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The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes
European Journal of Pharmacology 745 (2014) 117–122
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes Eiji Arimitsu a,b, Tomio Ogasawara c, Hiroyuki Takeda c, Tatsuya Sawasaki c, Yoshio Ikeda b, Yoichi Hiasa b, Kazutaka Maeyama a,n a
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan c Division of Cell-free Sciences, Proteo-Science Center, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 6 October 2014 Accepted 8 October 2014 Available online 16 October 2014
G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [3H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61 7 0.06 nM and 1.85 70.05 pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.6770.05 nM and Bmax of 0.7070.10 pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5–17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs. & 2014 Elsevier B.V. All rights reserved.
Keywords: Cell-free expression G-protein coupled receptors Binding assay Dopamine receptor
1. Introduction Dopamine is a neurotransmitter in the central and peripheral nervous system with a wide variety of physiological and behavioral functions, including movement, cognitive functions, reward and decision making. There are five dopamine receptors in mammals, which are grouped into the D1-like class (D1 and D5 receptors) and D2-like class (D2, D3, and D4 receptors) based on their biochemistry, pharmacology and amino acid sequence similarity. All of these receptors are members of the rhodopsin class A of GPCRs. GPCRs are major control units of cellular signal transduction events related to central processes such as development, proliferation, angiogenesis or cancer. Indeed GPCRs are estimated to comprise more than 40% of current drug targets (Filmore, 2004; Lagerstöm and Schiöth, 2008). The GPCR superfamily is generally divided into three main List of abbreviations: GPCRs, G-protein coupled receptors; Kd, dissociation constant; Bmax, maximum density n Corresponding author. Tel.: þ 81 89 960 5258; fax: þ 81 89 960 5263. E-mail address: [email protected] (K. Maeyama). http://dx.doi.org/10.1016/j.ejphar.2014.10.011 0014-2999/& 2014 Elsevier B.V. All rights reserved.
classes (A, B, and C) with no detectable shared sequence homology between classes (Hollenstein et al., 2014; Pal et al., 2012). Detailed knowledge of the 3-D structures of GPCR molecules is currently a major objective of research in this field as it is an indispensable prerequisite for directed drug targeting and rationally designed screening approaches used in the pharmaceutical industry. So far, most structural and functional approaches to GPCRs have been severely limited by difficulties in the production of sufficient amounts of receptor proteins using conventional expression systems. Recently, some groups have reported the cell-free synthesis of functional membrane proteins in the presence of liposomes (Nozawa et al., 2011; Proverbio et al., 2013; Schwarz et al., 2007). The productivity of cell-free protein expression systems using bacterial or wheat germ extracts has been optimized in recent years and preparative amounts of recombinant protein can be synthesized in a single milliliter of reaction mixture after just a few hours (Basu et al., 2013; Endo and Sawasaki, 2006; Sawasaki et al., 2002). Cell-free protein synthesis systems have now emerged as a promising tool for membrane protein production for structural and functional analyses. In addition to decoupling protein production from the toxic or
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inhibitory effects on host cell physiology, cell-free protein synthesis systems also offer a unique advantage in that protein synthesis can be easily modified by addition of accessory elements, such as detergents and lipids (Ishihara et al., 2005; Klammt et al., 2007). The addition of detergents and lipids to cell-free protein synthesis sys tems allows the synthesis of membrane protein/detergent and membrane protein/lipid complexes, respectively. In this study, we synthesized dopamine D1 receptors using a wheat germ cell-free protein synthesis system with liposomes and analyzed the receptor binding ability. The binding properties of a panel of agonists and antagonists to dopamine D1 receptors were also investigated.
2. Materials and methods 2.1. Drugs The specific D1 dopamine receptor antagonist [3H]SCH23390 (in saturation and competitive binding experiments, 80.0 Ci/mmol, Perkin-Elmer, Waltham, MA) was used to label dopamine receptors. The following drugs were used as cold competitors: SCH23390 hydrochloride, SKF83566 hydrobromide, LE300, A68930 hydrochloride, SKF38393 hydrobromide, remoxipride hydrochloride were obtained from TOCRIS Bioscience (Bristol, United Kingdom). Dopamine was obtained from Wako Pure Chemical Industries (Osaka, Japan). Norepinephrine and epinephrine were obtained from SigmaAldrich (St. Louis, MO).
antibody against human dopamine D1 receptor (LS-C50132) was purchased from Lifespan BioSciences (Seattle, WA). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody was purchased from Invitrogen. The blots were treated with a chemiluminescence substrate and determined using GE Image Quant TL (GE Healthcare, Uppsala, Sweden). 2.4. Preparation of rat striatal membranes Male Wistar rats (200–300 g) (Japan SLC, Hamamatsu, Japan) were housed at a constant temperature of 2272 1C with a humidity of 55710% on an automatically controlled 12:12 h light-dark cycle. Animal care and research protocols were in accordance with the principles and guidelines adopted by the Animal Care Committee of Ehime University and approved by the University Committee for Animal Research. After animals were euthanized, striatal tissues were quickly dissected, washed with ice-cold 0.9% NaCl solution, pooled and homogenized in 10 volumes of ice-cold 50 mM Tris–HCl buffer, pH 7.4, using a Potter glass homogenizer. The homogenate was then centrifuged at 1500g for 15 min and the resulting supernatant centrifuged at 20,000g for 10 min. All procedures were carried out at 4 1C. The final pellet was resuspended in 50 mM Tris–HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4. 2.5. Kinetics and saturation of [3H]SCH23390 binding
Details of the wheat cell-free reaction have been described previously (Nozawa et al., 2011). Cell-free protein synthesis was conducted using the WEPRO1240 Expression Kit (Cell-Free Sciences, Ehime, Japan). The open reading frame of human dopamine D1 receptor was amplified by PCR using full-length cDNA clones, obtained from Mammalian Gene Collection, as template (Strausberg et al., 1999). The amplified PCR product was subcloned into pEU-E01 vector (Takai, et al., 2010) using Gateway system (Life Technologies), and the resultant plasmid was used as transcription template. Translation reactions were performed using a bilayer method. For the bilayer systems, 3.5 ml of substrate mixture (SUB-AMIX, Cell-Free Science) was carefully overlaid on 500 μl of translation mixture containing wheat germ extract, mRNA, 40 μg/ml creatine kinase, 5% glycerol and 10 mg/ml asolectin liposome in 6-well titer plates and incubated at 26 1C for 14 h. In this system about 4 mg protein in liposomes in 4 ml of reaction mixture in a well was harvested.
