 1 mM dithiothreitol (DTT), 2/~g/ml aprotinin, and 1/zg/ml leupeptin. After Dounce homogenization (approximately 20 strokes), the mixture is layered over a 20-ml cushion consisting of 1.1 M sucrose in the same buffer. The homogenates are centrifuged at 103,900g (Beckman, Fullerton, CA; SW 28, 24,000 rpm) for 30 min. The membranes are collected at the interface between the 0.25 M and 1.1 M sucrose layers, diluted approximately ninefold with 50 mM HEPES, pH 6.5, 2 mM EDTA, 1 mM DTT and centrifuged at 41,200g (Beckman 70 Ti, 20,000 rpm) for 20 rain to remove the sucrose. The pellet containing the membranes is resuspended by Dounce homogenization in 50 mM HEPES, pH 6.5, 140 mM NaC1, 3 mM MgCI2, 2mM EDTA, 1 mM DTT and stored in aliquots at - 8 0 °. Approximately 100/xg of membrane protein per plate of cells is obtained using these methods. The level of rhodopsin expression is measured by Western blot analysis. Typically, 10/zg of HEK293 cell membrane protein is subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared with a standard curve of urea-stripped rod outer segment (ROS) membranes ranging from 50 to 300 ng of rhodopsin (Fig. 1). The urea-stripped ROS membranes are prepared from dark-adapted bovine retinas as described, 6 except that the ROS are isolated using a 25%/30% sucrose step gradient rather than a 25%/35% gradient. The level of rhodopsin in the urea-stripped ROS membranes is determined after solubilization in 1.5% octylglucoside by absorbance at 498 nm using a molar extinction coefficient of 42,700 M-1 cm-1.7 The gel also contains a lane of membranes isolated from nontransfected cells that is used to measure nonspecific binding of the anti-rhodopsin antibody. After electrophoretic transfer of the protein, the nitrocellulose membrane is incubated with an anti-rhodopsin monoclonal antibody, R2-15N (a gift from Drs. Grazyna Adamus and Paul 6 M. Wessling-Resnick and G. L. Johnson, J. Biol. Chem. 262, 3697 (1987). 7 K. Hong and W. L. Hubbell, Biochemistry 12, 4517 (1973).
414
PROTEINS THAT INTERACT WITH RHODOPSIN
Rhodopsin NT ROS (~tg) (~tg) (ng) amount , ~ ~ , of protein: 10 20 10 20 50 100 150 200 250
[9-8]
300
76--
49-4-- Rhodopsin 33-28--
FIG. 1. Western blot analysis of bovine rhodopsin transiently expressed in HEK293 cells. Membranes prepared from nontransfected (NT) or rhodopsin-transfected HEK293 cells (rhodopsin) and urea-stripped ROS membranes were subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane and blotted with the rhodopsin monoclonal antibody, R2-15N. The level of antibody binding was detected using I125-1abeled protein A and visualized by scanning with a Molecular Dynamics Phosphorlmager. Numbers on the left side represent molecular size markers in kilodaltons (Bio-Rad prestained markers).
Hargrave, University of Florida, Gainesville, FL) at a concentration of 2 ng//zl of blocking buffer composed of 10 mM Tris-HCl, pH 7.4, 150 mM NaC1, 5% nonfat dry milk, and 0.1% Tween 20. This antibody recognizes the NH2-terminal 15 amino acids of the rhodopsin polypeptide. 8 The level of antibody binding is detected with 125I-labeled protein A (0.1/~Ci/ml in blocking buffer). A Molecular Dynamics PhoshorImager (Sunnyvale, CA) is used to quantify the level of expression. Because rhodopsin is heterogeneously glycosylated when expressed in HEK293 cells, it appears as a series of multiple bands on Western blots (Fig. 1). Therefore, the entire lane above 35 kDa is used for quantification. Although expression varies for different transfections and different mutants, amounts of rhodopsin equaling 1.5-3% of the total membrane protein are typical. The level of rhodopsin expressed in these membranes can also be measured spectrophotometrically 8 G. Adamus, Z. S. Zam, A. Arendt, K. Palczewski, J. H. McDowell, and P. A. Hargrave, Vis. Res. 31, 17 (1991).
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
415
after reconstitution with 11-cis-retinal using methods described by Nathans.9 The amount of rhodopsin estimated by Western blot analysis correlates well with the amount of functional rhodopsin determined by absorbance. 5 Assay for Phosphorylation of Rhodopsin by Rhodopsin Kinase An assay method was developed to measure the ability of rhodopsin kinase to phosphorylate rhodopsin mutants expressed in HEK293 cell membranes. Wild-type and mutant rhodopsin may be expressed at different levels, which may affect the rate of phosphorylation (see later discussion). Therefore, it is critical that each reaction mixture contain equal amounts of rhodopsin and total protein. To achieve this, the membranes are adjusted so that 1 /zg of rhodopsin is used in each reaction. Nontransfected cell membranes are added to samples where necessary to equalize the amount of total protein. Before phosphorylation, the membranes must be reconstituted with ll-cis-retinal. Under Eastman Kodak (Rochester, NY) No. 2 safelights (dark red), membranes sufficient for duplicate samples are diluted into 0.5-1.0 ml phosphorylation buffer containing 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 6 mM MgC12, 1 mM DTF, 50 mM NaF, aprotinin (2 /zg/ml), leupeptin (1 /zg/ml), and 14 p~M ll-cis-retinal. The samples are rocked in the dark at room temperature for 60 rain. Because ll-cis-retinal is absorbed nonspecifically to the membranes, it is important to use at least 7 nmol/200/zg membrane protein to achieve maximum binding to rhodopsin. After reconstitution with ll-cis-retinal, the membranes are centrifuged at 12,000g (Beckman TLA, 14,000 rpm) for 10 rain at 4°. The pellet is resuspended in phosphorylation buffer and membranes corresponding to 1 /zg of rhodopsin are diluted into a final reaction volume of 250/zl containing phosphorylation buffer, 150/zM [~/-32p]ATP (50/zCi/ml), and approximately 60/zl of rhodopsin kinase extracted from dark-adapted bovine retinas using 200 mM Na-HEPES, pH 8.0, 20 mM EDTA, and 2 mM DTT as described. I° This amount of rhodopsin kinase is approximately the amount extracted from one bovine retina. The reaction is initiated by exposure to fluorescent room light at 30°. To compare the ability of wildtype and mutant rhodopsin to be phosphorylated by rhodopsin kinase, the reaction time is typically 8 min. The level of phosphorylation in the dark and phosphorylation of nontransfected cell membranes are included as controls. The reaction is terminated by incubating the assay mixture on ice and dilution with 500/zl of ice-cold 10 mM ATP neutralized with 0.1 M Tris-HC1, pH 7.5. The mixture is centrifuged for 15 rain at 38,500g (Beckman TLA-45, 25,000 rpm) at 4 ° to remove unincorporated radioactive ATP. J. Nathans, Biochemistry 29, 937 (1990). t0 D. J. Kelleher and G. L. Johnson, Z Biol. Chem. 265, 2632 (1990).