The specific binding of [3H]SCH23390 to dopamine D1 receptors in the liposome preparations and rat striatal membranes was measured by a filtration technique. For saturation binding studies, [3H]SCH23390 was used at a final concentration of 1.0–16.0 nM. Each assay was performed in triplicate using 0.25 ml aliquots containing about 50–100 μg protein which was measured according to the method of Lowry et al. (1951). Non-specific binding was measured in the presence of 1 μM unlabeled SCH23390. After the samples of liposome preparations and rat striatal membranes were incubated at 22 1C for 90 min, the reaction was terminated by filtering the assay mixture through polypropylene membrane filters (GH Polypro 0.45 μm 25 mm) pretreated with 50 mM Tris– HCl buffer, pH 7.4, at 4 1C, which were chosen to trap the liposomes instead of the glass filters. The filters were washed twice with 5 ml of 50 mM Tris–HCl buffer, then transferred into scintillation cocktail vials and 5 ml PICO-FLUOR PLUS liquid scintillation cocktail was added. Bound radioactivity was determined by counting (4 min) in an Aloka LSC-5100 liquid scintillation counter.
2.3. SDS-PAGE separation and Western blotting
2.6. Pharmacology of [3H]SCH23390 binding
Proteins in liposomes were separated by SDS-PAGE using a 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) and stained with Coomassieblue. The resolved proteins were then blotted onto a polyvinylidene difluoride membrane using the NuPAGE transfer buffer and XCell SureLock system (both from Invitrogen) according to the E-PAGE guide blotting protocol. The membrane was then blocked for 1 h in phosphate buffered saline supplemented with 5% skimmed milk powder and 0.1% Tween-20 followed by incubation with the first antibody (dilution 1:2000) at 4 1C overnight with gentle shaking in antibody incubation buffer (phosphate buffered saline supplemented with 1% skimmed milk powder and 0.1% Tween-20). After three times washing in the antibody incubation buffer, the secondary antibody conjugated with horseradish peroxidase was added to the antibody incubation buffer at a final concentration of 1:2000 for 1 h at room temperature. After extensive washing in the washing buffer, the membrane was analyzed by the Western Breeze Chromogenic Western Blot Immunodetection Kit (Invitrogen). The first rabbit
Eight concentrations of each cold competitor were used in the competitive binding studies. Increasing concentrations of the unlabeled ligands were incubated with 8 nM [3H]SCH23390 and liposome preparations or rat striatal membranes. The data presented are based on three separate experiments each performed in triplicate. The experimental conditions were the same as those used in the saturation studies.
2.2. Wheat cell-free protein synthesis
2.7. Data analysis The individual competition curve data were expressed as the percentage of decrease in specific binding of [3H]SCH23390 within each experiment. Saturation and competition curves were fitted by non-linear regression using Prism (Graph Pad Software Incorporated, La Jolla, CA). Inhibition constants (Ki) were determined using the Cheng–Prusoff equation in order to correct for receptor occupancy by the radioligand.
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We performed two-sample t-tests assuming unequal variance to evaluate the statistical significance of the differences in Ki values of ligands for synthesized and native receptors. P-values o0.01 were considered to be significant differences.
kDa
M
119
DA D1
FT
150
100 75
3. Results 3.1. Analysis of cell-free expressed dopamine D1 receptors In this study, we synthesized human dopamine D1 receptors using a wheat germ cell-free protein synthesis system with liposomes. In order to confirm that liposomes were trapped by the 0.45 μm of polypropylene membrane filter in the filtration assay, the lea ked proteins in the flow-through fraction were checked. The 0.25 ml liposome suspension containing 50–100 mg protein was filtered thr ough the polypropylene membrane filter and washed with 50 mM Tris–HCl buffer (pH 7.4). The flow-through fraction was collected and concentrated to the same volume of applied liposome suspension. SDS-PAGE followed by immunoblot analysis detected a band corresponding to the anticipated molecular mass (40 kDa) of dopamine D1 receptor in the sample prior to filtration (lane DAD1 in Fig. 1A and B). The ratio of synthetic dopamine D1 receptors to total protein produced by the wheat germ cell-free protein synthesis system was 32% based on densitometric determination. An extremely weak band corresponding to the expected size of the dopamine D1 receptor was detected in the flow-through fraction (lane FT in Fig. 1A and B). Finally, more than 94 % of liposomal dopamine D1 receptor protein was trapped by the polypropylene membrane filters. 3.2. Kinetics and saturation of [3H]SCH23390 binding 3
Next, we analyzed the specific binding of [ H]SCH23390 to the synthesized receptors and rat striatal membranes. The specific binding of [3H]SCH23390 was time-dependent, a plateau being reached after 90 min for the synthesized receptors (Fig. 2A). The specific binding of [3H]SCH23390 to the synthesized receptors and rat striatal membranes became saturated as the concentration of the radioligand increased (Fig. 2B and D). The apparent dissociation constants (Kd) and the maximum number of binding sites (Bmax) were calculated from Scatchard plot analysis (Fig. 2C and E). The Kd and Bmax of the synthesized receptors were 6.6170.06 nM and 1.8570.05 pmol/mg protein, respectively. The corresponding values for rat striatal membranes were 2.6770.05 nM and 0.7070.10 pmol/mg protein, respectively (Table 1). The saturation isotherms of [3H]SCH23390 binding to the synthesized receptors and rat striatal membranes are shown in Fig. 2F. These data show that a higher number of dopamine D1 receptors per protein(Po0.01) with less affinity (Po0.01) are obtained by the cell-free protein synthesis system by comparison to rat striatal membranes. 3.3. Pharmacological study of [3H]SCH23390 binding We also characterized the synthesized dopamine D1 receptors in liposomes. Here, a series of displacement binding experiments were performed using a fixed concentration (8 nM) of [3H]SCH23390 and increasing concentrations of dopamine D1 receptor antagonists (Fig. 3A and B) and agonists (Fig. 3C and D). Dopamine D1 antagonists, SCH23390, LE300 and SKF83566, produced concentration-dependent inhibition of the specific binding of [3H]SCH23390 to dopamine D1 binding sites in the synthesized receptors and rat striatal membranes. The most potent inhibitor of [3H]SCH23390 binding was cold SCH23390 with a Ki value of a few nM, followed by less potent antagonists, LE300 and SKF83566, in the synthesized receptors and rat striatal membranes (Ki in the tens of nM range; see Table 2). Rem oxipride, a dopamine D2 receptor antagonist, has no displacement
50
*
37
*
25
kDa 75
DA D1
FT
50
*
*
37 Fig. 1. SDS-PAGE separation and Western blotting of cell-free expressed dopamine D1 receptors. The 0.25 ml liposome suspension containing 50 mg protein (DAD1) was filtered through the polypropylene membrane filter and washed with 50 mM Tris–HCl buffer (pH 7.4). The flow-through fraction (FT) was collected and concentrated to the same volume of applied liposome suspension. (A) These samples were separated by 4–12% SDS-PAGE and the gel was stained with Coomassie-blue. (B) Western blotting analysis of dopamine D1 receptor (DAD1) and the flow-through fraction (FT) The samples were obtained in the same way, separated by 4–12% SDS-PAGE and immunoblotted with anti-human dopamine D1 antibodies. The anticipated molecular mass (40 kDa) of dopamine D1 receptor was shown (n).
ability for [3H]SCH23390 binding in the synthesized receptors and rat striatal membranes. In the displacement binding experiments with agonists, we first determined the agonists binding of catecholam ines, i.e., dopamine, epinephrine and norepinephrine. The Ki values of dopamine, epinephrine and norepinephrine were, respectively, 10.970.1 μM, 47.970.1 μM and 122.270.5 μM in the synthesized receptors, but 0.65270.104 μM, 8.5270.53 μM and 23.370.3 μM in rat striatal membranes. These findings show that dopamine binds to the synthesized dopamine D1 receptors with the highest affinity among these catecholamines (Table 2). We also compared the agonist binding affinity of A68930 and SKF38393. The Ki values of A68930 and SKF38393 to the synthesized receptors were 18476 nM and 944757 nM, respectively, which were 5–6 fold greater than those of rat striatal membranes. The Ki values of SCH23390 and all agonists to the synthesized receptors were significantly higher than those observed in rat striatal membranes (Po0.01).
4. Discussion Dopamine is a neurotransmitter in the central and peripheral nervous system with a wide variety of physiological and behavioral functions. The dopamine D1 receptor is the most abundant dopamine receptor in the central nervous system. This GPCR stimulates adenylyl
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Fig. 2. Binding characteristics of [3H]SCH23390 to the synthesized receptors from the cell-free protein synthesis system and rat striatal membranes. (A) Time course of [3H] SCH23390 binding to the synthesized receptors. [3H]SCH23390 saturation curve (B) and Scatchard analysis of [3H]SCH23390 binding (C) to the synthesized receptors. [3H] SCH23390 saturation curve (D) and Scatchard analysis of [3H]SCH23390 binding (E) on rat striatal membranes. Specific binding (▲) was calculated as the difference between total binding (●) and nonspecific binding (■) obtained in the absence and presence of 1 μM unlabeled, respectively. (F) Specific binding of [3H]SCH23390 on the synthesized receptors (●) and rat striatal membranes (▲) were compared. Each point represents the mean 7S.E.M. of three experiments performed in triplicate.
Table 1 Binding of [3H] SCH23390 to synthesized dopamine D1 receptors with liposomes and Rat striatal membranes.
Synthesized receptors Rat striatal membranes
Bmax (pmol/mg protein)
Kd (nM)
1.85 7 0.05a 0.707 0.10
6.617 0.06a 2.677 0.05
Each value of Bmax and Kd represents the mean 7 S.E.M. of values obtained by nonlinear regression of experimental data from saturation curves. a
P o0.01 vs value of rat striatal membranes.
cyclase and indirectly activates cyclic AMP-dependent protein kinases. Dopamine D1 receptors regulate neuronal growth and development, mediate some behavioral responses, and modulate dopamine receptor D2-mediated events. A dysfunction of dopaminergic neurons can lead to the onset of Parkinson's disease. L-DOPA has been clinically used in patients with Parkinson's disease to replenish the shortage of dopamine. Several dopamine D2 receptor agonists, such as bromocriptine and pergolide, have been used, but their efficacy is lower compared with L-DOPA treatment. However, dopamine D1 receptor agonists have been found to improve Parkinsonian symptoms (Rascol et al., 2001). Dopamine D1 and D2 receptors are co-localized as hetero-oligomers both in the striatum and in the cortex. These oligomeric receptors have a synergistic effect. Peripheral dopamine D1 receptors are important in the regulation of renal function and blood pressure. Dopamine released from dendritic cells has been shown to display a novel function in terms of inducing the differentiation of Th17 cells and causing experimental autoimmune encephalitis (Nakano et al., 2008) and antigen-induced neutrophilic airway inflammation (Nakagome et al., 2011). The drugs targeting dopamine D1 receptors may be useful in the treatment of a wide range of disorders, including Parkinson's disease (Rascol et al., 2001), schizophrenia (Mu et al., 2007), hypertension (Granda et al., 2014), allergy and autoimmune diseases (Nakagome et al., 2011; Nakano et al., 2008 ).
To investigate the natural function of dopamine via dopamine D1 receptors, several manipulation methods to reconstitute this kind receptor have been used after transfection of its cloned cDNA into eukaryotic cells. Since the cell-free protein synthesis system was developed (Endo and Sawasaki, 2006; Ogasawara et al., 1999; Spirin et al., 1988), membrane proteins such as GPCRs have been targeted for study. These in vitro expression systems usually employ Escherichia coli ribosomes (Schwarz et al., 2007) or wheat germ ribosomes (Nozawa et al., 2011). In this study, we obtained specific binding of dopamine D1 ligand, [3H]SCH23390 to human dopamine D1 receptors synthesized in liposomes using a wheat germ cell-free protein synthesis system. Using this method, dopamine D1 receptor protein was highly produced in the liposome membranes as confirmed by SDS-PAGE and Western blotting using anti human dopamine D1 receptor antibody (Fig. 1). In the radioligand binding experiments to dopamine D1 receptors with [3H]SCH23390, the free isotope ligand was separated after filtration through a polypropylene membrane of pore size 0.45 μm. Under these conditions, 494% of liposomes containing dopamine D1 receptors in the applied sample were trapped by the membrane. The specific binding of [3H] SCH23390 on the synthesized receptors showed radioactivity increase in a hyperbolic manner in accordance with the increase in [3H]SCH23390 concentration. From the Scatchard-plot analysis, the Kd value of SCH23390 to synthesized receptors,6.61 nM, was significantly higher than that of rat striatum (2.67 nM, Tables 1, Po0.01), but almost in the same order as the previously reported values for human dopamine D1 receptors. The Kd values of [3H] SCH23390 on human D1 receptors expressed in HEK cells and CHO cells were previously determined to be 2.41 nM and 0.58 nM, respectively (Kassack et al., 2002). Basu et al. (2013) succeeded in expressing the long isoform of human dopamine D2 receptor, which are located at the postsynaptic membrane, using two different cellfree protein synthesis systems with E. coli lysate and wheat germ lysate. The recovery of receptor proteins, having the specific binding activity of dopamine D2 antagonist, norpropylapomorphine, was 15 pmol/mg of total protein and 0.4 pmol/mg of total protein using
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Fig. 3. Pharmacological specificity of [3H]SCH23390 binding to the synthesized receptors with cell-free protein synthesis system and rat striatal membranes. Competitive inhibition curves of [3H]SCH23390 to the synthesized receptors (A) and rat striatal membranes (B) by dopamine D1 receptor antagonists, SCH23390 (●), SKF83566 (○), LE300 (△) and D2 receptor antagonist, remoxipride (◆). Competitive inhibition curves of [3H]SCH23390 to the synthesized receptors (C) and rat striatal membranes (D) by dopamine D1 receptor agonists, A68930 (○), SKF38393 (●), dopamine (△), epinephrine (▲) and norepinephrine (□). Apparent IC50 values were determined in competition assays with 8 nM [3H]SCH23390 preincubated with increasing concentrations of antagonists and agonists (Table 2). Each point represents the mean 7 S.E.M. of three experiments performed in triplicate.