416
PROTEINS THAT INTERACT WITH RHODOPSIN
[281
The pellets can be stored frozen at - 8 0 ° for later use or resuspended immediately for immunoprecipation of rhodopsin. The monoclonal antibody R2-15N is used to immunoprecipitate phosphorylated rhodopsin from HEK293 cell membranes. Microfuge tubes (1.5 ml) are pretreated with 10% y-globulin-free horse serum in Tris-buffeted saline (10 mM Tris-HC1, pH 7.5, 150 mM NaC1; TBS) for at least 1 hr, rocking at room temperature to reduce the nonspecific binding of protein to the walls. If 0.02% sodium azide is added, the tubes can be stored at 4 ° for future use. The blocking solution is removed just prior to the assay. Protein A-Sepharose CL-4B (Pharmacia, Piscataway, N J) beads are also pretreated with 10% y-globulin-free horse serum in TBS for 1 hr on a rocker, centrifuged at 600g (Sorvall H-1000B, 1,700 rpm) for 2 min and washed twice by centrifugation with TBS. The beads are stored as a 50% slurry in TBS containing 0.02% sodium azide. For immunoprecipitation of phosphorylated rhodopsin, HEK293 cell membranes are solubilized in 250/zl TBS containing 2 mM MgC12, 2 tzg/ml aprotinin, 1/zg/ml leupeptin, 1 mM DTT, 0.1 mM EDTA, 1.5% octylglucoside, 50 m M N a F and incubated for 1 hr at room temperature on a rocker. The mixture is centrifuged at 4 ° for 15 rain at 125,000g (Beckman TLA-45, 45,000 rpm). The supernatant is transferred to a pretreated microcentrifuge tube and incubated with the R2-15N monoclonal antibody at a ratio of 6 txg per microgram of rhodopsin at room temperature. After a 1-hr incubation, 50/zl of the pretreated protein A-Sepharose CL-4B bead slurry is added and the mixture is incubated for an additional 30 rain at room temperature on a rocker. After centrifugation at 600g (Sorvall H-1000B, 1700 rpm) for 2 min at 4 °, the beads are washed by centrifugation three times in TBS containing 0.1% sodium deoxycholate and once in 10 mM Tris-HCl, pH 6.8. The immune complexes are solubilized from the beads by incubation in 100 tzl Laemmli buffer 11 for 15-30 rain at room temperature on a rocker. The beads are pelleted by centrifugation as described earlier and the supernatant subjected to 10% SDS-PAGE. Phosphorimage analysis is used to quantify the level of phosphorylation of rhodopsin in the dark and in the light. The phosphorylation of nontransfected cell membranes is used as a measure of background and subtracted from values obtained for membranes expressing rhodopsin. The stoichiometry of phosphorylation, which is determined by excising the bands and measuring incorporated radioactivity by liquid scintillation spectroscopy, is approximately 0.5-1.0 mole of phosphate per mole of rhodopsin. 12 We have observed that the rate of rhodopsin phosphorylation in HEK293 cell membranes is much slower than that observed in ureastripped ROS membranes. The HEK293 cell membranes appear to suppress 11 U. K. Laemmli, Nature 227, 680 (1970). i2 W, Shi, S. Osawa, C. D. Dickerson, and E. R. Weiss, J. Biol. Chem. 270, 2112 (1995).
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H E T E R O L O G O U S E X P R E S S I O N A N D R E C O N S T I T U T I O N OF R H O D O P S I N
417
the activity of rhodopsin kinase, perhaps accounting for the low stoichiometry observed in our assay. As shown in Fig. 2, phosphorylation increases progressively but begins to plateau after 60 min. This is similar to the time course of phosphorylation observed for rhodopsin expressed in COS-1 cells. 13 The level at which the reaction reaches a plateau increases proportionately with the amount of rhodopsin kinase in the assay, suggesting that this does not represent saturation of the substrate, but rather a timedependent inactivation of an essential component of the assay. The most likely candidate is metarhodopsin-II, the active form of rhodopsin, which decays progressively in a temperature sensitive manner (see later section).
Assay for Arrestin Binding to Rhodopsin To measure arrestin binding, the membranes containing rhodopsin must first be phosphorylated by rhodopsin kinase. As described earlier, the amount of rhodopsin and the amount of total protein are adjusted so that they are the same in each reaction mixture. HEK293 cell membranes containing 1 ~g rhodopsin are incubated with l l-cis-retinal as described earlier, centrifuged at 12,000g (Beckman TLA-45, 14,000 rpm) at 4°, and resuspended in 100 ~tl of a buffer containing 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 6 mM MgCI2, 1 mM DTT, 10 mM NaF, aprotinin (2 ~g/ml), and leupeptin (1/.~g/ml). Approximately 30 ~1 of rhodopsin kinase extracted from bovine ROS as described earlier (equivalent to the amount isolated from 0.5 retina) and ATP at a final concentration of 2 mM are added to the membranes and incubated for 1 hr at 30° under fluorescent room light. After three 1-ml washes in the same buffer by centrifugation at 38,500g (Beckman TLA-45, 25,000 rpm) for 15 rain at 4°, the membranes are resuspended in 1 ml of 30 mM HEPES, pH 7.5, 2 mM MgC12,150 mM potassium acetate, 1 mM DTT, 10 mM NaF, aprotinin (2/~g/ml), leupeptin (1 ~g/ ml) and incubated with 14/~M ll-cis-retinal for 1 hr at room temperature in the dark to regenerate any rhodopsin that has decayed during the phosphorylation reaction. The membranes are again pelleted by centrifugation at 38,500g (Beckman TLA-45, 25,000 rpm) for 15 rain at 4° and resuspended at a rhodopsin concentration of 40 ng/~l in the buffer described earlier. The membranes can be stored at - 8 0 ° at this stage for later use. The bovine arrestin cDNA TM in pG2S6-I, a pGEM-based vector that contains an idealized 5' untranslated region to promote high-level in vitro expression, l~ was a generous gift from Dr. Vsevolod Gurevich (Sun Health 13S. Bhattacharya,K. D. Ridge, B. E. Knox, and H. G. Khorana,Z BioL Chem. 267, 6763 (1992). 14G. J. Wistow,A. Katial, C. Craft, and T. Shinohara,FEBS Lett. 196,23 (1986). 12V. V. Gurevich and J. L. Benovic,J. Biol. Chem. 267, 21919 (1992).