the E. coli and wheat germ cell-free protein synthesis systems, respectively. In our system, the dopamine D1 receptor was obtained as 1.85 pmol/mg of total protein (Bmax). In the [3H]SCH23390 competitive binding assay of the synthesized receptors, the most potent inhibitor of [3H]SCH23390 binding was SCH23390, followed by less potent antagonists, LE300 and SKF83566, and the Ki values of these antagonists were 0.895–1.43 fold of those estimated in the rat striatal membranes. LE300, a nanomolar dopamine receptor antagonist combining structural core elements of dopamine and serotonin, showed a 20-fold selectivity for human D1 receptors over D2 receptors (Kassack et al., 2002). However, a dopamine D2 antagonist was ineffective at suppressing [3H]SCH23390 binding. The Ki values of dopamine agonists, A68930 and SKF38393, using the synthesized receptors were 184 nM and 944 nM, respectively. Both these agonists showed 5–6 fold greater binding for the rat striatal membranes over that of the synthesized receptors (Table 2). The affinity of dopamine for the synthesized receptor (Ki of 10.9 μM) was greater than that of epinephrine or norepinephrine, but the affinity was 16.7-fold lower compared to the binding with rat striatal membrane. The binding affinity of antagonists for the synthesized dopamine D1 receptor was almost similar to that observed for the rat striatal membranes. However, the binding affinity of agonists, A68930, SKF38393 and dopamine, for the synthesized receptors was significantly weaker compared to that of the rat striatal membranes. The different binding patterns of antagonists and agonists to the synthetic receptors could be attributed to the lack of G proteins in our synthetic receptors, as suggested by the idea that the receptor in its active state results from the formation of agonist-receptor-G protein complex. Dopamine D1 receptors are mainly coupled with Gαs, an isoform of G protein, of which GDP is exchanged to GTP after agonist binding, resulting in dissociation from the βγ dimer and interaction with an appropriate effector, adenylyl cyclase. Treatment with cholera toxin, which disrupts the interaction between receptor and Gαs, reduced the
Table 2 Ability of ligands to displace [3H] SCH23390 binding to synthesized dopamine D1 receptor with liposomes and rat striatal membranes.
Displacer Antagonists R(þ )-SCH23390 LE300 SKF83566 Agonists A68930 ( 7)-SKF38393 Dopamine Epinephrine Norepinephrine
Synthesized receptors Ki (nM)
Rat striatal membranes Ki (nM)
3.62 7 0.15a 22.647 1.28 90.497 2.24
2.53 70.24 20.20 70.40 101.017 4.15
184.3 75.8a 9447 57a 10,860 7 86a 47,910 7146a 122,1727 524a
28.6 716.8 1827 38 652 7 104 8524 7529 23,308 7337
Data represent the average of three independent experiments performed in triplicates. a
Po 0.01 vs value of rat striatal membranes.
number of agonist high affinity binding sites of the dopamine D1 receptor in rat pituitary GH4C1 cells and SK-N-MC neuroblastoma cells (Kimura, et al., 1995). Dopamine D1 receptor supersensitivity was reported in striatal membranes from reserpine treated rats due to the increased high affinity agonist binding state (Butkerait, et al., 1994). Taken together, our results suggest that the synthesized dopamine D1 receptors in liposomes have a functional binding capacity. The Bmax was calculated according to the equation of Scatchard plot as normalized by concentration of liposomal protein. As shown in Fig. 1, liposomal proteins contained the receptor proteins, namely 32% of total protein ( 320 μg of receptor protein per mg total protein). Assuming the molecular weight of human dopamine D1 receptor estimated from amino acid numbers as 49,300, the product of dopamine D1 receptor was calculated to be 6.49 nmol/mg of total protein, whereas an extremely low Bmax value was obtained. There
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are several papers indicating that wild-type GPCRs expressed in a cell-free protein synthesis system lack ligand binding activity (Yang et al. 2011). Our experiments suggest that only a small fraction, that is, 0.02% of the total number of receptors, might participate in ligand binding. In the cell-free protein synthesis system used to produce membrane proteins, several factors, such as posttranslational modification and membrane composition, are required to ensure that the synthesized receptors adopt a proper conformation to enable them to bind their ligand. In particular, receptor status changes dynamically between inactivate and activate forms. To circumvent these problems, trials have been performed to insert T4 bacteriophage lysozyme sequences into the third intracellular loop in order to stabilize the receptor, which recovered the binding activities of β2adrenoceptor (Yang et al., 2011) and histamine H1 receptor (Shimamura et al., 2011). Yang et al. (2011) also pointed out that oxidative protein folding is required for ligand binding. In human β2adrenoceptor the extracellular disulfide bond between cysteines at position 184 and 190 is important to maintain binding activity. Intriguingly, β2-adrenoceptors produced in the absence of dithiothreitol using the cell-free protein synthesis system maintain ligand binding activity. To express fully functional receptors in liposomes, further consideration of the stability and conformation of the receptor protein will be necessary. The use of wheat germ ribosomes has the advantages of high production recovery (1.28 mg receptor protein in 4 ml reaction mixture after 14 h) of folded protein with a proper conformational structure owing to the characteristics of eukaryotic translation systems, in which their 80 S ribosomes produce proteins at a 10 times slower rate compared with prokaryotic ribosomes. The yield of mg order of GPCRs in liposomes will be an invaluable method for generating antibodies against targeted receptors. Recently, the crystal structural analysis of GPCRs and ion channels have produced important results and currently more than 14 GPCRs have been analyzed (Rasmussen et al., 2011). In the dopamine receptor family, the crystal analysis of human dopamine D3 receptor has already been completed (Chien et al., 2010). The wheat germ cellfree protein synthesis system will contribute to the crystal analysis of many GPCRs. In conclusion, we have demonstrated for the first time a pharmacological study of a human dopamine D1 receptor synthesized in liposomes using a wheat germ cell-free protein synthesis system. The observed selective binding activity of the receptor to [3H]SCH23390 infers that these synthesized receptors were expressed in their native conformation. This method will be useful in generating membrane proteins in vitro for insightful analysis of GPCRs, ion channels and transporters as well as for the development of new drugs to target these important proteins. Acknowledgments We thank professors Y. Endo and M. Onji (Professors emeritus, Ehime University) for advice on studies using cell-free protein synthesis system and helpful discussion and Dr. Y. Tanaka (Integrated Center for Sciences, Ehime University) for her technical supports. References Basu, D., Castellano, J.M., Thomas, N., Mishra, R.K., 2013. Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform. Biotechnol. Prog. 29, 601–608. Butkerait, P., Wang, H.Y., Friedman, E., 1994. Increases in guanine nucleotide binding to striatal G proteins is associated with dopamine receptor supersensitivity. J. Pharmacol. Exp. Ther. 271, 422–428. Chien, E.Y.T., Liu, W., Zhao, Q., Katritch, V., Han, G.W., Hanson, M.A., Shi, L., Newman, A.H., Javitch, J.A., Cherezov, V., Stevens, R., 2010. Structure of the human
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes Eiji Arimitsu a,b, Tomio Ogasawara c, Hiroyuki Takeda c, Tatsuya Sawasaki c, Yoshio Ikeda b, Yoichi Hiasa b, Kazutaka Maeyama a,n a
Department of Pharmacology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan c Division of Cell-free Sciences, Proteo-Science Center, Ehime University, 3 Bunkyo-Cho, Matsuyama, Ehime 790-8577, Japan b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 June 2014 Received in revised form 6 October 2014 Accepted 8 October 2014 Available online 16 October 2014
G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [3H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61 7 0.06 nM and 1.85 70.05 pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.6770.05 nM and Bmax of 0.7070.10 pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5–17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs. & 2014 Elsevier B.V. All rights reserved.
Keywords: Cell-free expression G-protein coupled receptors Binding assay Dopamine receptor
1. Introduction Dopamine is a neurotransmitter in the central and peripheral nervous system with a wide variety of physiological and behavioral functions, including movement, cognitive functions, reward and decision making. There are five dopamine receptors in mammals, which are grouped into the D1-like class (D1 and D5 receptors) and D2-like class (D2, D3, and D4 receptors) based on their biochemistry, pharmacology and amino acid sequence similarity. All of these receptors are members of the rhodopsin class A of GPCRs. GPCRs are major control units of cellular signal transduction events related to central processes such as development, proliferation, angiogenesis or cancer. Indeed GPCRs are estimated to comprise more than 40% of current drug targets (Filmore, 2004; Lagerstöm and Schiöth, 2008). The GPCR superfamily is generally divided into three main List of abbreviations: GPCRs, G-protein coupled receptors; Kd, dissociation constant; Bmax, maximum density n Corresponding author. Tel.: þ 81 89 960 5258; fax: þ 81 89 960 5263. E-mail address: [email protected] (K. Maeyama). http://dx.doi.org/10.1016/j.ejphar.2014.10.011 0014-2999/& 2014 Elsevier B.V. All rights reserved.
classes (A, B, and C) with no detectable shared sequence homology between classes (Hollenstein et al., 2014; Pal et al., 2012). Detailed knowledge of the 3-D structures of GPCR molecules is currently a major objective of research in this field as it is an indispensable prerequisite for directed drug targeting and rationally designed screening approaches used in the pharmaceutical industry. So far, most structural and functional approaches to GPCRs have been severely limited by difficulties in the production of sufficient amounts of receptor proteins using conventional expression systems. Recently, some groups have reported the cell-free synthesis of functional membrane proteins in the presence of liposomes (Nozawa et al., 2011; Proverbio et al., 2013; Schwarz et al., 2007). The productivity of cell-free protein expression systems using bacterial or wheat germ extracts has been optimized in recent years and preparative amounts of recombinant protein can be synthesized in a single milliliter of reaction mixture after just a few hours (Basu et al., 2013; Endo and Sawasaki, 2006; Sawasaki et al., 2002). Cell-free protein synthesis systems have now emerged as a promising tool for membrane protein production for structural and functional analyses. In addition to decoupling protein production from the toxic or
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inhibitory effects on host cell physiology, cell-free protein synthesis systems also offer a unique advantage in that protein synthesis can be easily modified by addition of accessory elements, such as detergents and lipids (Ishihara et al., 2005; Klammt et al., 2007). The addition of detergents and lipids to cell-free protein synthesis sys tems allows the synthesis of membrane protein/detergent and membrane protein/lipid complexes, respectively. In this study, we synthesized dopamine D1 receptors using a wheat germ cell-free protein synthesis system with liposomes and analyzed the receptor binding ability. The binding properties of a panel of agonists and antagonists to dopamine D1 receptors were also investigated.
2. Materials and methods 2.1. Drugs The specific D1 dopamine receptor antagonist [3H]SCH23390 (in saturation and competitive binding experiments, 80.0 Ci/mmol, Perkin-Elmer, Waltham, MA) was used to label dopamine receptors. The following drugs were used as cold competitors: SCH23390 hydrochloride, SKF83566 hydrobromide, LE300, A68930 hydrochloride, SKF38393 hydrobromide, remoxipride hydrochloride were obtained from TOCRIS Bioscience (Bristol, United Kingdom). Dopamine was obtained from Wako Pure Chemical Industries (Osaka, Japan). Norepinephrine and epinephrine were obtained from SigmaAldrich (St. Louis, MO).
antibody against human dopamine D1 receptor (LS-C50132) was purchased from Lifespan BioSciences (Seattle, WA). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody was purchased from Invitrogen. The blots were treated with a chemiluminescence substrate and determined using GE Image Quant TL (GE Healthcare, Uppsala, Sweden). 2.4. Preparation of rat striatal membranes Male Wistar rats (200–300 g) (Japan SLC, Hamamatsu, Japan) were housed at a constant temperature of 2272 1C with a humidity of 55710% on an automatically controlled 12:12 h light-dark cycle. Animal care and research protocols were in accordance with the principles and guidelines adopted by the Animal Care Committee of Ehime University and approved by the University Committee for Animal Research. After animals were euthanized, striatal tissues were quickly dissected, washed with ice-cold 0.9% NaCl solution, pooled and homogenized in 10 volumes of ice-cold 50 mM Tris–HCl buffer, pH 7.4, using a Potter glass homogenizer. The homogenate was then centrifuged at 1500g for 15 min and the resulting supernatant centrifuged at 20,000g for 10 min. All procedures were carried out at 4 1C. The final pellet was resuspended in 50 mM Tris–HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4. 2.5. Kinetics and saturation of [3H]SCH23390 binding
Details of the wheat cell-free reaction have been described previously (Nozawa et al., 2011). Cell-free protein synthesis was conducted using the WEPRO1240 Expression Kit (Cell-Free Sciences, Ehime, Japan). The open reading frame of human dopamine D1 receptor was amplified by PCR using full-length cDNA clones, obtained from Mammalian Gene Collection, as template (Strausberg et al., 1999). The amplified PCR product was subcloned into pEU-E01 vector (Takai, et al., 2010) using Gateway system (Life Technologies), and the resultant plasmid was used as transcription template. Translation reactions were performed using a bilayer method. For the bilayer systems, 3.5 ml of substrate mixture (SUB-AMIX, Cell-Free Science) was carefully overlaid on 500 μl of translation mixture containing wheat germ extract, mRNA, 40 μg/ml creatine kinase, 5% glycerol and 10 mg/ml asolectin liposome in 6-well titer plates and incubated at 26 1C for 14 h. In this system about 4 mg protein in liposomes in 4 ml of reaction mixture in a well was harvested.