418
PROTEINS THAT INTERACTWITH RHODOPSIN
[28]
FIG. 2. Phosphorylation of bovine rhodopsin expressed in HEK293 cells by rhodopsin kinase. (A) Time course of rhodopsin phosphorylated in the dark and in light. HEK293 cell membranes containing rhodopsin were phosphorylated by rhodopsin kinase, immunoprecipitared with the rhodopsin monoclonal antibody, R2-215N, and subjected to 10% SDS-PAGE. The radioactivity was visualized by autoradiography. (B) Quantitation of the gel from (A) using a Molecular Dynamics PhosphorImager. Open circles, samples phosphorylated in the light; closed circles, samples phosphorylated in the dark. [Reprinted from W. Shi, S. Osawa, C. D. Dickerson, and E. R. Weiss, J. Biol. Chem. 270, 2112 (1995).]
[9~8]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
419
Research Institute, Sun City, AZ). The plasmid is linearized with HindlII and transcribed in vitro using SP6 R N A polymerase (New England Biolabs) in the presence of the cap, P1-5'-(7-methyl)guanosine-p3-5'-guanosine triphosphate [mTG(5')ppp(5')G; Boehringer Mannheim] to increase mRNA stability and translation efficiency. Approximately 2-4/xg of the synthesized R N A is translated in vitro using 35/zl of rabbit reticulocyte lysate (micrococcal nuclease-treated; Promega, Madison, WI), 2/zl of 1 mM amino acid mix (methionine-free), 1/zl RNAsin, and 4 tzl [3SS]methionine (final concentration = 1200 /zCi/ml) in a 50-/zl volume at 30° for 1 hr according to procedures supplied by the manufacturer. The reaction mixture is centrifuged at ll00g (Sorvall H-1000B, 2250 rpm) through a Bio-Spin 6 column (Bio-Rad Laboratories, Hercules, CA) into a microfuge tube containing 5 /zl of 300 mM HEPES, pH 7.5, 20 mM MgCI2 and 1.5 M potassium acetate (a 10× concentration of the arrestin binding buffer described in the next paragraph) to remove unincorporated [3SS]methionine and exchange the buffer. The amount of in vitro-translated protein synthesized using these procedures is measured as incorporation of [3SS]methionine into a hot trichloroacetic acid (TCA)-insoluble fraction. 16 The reaction mixture (2.5 /zl) is spotted onto 1- × 1-cm 3MMChr paper (Millipore, Bedford, MA). The paper is boiled in a beaker containing 5% TCA for 10 min, then rinsed three times with 5% TCA and four times with 95% ethanol at room temperature. The filters are dried and the level of radioactivity is quantified by liquid scintillation spectroscopy. To calculate the specific activity of the preparation, the concentration of nonradioactive methionine must be obtained from Promega for each batch of reticulocyte lysate. The concentration of methionine is approximately 5/zM for most batches of reticulocyte lysate. Taking into account that bovine arrestin has 8 methionines, this value can be used to calculate the yield of in vitro-translated arrestin. Typically, 800 fmol are obtained from a 50-/zl reaction. Approximately 10 fmol of in vitro-translated arrestin is used in the arrestin binding assay for each sample. The arrestin binding assay is a modification of procedures originally developed by Gurevich and Benovic. ~5'17 It is performed in a reaction volume of 35 /zl. The arrestin (10 fmol) is diluted into 25 tzl of ice-cold arrestin binding assay buffer containing 30 mM HEPES, 2 mM MgC12 and 150 mM potassium acetate, pH 7.5. Ten microliters of HEK293 cell membranes containing 0.4/zg of phosphorylated, 11-cis-retinal-regenerated rhodopsin are added to the arrestin in the dark at 4°. The mixture is incubated in the light at 4° for 5 min, then transferred to a 37° water bath ~ F. J. Bollum, Methods Enzymol. 12, 169 (1968). ~7 V. V. Gurevich and J. L. Benovic, J. Biol. Chem, 268, 11628 (1993).
A Rhodopsin
NT II
Phosphorylated!
Arrestin---*
B 70 60~ ---g 50 i
~ 40o en
-
'- 30--
~
J
~ 2o-
+
Phosphorylated: NT
Rhodopsin
Std
i I--'-I
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HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
421
for 5 min to allow arrestin to bind rhodopsin. To terminate the reaction, the assay mixture is placed on ice and 200/zl of ice-cold arrestin binding buffer is added. The diluted sample is loaded onto a 200/xl cushion of icecold 0.2 M sucrose in arrestin binding buffer and immediately centrifuged at 2° at 125,000g (Beckman TLA-45, 45,000 rpm) to separate free arrestin from arrestin bound to rhodopsin. The length of centrifugation time has been varied from 3 to 30 min without significant changes in the level of arrestin bound to rhodopsin. The pellets are washed once with 200/zl of ice-cold arrestin binding buffer, solubilized in Laemmli buffer, and analyzed by 10% SDS-PAGE and phosphorimage analysis. A sample consisting of 10 fmol of in vitro-translated arrestin is applied to the gel as a standard so that the amount of arrestin bound to rhodopsin can be estimated. Arrestin binding to nontransfected cell membranes is used as a measure of nonspecific binding. Approximately 40-65% of the arrestin in the assay binds to HEK293 cell membranes containing wild-type rhodopsin under these conditions (Fig. 3B). Our laboratory has used this method to characterize the phosphorylation sites on rhodopsin that play a role in arrestin binding in vitro. 2 We have measured an apparent KD of approximately 0.74 nM for arrestin binding to HEK293 cell membranes expressing rhodopsin. However, the active form of rhodopsin, metarhodopsin-II, is known to decay in a temperaturesensitive manner. For example, detergent-extracted rhodopsin isolated from COS-1 cells decays with a half-time of 18 rain at 20°. TM Therefore, equilibrium binding studies cannot accurately be performed. Nevertheless, the value that we reported is significantly lower than the previously published value of 50 nM, obtained from light scattering studies 19 and not very different from the value of 2 nM reported for the binding of visual arrestin to the/32-adrenergic receptor. 2° This may be due in part to the ability of arrestin to stabilize metarhodopsin-II in a manner similar to transducin, 1~ is T. Sakamoto and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 92, 249 (1995). 19A. Schleicher, H. Kilhn, and K. P. Hofmann, Biochemistry 28, 1770 (1989). 20V. V. Gurevich, S. B. Dion, J. J. Onorato, J. Ptasienski, C. M. Kim, R. Sterne-Marr, M. M. Hosey, and J. L. Benovic, J. BioL Chem. 270, 720 (1995).