The specific binding of [3H]SCH23390 to dopamine D1 receptors in the liposome preparations and rat striatal membranes was measured by a filtration technique. For saturation binding studies, [3H]SCH23390 was used at a final concentration of 1.0–16.0 nM. Each assay was performed in triplicate using 0.25 ml aliquots containing about 50–100 μg protein which was measured according to the method of Lowry et al. (1951). Non-specific binding was measured in the presence of 1 μM unlabeled SCH23390. After the samples of liposome preparations and rat striatal membranes were incubated at 22 1C for 90 min, the reaction was terminated by filtering the assay mixture through polypropylene membrane filters (GH Polypro 0.45 μm 25 mm) pretreated with 50 mM Tris– HCl buffer, pH 7.4, at 4 1C, which were chosen to trap the liposomes instead of the glass filters. The filters were washed twice with 5 ml of 50 mM Tris–HCl buffer, then transferred into scintillation cocktail vials and 5 ml PICO-FLUOR PLUS liquid scintillation cocktail was added. Bound radioactivity was determined by counting (4 min) in an Aloka LSC-5100 liquid scintillation counter.
2.3. SDS-PAGE separation and Western blotting
2.6. Pharmacology of [3H]SCH23390 binding
Proteins in liposomes were separated by SDS-PAGE using a 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) and stained with Coomassieblue. The resolved proteins were then blotted onto a polyvinylidene difluoride membrane using the NuPAGE transfer buffer and XCell SureLock system (both from Invitrogen) according to the E-PAGE guide blotting protocol. The membrane was then blocked for 1 h in phosphate buffered saline supplemented with 5% skimmed milk powder and 0.1% Tween-20 followed by incubation with the first antibody (dilution 1:2000) at 4 1C overnight with gentle shaking in antibody incubation buffer (phosphate buffered saline supplemented with 1% skimmed milk powder and 0.1% Tween-20). After three times washing in the antibody incubation buffer, the secondary antibody conjugated with horseradish peroxidase was added to the antibody incubation buffer at a final concentration of 1:2000 for 1 h at room temperature. After extensive washing in the washing buffer, the membrane was analyzed by the Western Breeze Chromogenic Western Blot Immunodetection Kit (Invitrogen). The first rabbit
Eight concentrations of each cold competitor were used in the competitive binding studies. Increasing concentrations of the unlabeled ligands were incubated with 8 nM [3H]SCH23390 and liposome preparations or rat striatal membranes. The data presented are based on three separate experiments each performed in triplicate. The experimental conditions were the same as those used in the saturation studies.
2.2. Wheat cell-free protein synthesis
2.7. Data analysis The individual competition curve data were expressed as the percentage of decrease in specific binding of [3H]SCH23390 within each experiment. Saturation and competition curves were fitted by non-linear regression using Prism (Graph Pad Software Incorporated, La Jolla, CA). Inhibition constants (Ki) were determined using the Cheng–Prusoff equation in order to correct for receptor occupancy by the radioligand.
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We performed two-sample t-tests assuming unequal variance to evaluate the statistical significance of the differences in Ki values of ligands for synthesized and native receptors. P-values o0.01 were considered to be significant differences.
kDa
M
119
DA D1
FT
150
100 75
3. Results 3.1. Analysis of cell-free expressed dopamine D1 receptors In this study, we synthesized human dopamine D1 receptors using a wheat germ cell-free protein synthesis system with liposomes. In order to confirm that liposomes were trapped by the 0.45 μm of polypropylene membrane filter in the filtration assay, the lea ked proteins in the flow-through fraction were checked. The 0.25 ml liposome suspension containing 50–100 mg protein was filtered thr ough the polypropylene membrane filter and washed with 50 mM Tris–HCl buffer (pH 7.4). The flow-through fraction was collected and concentrated to the same volume of applied liposome suspension. SDS-PAGE followed by immunoblot analysis detected a band corresponding to the anticipated molecular mass (40 kDa) of dopamine D1 receptor in the sample prior to filtration (lane DAD1 in Fig. 1A and B). The ratio of synthetic dopamine D1 receptors to total protein produced by the wheat germ cell-free protein synthesis system was 32% based on densitometric determination. An extremely weak band corresponding to the expected size of the dopamine D1 receptor was detected in the flow-through fraction (lane FT in Fig. 1A and B). Finally, more than 94 % of liposomal dopamine D1 receptor protein was trapped by the polypropylene membrane filters. 3.2. Kinetics and saturation of [3H]SCH23390 binding 3
Next, we analyzed the specific binding of [ H]SCH23390 to the synthesized receptors and rat striatal membranes. The specific binding of [3H]SCH23390 was time-dependent, a plateau being reached after 90 min for the synthesized receptors (Fig. 2A). The specific binding of [3H]SCH23390 to the synthesized receptors and rat striatal membranes became saturated as the concentration of the radioligand increased (Fig. 2B and D). The apparent dissociation constants (Kd) and the maximum number of binding sites (Bmax) were calculated from Scatchard plot analysis (Fig. 2C and E). The Kd and Bmax of the synthesized receptors were 6.6170.06 nM and 1.8570.05 pmol/mg protein, respectively. The corresponding values for rat striatal membranes were 2.6770.05 nM and 0.7070.10 pmol/mg protein, respectively (Table 1). The saturation isotherms of [3H]SCH23390 binding to the synthesized receptors and rat striatal membranes are shown in Fig. 2F. These data show that a higher number of dopamine D1 receptors per protein(Po0.01) with less affinity (Po0.01) are obtained by the cell-free protein synthesis system by comparison to rat striatal membranes. 3.3. Pharmacological study of [3H]SCH23390 binding We also characterized the synthesized dopamine D1 receptors in liposomes. Here, a series of displacement binding experiments were performed using a fixed concentration (8 nM) of [3H]SCH23390 and increasing concentrations of dopamine D1 receptor antagonists (Fig. 3A and B) and agonists (Fig. 3C and D). Dopamine D1 antagonists, SCH23390, LE300 and SKF83566, produced concentration-dependent inhibition of the specific binding of [3H]SCH23390 to dopamine D1 binding sites in the synthesized receptors and rat striatal membranes. The most potent inhibitor of [3H]SCH23390 binding was cold SCH23390 with a Ki value of a few nM, followed by less potent antagonists, LE300 and SKF83566, in the synthesized receptors and rat striatal membranes (Ki in the tens of nM range; see Table 2). Rem oxipride, a dopamine D2 receptor antagonist, has no displacement
50
*
37
*
25
kDa 75
DA D1
FT
50
*
*
37 Fig. 1. SDS-PAGE separation and Western blotting of cell-free expressed dopamine D1 receptors. The 0.25 ml liposome suspension containing 50 mg protein (DAD1) was filtered through the polypropylene membrane filter and washed with 50 mM Tris–HCl buffer (pH 7.4). The flow-through fraction (FT) was collected and concentrated to the same volume of applied liposome suspension. (A) These samples were separated by 4–12% SDS-PAGE and the gel was stained with Coomassie-blue. (B) Western blotting analysis of dopamine D1 receptor (DAD1) and the flow-through fraction (FT) The samples were obtained in the same way, separated by 4–12% SDS-PAGE and immunoblotted with anti-human dopamine D1 antibodies. The anticipated molecular mass (40 kDa) of dopamine D1 receptor was shown (n).
ability for [3H]SCH23390 binding in the synthesized receptors and rat striatal membranes. In the displacement binding experiments with agonists, we first determined the agonists binding of catecholam ines, i.e., dopamine, epinephrine and norepinephrine. The Ki values of dopamine, epinephrine and norepinephrine were, respectively, 10.970.1 μM, 47.970.1 μM and 122.270.5 μM in the synthesized receptors, but 0.65270.104 μM, 8.5270.53 μM and 23.370.3 μM in rat striatal membranes. These findings show that dopamine binds to the synthesized dopamine D1 receptors with the highest affinity among these catecholamines (Table 2). We also compared the agonist binding affinity of A68930 and SKF38393. The Ki values of A68930 and SKF38393 to the synthesized receptors were 18476 nM and 944757 nM, respectively, which were 5–6 fold greater than those of rat striatal membranes. The Ki values of SCH23390 and all agonists to the synthesized receptors were significantly higher than those observed in rat striatal membranes (Po0.01).