FIG. 3. Arrestin binding to bovine rhodopsin expressed in HEK293 cells. (A) Nontransfeeted (NT) and rhodopsin-transfected HEK293 cell membranes (Rhodopsin) were incubated with [35S]methionine-labeled, in vitro-translated arrestin, centrifuged to isolate the arrestin/rhodopsin complexes, and subjected to 10% SDS-PAGE. A lane of arrestin representing 10 fmol (Std) was also applied to the gel. The samples were analyzed using a Molecular Dynamics PhosphorImager. -, Nonphosphorylated; +, phosphorylated. (B) Quantitation of the polyacrylamide gel shown in (A). Labels are as described in (A). The label Arrestin Bound (%) refers to the fraction of arrestin in the assay that binds to rhodopsin.
422
PROTEINS THAT INTERACT WITH RHODOPSIN
[29]
allowing the isolation of arrestin/rhodopsin complexes without appreciable dissociation during centrifugation. These observations suggest that this arrestin binding assay is a reasonable method for determining relative affinities for rhodopsin mutants expressed in cultured cells.
[29] A r r e s t i n : M u t a g e n e s i s , E x p r e s s i o n , P u r i f i c a t i o n , a n d Functional Characterization B y V S E V O L O D V . G U R E V I C H a n d J E F F R E Y L. B E N O V I C
Introduction In the photoreceptor cell the activation of rhodopsin by a photon of light initiates two cascades of events: signal transduction and signal termination. The visual amplification cascade (rhodopsin ~ transducin ~ cGMP phosphodiesterase) has long served as an archetypal G-protein-coupled receptor signaling system. 1 A single light-activated rhodopsin (Rh*) can sequentially activate hundreds of transducin molecules, each of which in turn activates a cGMP phosphodiesterase resulting in the hydrolysis of thousands of cGMP molecules. Thus, the potential for signal amplification is enormous, providing for very high sensitivity. However, because relatively modest local changes in the cGMP concentration are sufficient for a full cellular response, the signaling machinery needs to be turned off as soon as the signal goes through. A parallel chain of events brings this about, e The initial step in this turn off is the recognition and phosphorylation of Rh* by the enzyme rhodopsin kinase. Rhodopsin phosphorylation attenuates its ability to activate transducin and increases its affinity for the retinal protein, arrestin. Arrestin binds to phosphorylated Rh* (P-Rh*) and effectively shuts down the phototransduction cascade by blocking further transducin activation. Arrestin appears to stay bound to P-Rh* until it decays into phosphoopsin after which arrestin dissociates and phosphoopsin is dephosphorylated by a type IIA protein phosphatase. Retinal arrestin, also termed 48-kDa protein or S-antigen, was initially identified as a protein that binds to disk membranes following light activation of rhodopsin. 3 Arrestin binding and its ability to terminate signaling require rhodopsin phosphorylation? In fact, although arrestin can indepen1 p. A. Hargrave and J. H. McDowell, FASEB J. 6, 2323 (1992). 2 U. Wilden, Biochemistry 34, 1446 (1995). 3 U. Wilden, S. W. Hall, and H. Kuhn, Proc. Natl. Acad. Sci. U.S.A. 83, 1174 (1986).
METHODS IN ENZYMOLOGY,VOL. 315
Copyright © 2000 by AcademicPress All rights of reproductionin any form reserved. 0076-6879/00 $30.00
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
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[281 H e t e r o l o g o u s E x p r e s s i o n a n d R e c o n s t i t u t i o n o f Rhodopsin with Rhodopsin Kinase and Arrestin By
SHOJI O S A W A , D A Y A N I D H I R A M A N ,
and
ELLEN R. WEISS
Introduction Rhodopsin, the light receptor of the vertebrate rod cell. mediates a rapid but controlled response to light through its interactions with transducin (the rod cell G protein), rhodopsin kinase (also known as GRKI), and arre~.tin. The phosphorylation of rhodopsin by rhodopsin kinase and the subsequent binding of arrestin mediate desensitization, a rapid turnoff mechanism that limits the lifetime of photoactivated rhodopsin. Seven serine and threonine residues at the rhodopsin COOH terminus can serve as substrates in vitro for phosphorylation by rhodopsin kinase. Rhodopsin kinase belongs to a unique family of G-protein-coupled receptor kinases (GRKs) that phosphorylate only activated receptors. This may be explained by the observation that the cytoplasmic loops of light-activated rhodopsin stimulate rhodopsin kinase activity, promoting phosphorylation of the rhodopsin COOH terminus. 1 Although phosphorylation at multiple sites can support arrestin binding, 2 only one or two sites are thought to be necessary for arrestin binding in vivo.1 The interaction of arrestin with the phosphorylated COOH terminus of rhodopsin has been shown to induce a conformational change in arrestin that promotes its stable binding to a site presumed to include the cytoplasmic loops. 3 Therefore, multiple domains of rhodopsin participate in its interactions with both rhodopsin kinase and arrestin. Our laboratory is interested in identifying these domains as a step toward understanding the complex process of desensitization. This chapter describes procedures developed to express wild-type and site-directed mutants of rhodopsin in HEK293 cells and to measure the ability of these recombinant proteins to be phosphorylated by rhodopsin kinase and to bind arrestin. Expression of Bovine Rhodopsin in HEK293 Cells The cDNA for bovine rhodopsin, 4 obtained from Dr. Jeremy Nathans (Johns Hopkins University, Baltimore, MD), was truncated to remove part l K. Palczewski, Eur. J. Biochem. 2,48, 261 (1997). 2 L. Zhang, C. D. Sports, S. Osawa, and E. R. Weiss, J. BioL Chem. 272, 14762 (1997). 3 R. Sterne-Marr and J. L. Benovic, Vit. Hormones 51, 193 (1995). 4 j. N a t h a n s and D. S. Hogness, Cell 34, 807 (1983).
METHODS IN ENZYMOLOGY, VOL. 315
Copyright © 2000 by AcademicPress All rights of reproduction in any form reserved. 0076-6879/00$30.00
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PROTEINS THAT INTERACT WITH RHODOPSIN
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of the 5' and 3' noncoding domains using the restriction enzyme Sinai, creating a 1.3-kb fragment. Truncation of the cDNA significantly enhances the level of rhodopsin expression in cultured mammalian cells. After the addition of HindlII linkers, the cDNA is ligated into the vector pcDNAI/ Amp (Invitrogen, Carlsbad, CA). The construct is transfected into HEK293 cells using DEAE-dextran following procedures similar to those described previously. 5 HEK293 cells are plated at approximately 75% confluence in 10-cm dishes in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 10% fetal calf serum (complete medium) 1 day prior to transfection. On the day of transfection, the cells are rinsed once with phosphate-buffered saline (PBS; 4.3 mM Na2HPO4" 7H20, 1.4 mM KH2PO4, 2.7 mM KCI, 137 mM NaCI) and incubated with 4 ml of DMEM/F12 containing either 10% (v/v) NuSerum (Collaborative Biomedical Products) or 2.5% newborn calf serum. This prevents heavy protein precipitation during the transfection. Each 10-cm dish of cells is cotransfected with 4/zg of pcDNAI/Amprhodopsin and 2 /zg of pRSV-TAg (T antigen; a gift from Dr. Jeremy Nathans). Transfection with pRSV-TAg increases the level of rhodopsin expression in the HEK293 cell line. The solutions are prepared as described next. The transfection buffer is composed of 25 mM Tris-HC1, pH 7.4, 137 mM NaCI, 5 mM KCI, 1.4 mM Na2 HPO4, 10 mM CaCI2, and 5 mM MgC12. To prepare 100 ml of the transfection buffer, mix 10 ml of solution A (250 mM Tris-HC1, pH 7.4, 1.37 M NaC1, 50 mM KC1, 14 mM Na2 HPO4) and 1 ml of solution B (1 M CaCI2, 0.5 M MgClz) with 89 ml of distilled, deionized water and filter sterilize. The D N A needed for transfection is resuspended at a concentration of 0.2 mg/ml in the transfection buffer. A 10 mg/ml solution of DEAE-dextran (500,000 MW; Pharmacia, Piscataway, NJ) and a 10 mM solution of chloroquine are also prepared in the transfection buffer and filter sterilized. For each dish, 20/zl of pcDNAI/Amp-rhodopsin and 10/zl of pRSVTAg are mixed with 200/zl of the DEAE-dextran solution at room temperature. Chloroquine (40/zl) is also added to the DNA/DEAE-dextran solution and the entire mixture is added dropwise to the plate, swirling slowly to distribute evenly. The cells are incubated for 3 hr in a 5% (v/v) CO2 incubator at 37 °, then rinsed with 2 ml PBS and incubated in 4 ml 10% dimethyl sulfoxide (DMSO) in PBS for 2 min. Because prolonged exposure to DMSO is highly toxic to the cells, the DMSO solution is removed immediately and each dish is gently rinsed with 2-4 ml complete medium. Care must be taken during this step not to dislodge the cells, which are very loosely attached. The cells are refed with 10 ml complete medium 5 E. R. Weiss, S. Osawa, W. Shi, and C. D. Dickerson,
Biochemistry 33, 7587 (1994).
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
and incubated in a 5% before harvesting.
CO 2
413
incubator at 37 ° for approximately 3 days
Preparation of Plasma Membranes Plates of transfected cells are rinsed with 2 ml ice-cold PBS, scraped in 2 ml PBS, then transferred to a chilled 50-ml conical centrifuge tube. Typically, the cells from 5-10 plates are pooled in one centrifuge tube. The cells are pelleted by centrifugation at 400g (Sorvall, Newtown, CA; H1000B, 1,500 rpm) for 5 rain at 4°. The cell pellet is frozen at - 8 0 ° to improve cell disruption, thawed on ice, and resuspended in 18 ml of 0.25 M sucrose in buffer containing 0.1 M sodium phosphate, pH 6.5, 1 mM EDTA, 1 mM dithiothreitol (DTT), 2/~g/ml aprotinin, and 1/zg/ml leupeptin. After Dounce homogenization (approximately 20 strokes), the mixture is layered over a 20-ml cushion consisting of 1.1 M sucrose in the same buffer. The homogenates are centrifuged at 103,900g (Beckman, Fullerton, CA; SW 28, 24,000 rpm) for 30 min. The membranes are collected at the interface between the 0.25 M and 1.1 M sucrose layers, diluted approximately ninefold with 50 mM HEPES, pH 6.5, 2 mM EDTA, 1 mM DTT and centrifuged at 41,200g (Beckman 70 Ti, 20,000 rpm) for 20 rain to remove the sucrose. The pellet containing the membranes is resuspended by Dounce homogenization in 50 mM HEPES, pH 6.5, 140 mM NaC1, 3 mM MgCI2, 2mM EDTA, 1 mM DTT and stored in aliquots at - 8 0 °. Approximately 100/xg of membrane protein per plate of cells is obtained using these methods. The level of rhodopsin expression is measured by Western blot analysis. Typically, 10/zg of HEK293 cell membrane protein is subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared with a standard curve of urea-stripped rod outer segment (ROS) membranes ranging from 50 to 300 ng of rhodopsin (Fig. 1). The urea-stripped ROS membranes are prepared from dark-adapted bovine retinas as described, 6 except that the ROS are isolated using a 25%/30% sucrose step gradient rather than a 25%/35% gradient. The level of rhodopsin in the urea-stripped ROS membranes is determined after solubilization in 1.5% octylglucoside by absorbance at 498 nm using a molar extinction coefficient of 42,700 M-1 cm-1.7 The gel also contains a lane of membranes isolated from nontransfected cells that is used to measure nonspecific binding of the anti-rhodopsin antibody. After electrophoretic transfer of the protein, the nitrocellulose membrane is incubated with an anti-rhodopsin monoclonal antibody, R2-15N (a gift from Drs. Grazyna Adamus and Paul 6 M. Wessling-Resnick and G. L. Johnson, J. Biol. Chem. 262, 3697 (1987). 7 K. Hong and W. L. Hubbell, Biochemistry 12, 4517 (1973).
414
PROTEINS THAT INTERACT WITH RHODOPSIN
Rhodopsin NT ROS (~tg) (~tg) (ng) amount , ~ ~ , of protein: 10 20 10 20 50 100 150 200 250
[9-8]
300
76--
49-4-- Rhodopsin 33-28--
FIG. 1. Western blot analysis of bovine rhodopsin transiently expressed in HEK293 cells. Membranes prepared from nontransfected (NT) or rhodopsin-transfected HEK293 cells (rhodopsin) and urea-stripped ROS membranes were subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane and blotted with the rhodopsin monoclonal antibody, R2-15N. The level of antibody binding was detected using I125-1abeled protein A and visualized by scanning with a Molecular Dynamics Phosphorlmager. Numbers on the left side represent molecular size markers in kilodaltons (Bio-Rad prestained markers).