4. Discussion Dopamine is a neurotransmitter in the central and peripheral nervous system with a wide variety of physiological and behavioral functions. The dopamine D1 receptor is the most abundant dopamine receptor in the central nervous system. This GPCR stimulates adenylyl
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Fig. 2. Binding characteristics of [3H]SCH23390 to the synthesized receptors from the cell-free protein synthesis system and rat striatal membranes. (A) Time course of [3H] SCH23390 binding to the synthesized receptors. [3H]SCH23390 saturation curve (B) and Scatchard analysis of [3H]SCH23390 binding (C) to the synthesized receptors. [3H] SCH23390 saturation curve (D) and Scatchard analysis of [3H]SCH23390 binding (E) on rat striatal membranes. Specific binding (▲) was calculated as the difference between total binding (●) and nonspecific binding (■) obtained in the absence and presence of 1 μM unlabeled, respectively. (F) Specific binding of [3H]SCH23390 on the synthesized receptors (●) and rat striatal membranes (▲) were compared. Each point represents the mean 7S.E.M. of three experiments performed in triplicate.
Table 1 Binding of [3H] SCH23390 to synthesized dopamine D1 receptors with liposomes and Rat striatal membranes.
Synthesized receptors Rat striatal membranes
Bmax (pmol/mg protein)
Kd (nM)
1.85 7 0.05a 0.707 0.10
6.617 0.06a 2.677 0.05
Each value of Bmax and Kd represents the mean 7 S.E.M. of values obtained by nonlinear regression of experimental data from saturation curves. a
P o0.01 vs value of rat striatal membranes.
cyclase and indirectly activates cyclic AMP-dependent protein kinases. Dopamine D1 receptors regulate neuronal growth and development, mediate some behavioral responses, and modulate dopamine receptor D2-mediated events. A dysfunction of dopaminergic neurons can lead to the onset of Parkinson's disease. L-DOPA has been clinically used in patients with Parkinson's disease to replenish the shortage of dopamine. Several dopamine D2 receptor agonists, such as bromocriptine and pergolide, have been used, but their efficacy is lower compared with L-DOPA treatment. However, dopamine D1 receptor agonists have been found to improve Parkinsonian symptoms (Rascol et al., 2001). Dopamine D1 and D2 receptors are co-localized as hetero-oligomers both in the striatum and in the cortex. These oligomeric receptors have a synergistic effect. Peripheral dopamine D1 receptors are important in the regulation of renal function and blood pressure. Dopamine released from dendritic cells has been shown to display a novel function in terms of inducing the differentiation of Th17 cells and causing experimental autoimmune encephalitis (Nakano et al., 2008) and antigen-induced neutrophilic airway inflammation (Nakagome et al., 2011). The drugs targeting dopamine D1 receptors may be useful in the treatment of a wide range of disorders, including Parkinson's disease (Rascol et al., 2001), schizophrenia (Mu et al., 2007), hypertension (Granda et al., 2014), allergy and autoimmune diseases (Nakagome et al., 2011; Nakano et al., 2008 ).
To investigate the natural function of dopamine via dopamine D1 receptors, several manipulation methods to reconstitute this kind receptor have been used after transfection of its cloned cDNA into eukaryotic cells. Since the cell-free protein synthesis system was developed (Endo and Sawasaki, 2006; Ogasawara et al., 1999; Spirin et al., 1988), membrane proteins such as GPCRs have been targeted for study. These in vitro expression systems usually employ Escherichia coli ribosomes (Schwarz et al., 2007) or wheat germ ribosomes (Nozawa et al., 2011). In this study, we obtained specific binding of dopamine D1 ligand, [3H]SCH23390 to human dopamine D1 receptors synthesized in liposomes using a wheat germ cell-free protein synthesis system. Using this method, dopamine D1 receptor protein was highly produced in the liposome membranes as confirmed by SDS-PAGE and Western blotting using anti human dopamine D1 receptor antibody (Fig. 1). In the radioligand binding experiments to dopamine D1 receptors with [3H]SCH23390, the free isotope ligand was separated after filtration through a polypropylene membrane of pore size 0.45 μm. Under these conditions, 494% of liposomes containing dopamine D1 receptors in the applied sample were trapped by the membrane. The specific binding of [3H] SCH23390 on the synthesized receptors showed radioactivity increase in a hyperbolic manner in accordance with the increase in [3H]SCH23390 concentration. From the Scatchard-plot analysis, the Kd value of SCH23390 to synthesized receptors,6.61 nM, was significantly higher than that of rat striatum (2.67 nM, Tables 1, Po0.01), but almost in the same order as the previously reported values for human dopamine D1 receptors. The Kd values of [3H] SCH23390 on human D1 receptors expressed in HEK cells and CHO cells were previously determined to be 2.41 nM and 0.58 nM, respectively (Kassack et al., 2002). Basu et al. (2013) succeeded in expressing the long isoform of human dopamine D2 receptor, which are located at the postsynaptic membrane, using two different cellfree protein synthesis systems with E. coli lysate and wheat germ lysate. The recovery of receptor proteins, having the specific binding activity of dopamine D2 antagonist, norpropylapomorphine, was 15 pmol/mg of total protein and 0.4 pmol/mg of total protein using
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Fig. 3. Pharmacological specificity of [3H]SCH23390 binding to the synthesized receptors with cell-free protein synthesis system and rat striatal membranes. Competitive inhibition curves of [3H]SCH23390 to the synthesized receptors (A) and rat striatal membranes (B) by dopamine D1 receptor antagonists, SCH23390 (●), SKF83566 (○), LE300 (△) and D2 receptor antagonist, remoxipride (◆). Competitive inhibition curves of [3H]SCH23390 to the synthesized receptors (C) and rat striatal membranes (D) by dopamine D1 receptor agonists, A68930 (○), SKF38393 (●), dopamine (△), epinephrine (▲) and norepinephrine (□). Apparent IC50 values were determined in competition assays with 8 nM [3H]SCH23390 preincubated with increasing concentrations of antagonists and agonists (Table 2). Each point represents the mean 7 S.E.M. of three experiments performed in triplicate.