Hargrave, University of Florida, Gainesville, FL) at a concentration of 2 ng//zl of blocking buffer composed of 10 mM Tris-HCl, pH 7.4, 150 mM NaC1, 5% nonfat dry milk, and 0.1% Tween 20. This antibody recognizes the NH2-terminal 15 amino acids of the rhodopsin polypeptide. 8 The level of antibody binding is detected with 125I-labeled protein A (0.1/~Ci/ml in blocking buffer). A Molecular Dynamics PhoshorImager (Sunnyvale, CA) is used to quantify the level of expression. Because rhodopsin is heterogeneously glycosylated when expressed in HEK293 cells, it appears as a series of multiple bands on Western blots (Fig. 1). Therefore, the entire lane above 35 kDa is used for quantification. Although expression varies for different transfections and different mutants, amounts of rhodopsin equaling 1.5-3% of the total membrane protein are typical. The level of rhodopsin expressed in these membranes can also be measured spectrophotometrically 8 G. Adamus, Z. S. Zam, A. Arendt, K. Palczewski, J. H. McDowell, and P. A. Hargrave, Vis. Res. 31, 17 (1991).
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
415
after reconstitution with 11-cis-retinal using methods described by Nathans.9 The amount of rhodopsin estimated by Western blot analysis correlates well with the amount of functional rhodopsin determined by absorbance. 5 Assay for Phosphorylation of Rhodopsin by Rhodopsin Kinase An assay method was developed to measure the ability of rhodopsin kinase to phosphorylate rhodopsin mutants expressed in HEK293 cell membranes. Wild-type and mutant rhodopsin may be expressed at different levels, which may affect the rate of phosphorylation (see later discussion). Therefore, it is critical that each reaction mixture contain equal amounts of rhodopsin and total protein. To achieve this, the membranes are adjusted so that 1 /zg of rhodopsin is used in each reaction. Nontransfected cell membranes are added to samples where necessary to equalize the amount of total protein. Before phosphorylation, the membranes must be reconstituted with ll-cis-retinal. Under Eastman Kodak (Rochester, NY) No. 2 safelights (dark red), membranes sufficient for duplicate samples are diluted into 0.5-1.0 ml phosphorylation buffer containing 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 6 mM MgC12, 1 mM DTF, 50 mM NaF, aprotinin (2 /zg/ml), leupeptin (1 /zg/ml), and 14 p~M ll-cis-retinal. The samples are rocked in the dark at room temperature for 60 rain. Because ll-cis-retinal is absorbed nonspecifically to the membranes, it is important to use at least 7 nmol/200/zg membrane protein to achieve maximum binding to rhodopsin. After reconstitution with ll-cis-retinal, the membranes are centrifuged at 12,000g (Beckman TLA, 14,000 rpm) for 10 rain at 4°. The pellet is resuspended in phosphorylation buffer and membranes corresponding to 1 /zg of rhodopsin are diluted into a final reaction volume of 250/zl containing phosphorylation buffer, 150/zM [~/-32p]ATP (50/zCi/ml), and approximately 60/zl of rhodopsin kinase extracted from dark-adapted bovine retinas using 200 mM Na-HEPES, pH 8.0, 20 mM EDTA, and 2 mM DTT as described. I° This amount of rhodopsin kinase is approximately the amount extracted from one bovine retina. The reaction is initiated by exposure to fluorescent room light at 30°. To compare the ability of wildtype and mutant rhodopsin to be phosphorylated by rhodopsin kinase, the reaction time is typically 8 min. The level of phosphorylation in the dark and phosphorylation of nontransfected cell membranes are included as controls. The reaction is terminated by incubating the assay mixture on ice and dilution with 500/zl of ice-cold 10 mM ATP neutralized with 0.1 M Tris-HC1, pH 7.5. The mixture is centrifuged for 15 rain at 38,500g (Beckman TLA-45, 25,000 rpm) at 4 ° to remove unincorporated radioactive ATP. J. Nathans, Biochemistry 29, 937 (1990). t0 D. J. Kelleher and G. L. Johnson, Z Biol. Chem. 265, 2632 (1990).
416
PROTEINS THAT INTERACT WITH RHODOPSIN
[281
The pellets can be stored frozen at - 8 0 ° for later use or resuspended immediately for immunoprecipation of rhodopsin. The monoclonal antibody R2-15N is used to immunoprecipitate phosphorylated rhodopsin from HEK293 cell membranes. Microfuge tubes (1.5 ml) are pretreated with 10% y-globulin-free horse serum in Tris-buffeted saline (10 mM Tris-HC1, pH 7.5, 150 mM NaC1; TBS) for at least 1 hr, rocking at room temperature to reduce the nonspecific binding of protein to the walls. If 0.02% sodium azide is added, the tubes can be stored at 4 ° for future use. The blocking solution is removed just prior to the assay. Protein A-Sepharose CL-4B (Pharmacia, Piscataway, N J) beads are also pretreated with 10% y-globulin-free horse serum in TBS for 1 hr on a rocker, centrifuged at 600g (Sorvall H-1000B, 1,700 rpm) for 2 min and washed twice by centrifugation with TBS. The beads are stored as a 50% slurry in TBS containing 0.02% sodium azide. For immunoprecipitation of phosphorylated rhodopsin, HEK293 cell membranes are solubilized in 250/zl TBS containing 2 mM MgC12, 2 tzg/ml aprotinin, 1/zg/ml leupeptin, 1 mM DTT, 0.1 mM EDTA, 1.5% octylglucoside, 50 m M N a F and incubated for 1 hr at room temperature on a rocker. The mixture is centrifuged at 4 ° for 15 rain at 125,000g (Beckman TLA-45, 45,000 rpm). The supernatant is transferred to a pretreated microcentrifuge tube and incubated with the R2-15N monoclonal antibody at a ratio of 6 txg per microgram of rhodopsin at room temperature. After a 1-hr incubation, 50/zl of the pretreated protein A-Sepharose CL-4B bead slurry is added and the mixture is incubated for an additional 30 rain at room temperature on a rocker. After centrifugation at 600g (Sorvall H-1000B, 1700 rpm) for 2 min at 4 °, the beads are washed by centrifugation three times in TBS containing 0.1% sodium deoxycholate and once in 10 mM Tris-HCl, pH 6.8. The immune complexes are solubilized from the beads by incubation in 100 tzl Laemmli buffer 11 for 15-30 rain at room temperature on a rocker. The beads are pelleted by centrifugation as described earlier and the supernatant subjected to 10% SDS-PAGE. Phosphorimage analysis is used to quantify the level of phosphorylation of rhodopsin in the dark and in the light. The phosphorylation of nontransfected cell membranes is used as a measure of background and subtracted from values obtained for membranes expressing rhodopsin. The stoichiometry of phosphorylation, which is determined by excising the bands and measuring incorporated radioactivity by liquid scintillation spectroscopy, is approximately 0.5-1.0 mole of phosphate per mole of rhodopsin. 12 We have observed that the rate of rhodopsin phosphorylation in HEK293 cell membranes is much slower than that observed in ureastripped ROS membranes. The HEK293 cell membranes appear to suppress 11 U. K. Laemmli, Nature 227, 680 (1970). i2 W, Shi, S. Osawa, C. D. Dickerson, and E. R. Weiss, J. Biol. Chem. 270, 2112 (1995).
[281
H E T E R O L O G O U S E X P R E S S I O N A N D R E C O N S T I T U T I O N OF R H O D O P S I N
417
the activity of rhodopsin kinase, perhaps accounting for the low stoichiometry observed in our assay. As shown in Fig. 2, phosphorylation increases progressively but begins to plateau after 60 min. This is similar to the time course of phosphorylation observed for rhodopsin expressed in COS-1 cells. 13 The level at which the reaction reaches a plateau increases proportionately with the amount of rhodopsin kinase in the assay, suggesting that this does not represent saturation of the substrate, but rather a timedependent inactivation of an essential component of the assay. The most likely candidate is metarhodopsin-II, the active form of rhodopsin, which decays progressively in a temperature sensitive manner (see later section).
Assay for Arrestin Binding to Rhodopsin To measure arrestin binding, the membranes containing rhodopsin must first be phosphorylated by rhodopsin kinase. As described earlier, the amount of rhodopsin and the amount of total protein are adjusted so that they are the same in each reaction mixture. HEK293 cell membranes containing 1 ~g rhodopsin are incubated with l l-cis-retinal as described earlier, centrifuged at 12,000g (Beckman TLA-45, 14,000 rpm) at 4°, and resuspended in 100 ~tl of a buffer containing 20 mM Tris-HC1, pH 7.5, 2 mM EDTA, 6 mM MgCI2, 1 mM DTT, 10 mM NaF, aprotinin (2 ~g/ml), and leupeptin (1/.~g/ml). Approximately 30 ~1 of rhodopsin kinase extracted from bovine ROS as described earlier (equivalent to the amount isolated from 0.5 retina) and ATP at a final concentration of 2 mM are added to the membranes and incubated for 1 hr at 30° under fluorescent room light. After three 1-ml washes in the same buffer by centrifugation at 38,500g (Beckman TLA-45, 25,000 rpm) for 15 rain at 4°, the membranes are resuspended in 1 ml of 30 mM HEPES, pH 7.5, 2 mM MgC12,150 mM potassium acetate, 1 mM DTT, 10 mM NaF, aprotinin (2/~g/ml), leupeptin (1 ~g/ ml) and incubated with 14/~M ll-cis-retinal for 1 hr at room temperature in the dark to regenerate any rhodopsin that has decayed during the phosphorylation reaction. The membranes are again pelleted by centrifugation at 38,500g (Beckman TLA-45, 25,000 rpm) for 15 rain at 4° and resuspended at a rhodopsin concentration of 40 ng/~l in the buffer described earlier. The membranes can be stored at - 8 0 ° at this stage for later use. The bovine arrestin cDNA TM in pG2S6-I, a pGEM-based vector that contains an idealized 5' untranslated region to promote high-level in vitro expression, l~ was a generous gift from Dr. Vsevolod Gurevich (Sun Health 13S. Bhattacharya,K. D. Ridge, B. E. Knox, and H. G. Khorana,Z BioL Chem. 267, 6763 (1992). 14G. J. Wistow,A. Katial, C. Craft, and T. Shinohara,FEBS Lett. 196,23 (1986). 12V. V. Gurevich and J. L. Benovic,J. Biol. Chem. 267, 21919 (1992).
418
PROTEINS THAT INTERACTWITH RHODOPSIN
[28]
FIG. 2. Phosphorylation of bovine rhodopsin expressed in HEK293 cells by rhodopsin kinase. (A) Time course of rhodopsin phosphorylated in the dark and in light. HEK293 cell membranes containing rhodopsin were phosphorylated by rhodopsin kinase, immunoprecipitared with the rhodopsin monoclonal antibody, R2-215N, and subjected to 10% SDS-PAGE. The radioactivity was visualized by autoradiography. (B) Quantitation of the gel from (A) using a Molecular Dynamics PhosphorImager. Open circles, samples phosphorylated in the light; closed circles, samples phosphorylated in the dark. [Reprinted from W. Shi, S. Osawa, C. D. Dickerson, and E. R. Weiss, J. Biol. Chem. 270, 2112 (1995).]
[9~8]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
419
Research Institute, Sun City, AZ). The plasmid is linearized with HindlII and transcribed in vitro using SP6 R N A polymerase (New England Biolabs) in the presence of the cap, P1-5'-(7-methyl)guanosine-p3-5'-guanosine triphosphate [mTG(5')ppp(5')G; Boehringer Mannheim] to increase mRNA stability and translation efficiency. Approximately 2-4/xg of the synthesized R N A is translated in vitro using 35/zl of rabbit reticulocyte lysate (micrococcal nuclease-treated; Promega, Madison, WI), 2/zl of 1 mM amino acid mix (methionine-free), 1/zl RNAsin, and 4 tzl [3SS]methionine (final concentration = 1200 /zCi/ml) in a 50-/zl volume at 30° for 1 hr according to procedures supplied by the manufacturer. The reaction mixture is centrifuged at ll00g (Sorvall H-1000B, 2250 rpm) through a Bio-Spin 6 column (Bio-Rad Laboratories, Hercules, CA) into a microfuge tube containing 5 /zl of 300 mM HEPES, pH 7.5, 20 mM MgCI2 and 1.5 M potassium acetate (a 10× concentration of the arrestin binding buffer described in the next paragraph) to remove unincorporated [3SS]methionine and exchange the buffer. The amount of in vitro-translated protein synthesized using these procedures is measured as incorporation of [3SS]methionine into a hot trichloroacetic acid (TCA)-insoluble fraction. 16 The reaction mixture (2.5 /zl) is spotted onto 1- × 1-cm 3MMChr paper (Millipore, Bedford, MA). The paper is boiled in a beaker containing 5% TCA for 10 min, then rinsed three times with 5% TCA and four times with 95% ethanol at room temperature. The filters are dried and the level of radioactivity is quantified by liquid scintillation spectroscopy. To calculate the specific activity of the preparation, the concentration of nonradioactive methionine must be obtained from Promega for each batch of reticulocyte lysate. The concentration of methionine is approximately 5/zM for most batches of reticulocyte lysate. Taking into account that bovine arrestin has 8 methionines, this value can be used to calculate the yield of in vitro-translated arrestin. Typically, 800 fmol are obtained from a 50-/zl reaction. Approximately 10 fmol of in vitro-translated arrestin is used in the arrestin binding assay for each sample. The arrestin binding assay is a modification of procedures originally developed by Gurevich and Benovic. ~5'17 It is performed in a reaction volume of 35 /zl. The arrestin (10 fmol) is diluted into 25 tzl of ice-cold arrestin binding assay buffer containing 30 mM HEPES, 2 mM MgC12 and 150 mM potassium acetate, pH 7.5. Ten microliters of HEK293 cell membranes containing 0.4/zg of phosphorylated, 11-cis-retinal-regenerated rhodopsin are added to the arrestin in the dark at 4°. The mixture is incubated in the light at 4° for 5 min, then transferred to a 37° water bath ~ F. J. Bollum, Methods Enzymol. 12, 169 (1968). ~7 V. V. Gurevich and J. L. Benovic, J. Biol. Chem, 268, 11628 (1993).