the E. coli and wheat germ cell-free protein synthesis systems, respectively. In our system, the dopamine D1 receptor was obtained as 1.85 pmol/mg of total protein (Bmax). In the [3H]SCH23390 competitive binding assay of the synthesized receptors, the most potent inhibitor of [3H]SCH23390 binding was SCH23390, followed by less potent antagonists, LE300 and SKF83566, and the Ki values of these antagonists were 0.895–1.43 fold of those estimated in the rat striatal membranes. LE300, a nanomolar dopamine receptor antagonist combining structural core elements of dopamine and serotonin, showed a 20-fold selectivity for human D1 receptors over D2 receptors (Kassack et al., 2002). However, a dopamine D2 antagonist was ineffective at suppressing [3H]SCH23390 binding. The Ki values of dopamine agonists, A68930 and SKF38393, using the synthesized receptors were 184 nM and 944 nM, respectively. Both these agonists showed 5–6 fold greater binding for the rat striatal membranes over that of the synthesized receptors (Table 2). The affinity of dopamine for the synthesized receptor (Ki of 10.9 μM) was greater than that of epinephrine or norepinephrine, but the affinity was 16.7-fold lower compared to the binding with rat striatal membrane. The binding affinity of antagonists for the synthesized dopamine D1 receptor was almost similar to that observed for the rat striatal membranes. However, the binding affinity of agonists, A68930, SKF38393 and dopamine, for the synthesized receptors was significantly weaker compared to that of the rat striatal membranes. The different binding patterns of antagonists and agonists to the synthetic receptors could be attributed to the lack of G proteins in our synthetic receptors, as suggested by the idea that the receptor in its active state results from the formation of agonist-receptor-G protein complex. Dopamine D1 receptors are mainly coupled with Gαs, an isoform of G protein, of which GDP is exchanged to GTP after agonist binding, resulting in dissociation from the βγ dimer and interaction with an appropriate effector, adenylyl cyclase. Treatment with cholera toxin, which disrupts the interaction between receptor and Gαs, reduced the
Table 2 Ability of ligands to displace [3H] SCH23390 binding to synthesized dopamine D1 receptor with liposomes and rat striatal membranes.
Displacer Antagonists R(þ )-SCH23390 LE300 SKF83566 Agonists A68930 ( 7)-SKF38393 Dopamine Epinephrine Norepinephrine
Synthesized receptors Ki (nM)
Rat striatal membranes Ki (nM)
3.62 7 0.15a 22.647 1.28 90.497 2.24
2.53 70.24 20.20 70.40 101.017 4.15
184.3 75.8a 9447 57a 10,860 7 86a 47,910 7146a 122,1727 524a
28.6 716.8 1827 38 652 7 104 8524 7529 23,308 7337
Data represent the average of three independent experiments performed in triplicates. a
Po 0.01 vs value of rat striatal membranes.
number of agonist high affinity binding sites of the dopamine D1 receptor in rat pituitary GH4C1 cells and SK-N-MC neuroblastoma cells (Kimura, et al., 1995). Dopamine D1 receptor supersensitivity was reported in striatal membranes from reserpine treated rats due to the increased high affinity agonist binding state (Butkerait, et al., 1994). Taken together, our results suggest that the synthesized dopamine D1 receptors in liposomes have a functional binding capacity. The Bmax was calculated according to the equation of Scatchard plot as normalized by concentration of liposomal protein. As shown in Fig. 1, liposomal proteins contained the receptor proteins, namely 32% of total protein ( 320 μg of receptor protein per mg total protein). Assuming the molecular weight of human dopamine D1 receptor estimated from amino acid numbers as 49,300, the product of dopamine D1 receptor was calculated to be 6.49 nmol/mg of total protein, whereas an extremely low Bmax value was obtained. There
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are several papers indicating that wild-type GPCRs expressed in a cell-free protein synthesis system lack ligand binding activity (Yang et al. 2011). Our experiments suggest that only a small fraction, that is, 0.02% of the total number of receptors, might participate in ligand binding. In the cell-free protein synthesis system used to produce membrane proteins, several factors, such as posttranslational modification and membrane composition, are required to ensure that the synthesized receptors adopt a proper conformation to enable them to bind their ligand. In particular, receptor status changes dynamically between inactivate and activate forms. To circumvent these problems, trials have been performed to insert T4 bacteriophage lysozyme sequences into the third intracellular loop in order to stabilize the receptor, which recovered the binding activities of β2adrenoceptor (Yang et al., 2011) and histamine H1 receptor (Shimamura et al., 2011). Yang et al. (2011) also pointed out that oxidative protein folding is required for ligand binding. In human β2adrenoceptor the extracellular disulfide bond between cysteines at position 184 and 190 is important to maintain binding activity. Intriguingly, β2-adrenoceptors produced in the absence of dithiothreitol using the cell-free protein synthesis system maintain ligand binding activity. To express fully functional receptors in liposomes, further consideration of the stability and conformation of the receptor protein will be necessary. The use of wheat germ ribosomes has the advantages of high production recovery (1.28 mg receptor protein in 4 ml reaction mixture after 14 h) of folded protein with a proper conformational structure owing to the characteristics of eukaryotic translation systems, in which their 80 S ribosomes produce proteins at a 10 times slower rate compared with prokaryotic ribosomes. The yield of mg order of GPCRs in liposomes will be an invaluable method for generating antibodies against targeted receptors. Recently, the crystal structural analysis of GPCRs and ion channels have produced important results and currently more than 14 GPCRs have been analyzed (Rasmussen et al., 2011). In the dopamine receptor family, the crystal analysis of human dopamine D3 receptor has already been completed (Chien et al., 2010). The wheat germ cellfree protein synthesis system will contribute to the crystal analysis of many GPCRs. In conclusion, we have demonstrated for the first time a pharmacological study of a human dopamine D1 receptor synthesized in liposomes using a wheat germ cell-free protein synthesis system. The observed selective binding activity of the receptor to [3H]SCH23390 infers that these synthesized receptors were expressed in their native conformation. This method will be useful in generating membrane proteins in vitro for insightful analysis of GPCRs, ion channels and transporters as well as for the development of new drugs to target these important proteins. Acknowledgments We thank professors Y. Endo and M. Onji (Professors emeritus, Ehime University) for advice on studies using cell-free protein synthesis system and helpful discussion and Dr. Y. Tanaka (Integrated Center for Sciences, Ehime University) for her technical supports. References Basu, D., Castellano, J.M., Thomas, N., Mishra, R.K., 2013. Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform. Biotechnol. Prog. 29, 601–608. Butkerait, P., Wang, H.Y., Friedman, E., 1994. Increases in guanine nucleotide binding to striatal G proteins is associated with dopamine receptor supersensitivity. J. Pharmacol. Exp. Ther. 271, 422–428. Chien, E.Y.T., Liu, W., Zhao, Q., Katritch, V., Han, G.W., Hanson, M.A., Shi, L., Newman, A.H., Javitch, J.A., Cherezov, V., Stevens, R., 2010. Structure of the human
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