A Rhodopsin
NT II
Phosphorylated!
Arrestin---*
B 70 60~ ---g 50 i
~ 40o en
-
'- 30--
~
J
~ 2o-
+
Phosphorylated: NT
Rhodopsin
Std
i I--'-I
[28]
HETEROLOGOUS EXPRESSION AND RECONSTITUTION OF RHODOPSIN
421
for 5 min to allow arrestin to bind rhodopsin. To terminate the reaction, the assay mixture is placed on ice and 200/zl of ice-cold arrestin binding buffer is added. The diluted sample is loaded onto a 200/xl cushion of icecold 0.2 M sucrose in arrestin binding buffer and immediately centrifuged at 2° at 125,000g (Beckman TLA-45, 45,000 rpm) to separate free arrestin from arrestin bound to rhodopsin. The length of centrifugation time has been varied from 3 to 30 min without significant changes in the level of arrestin bound to rhodopsin. The pellets are washed once with 200/zl of ice-cold arrestin binding buffer, solubilized in Laemmli buffer, and analyzed by 10% SDS-PAGE and phosphorimage analysis. A sample consisting of 10 fmol of in vitro-translated arrestin is applied to the gel as a standard so that the amount of arrestin bound to rhodopsin can be estimated. Arrestin binding to nontransfected cell membranes is used as a measure of nonspecific binding. Approximately 40-65% of the arrestin in the assay binds to HEK293 cell membranes containing wild-type rhodopsin under these conditions (Fig. 3B). Our laboratory has used this method to characterize the phosphorylation sites on rhodopsin that play a role in arrestin binding in vitro. 2 We have measured an apparent KD of approximately 0.74 nM for arrestin binding to HEK293 cell membranes expressing rhodopsin. However, the active form of rhodopsin, metarhodopsin-II, is known to decay in a temperaturesensitive manner. For example, detergent-extracted rhodopsin isolated from COS-1 cells decays with a half-time of 18 rain at 20°. TM Therefore, equilibrium binding studies cannot accurately be performed. Nevertheless, the value that we reported is significantly lower than the previously published value of 50 nM, obtained from light scattering studies 19 and not very different from the value of 2 nM reported for the binding of visual arrestin to the/32-adrenergic receptor. 2° This may be due in part to the ability of arrestin to stabilize metarhodopsin-II in a manner similar to transducin, 1~ is T. Sakamoto and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 92, 249 (1995). 19A. Schleicher, H. Kilhn, and K. P. Hofmann, Biochemistry 28, 1770 (1989). 20V. V. Gurevich, S. B. Dion, J. J. Onorato, J. Ptasienski, C. M. Kim, R. Sterne-Marr, M. M. Hosey, and J. L. Benovic, J. BioL Chem. 270, 720 (1995).
FIG. 3. Arrestin binding to bovine rhodopsin expressed in HEK293 cells. (A) Nontransfeeted (NT) and rhodopsin-transfected HEK293 cell membranes (Rhodopsin) were incubated with [35S]methionine-labeled, in vitro-translated arrestin, centrifuged to isolate the arrestin/rhodopsin complexes, and subjected to 10% SDS-PAGE. A lane of arrestin representing 10 fmol (Std) was also applied to the gel. The samples were analyzed using a Molecular Dynamics PhosphorImager. -, Nonphosphorylated; +, phosphorylated. (B) Quantitation of the polyacrylamide gel shown in (A). Labels are as described in (A). The label Arrestin Bound (%) refers to the fraction of arrestin in the assay that binds to rhodopsin.
422
PROTEINS THAT INTERACT WITH RHODOPSIN
[29]
allowing the isolation of arrestin/rhodopsin complexes without appreciable dissociation during centrifugation. These observations suggest that this arrestin binding assay is a reasonable method for determining relative affinities for rhodopsin mutants expressed in cultured cells.
[29] A r r e s t i n : M u t a g e n e s i s , E x p r e s s i o n , P u r i f i c a t i o n , a n d Functional Characterization B y V S E V O L O D V . G U R E V I C H a n d J E F F R E Y L. B E N O V I C
Introduction In the photoreceptor cell the activation of rhodopsin by a photon of light initiates two cascades of events: signal transduction and signal termination. The visual amplification cascade (rhodopsin ~ transducin ~ cGMP phosphodiesterase) has long served as an archetypal G-protein-coupled receptor signaling system. 1 A single light-activated rhodopsin (Rh*) can sequentially activate hundreds of transducin molecules, each of which in turn activates a cGMP phosphodiesterase resulting in the hydrolysis of thousands of cGMP molecules. Thus, the potential for signal amplification is enormous, providing for very high sensitivity. However, because relatively modest local changes in the cGMP concentration are sufficient for a full cellular response, the signaling machinery needs to be turned off as soon as the signal goes through. A parallel chain of events brings this about, e The initial step in this turn off is the recognition and phosphorylation of Rh* by the enzyme rhodopsin kinase. Rhodopsin phosphorylation attenuates its ability to activate transducin and increases its affinity for the retinal protein, arrestin. Arrestin binds to phosphorylated Rh* (P-Rh*) and effectively shuts down the phototransduction cascade by blocking further transducin activation. Arrestin appears to stay bound to P-Rh* until it decays into phosphoopsin after which arrestin dissociates and phosphoopsin is dephosphorylated by a type IIA protein phosphatase. Retinal arrestin, also termed 48-kDa protein or S-antigen, was initially identified as a protein that binds to disk membranes following light activation of rhodopsin. 3 Arrestin binding and its ability to terminate signaling require rhodopsin phosphorylation? In fact, although arrestin can indepen1 p. A. Hargrave and J. H. McDowell, FASEB J. 6, 2323 (1992). 2 U. Wilden, Biochemistry 34, 1446 (1995). 3 U. Wilden, S. W. Hall, and H. Kuhn, Proc. Natl. Acad. Sci. U.S.A. 83, 1174 (1986).
METHODS IN ENZYMOLOGY,VOL. 315
Copyright © 2000 by AcademicPress All rights of reproductionin any form reserved. 0076-6879/00 $30.00