Hetero-oligomerization and Specificity Changes of G Protein-Coupled Purinergic Receptors

CHAPTER THIRTEEN

Hetero-oligomerization and Specificity Changes of G Protein-Coupled Purinergic Receptors: Novel Insight into Diversification of Signal Transduction Tokiko Suzuki*, Kazunori Namba†, Natsumi Mizuno*,‡, Hiroyasu Nakata},1

*Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan † Otolaryngology/Laboratory of Auditory Disorders, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan ‡ Department of Pharmacotherapy of Lifestyle Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan } Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Measurement of GPCR Dimerization 2.1 Coimmunoprecipitation 2.2 Bioluminescence resonance energy transfer2 2.3 Immunoelectron microscopy 3. Receptor Pharmacology 3.1 Ligand binding assay 3.2 cAMP assay 3.3 Ca2 þ assay 4. Conclusion References

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Abstract The formation of homo- and hetero-oligomers between various G protein-coupled receptors (GPCRs) has been demonstrated over the past decade. In most cases, GPCR heterodimerization increases the diversity of intracellular signaling. GPCR-type purinergic receptors (adenosine and P2Y receptors) are actively reported to form hetero-oligomers with each other, with GPCRs belonging to the same group (type 1,

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rhodopsin-like), and even with GPCRs from another group. This chapter describes common strategies to identify dimerization of purinergic receptors (coimmunoprecipitation, bioluminescence resonance energy transfer (BRET), and immunoelectron microscopy) and to assess the alteration of their pharmacology (ligand binding, intracellular cAMP, and intracellular Ca2þ assays). We have reported dimerization of purinergic receptors using these strategies in transfected human embryonic kidney 293T cells and native brain tissue. Our data suggest that homo- and hetero-oligomerization between purinergic receptors exert unique pharmacology in this receptor group. According to these discoveries, heterodimerization is likely to be employed for the “fine-tuning” of purinergic receptor signaling.

1. INTRODUCTION Posttranslational processing of G protein-coupled receptors (GPCRs), including multimerization, generates diversity of cellular signaling transduction that cannot be obtained from genomic sequences. In the past decade, many GPCRs have been reported to form homo- and hetero-oligomers, which generate various pharmacological effects (Birdsall, 2010). Information concerning interacting partners and predicted dimerization interfaces are available in GRIPDB (G Protein-Coupled Receptor Interaction Partner Database; http://grip.cbrc.jp/; Nemoto, Fukui, & Toh, 2011). This database presents information about experimentally identified GPCR oligomers and their annotations, experimentally suggested interfaces for the oligomerization, and the oligomerization interfaces predicted using computational methods. Though increasing evidence has accumulated to indicate that various GPCRs form homo- and hetero-oligomers, there are limited data that reveal the physiological significance of oligomerization in vivo. Most studies are focused on the exploration of pharmacological changes following GPCR dimerization in vitro. As a result, the significance of heterodimerization can be classified into three categories: (1) diversification of signal transduction, (2) requirement for membrane trafficking, and (3) altering ligand specificity. 1. Diversification of signal transduction: Most GPCR heterodimers alter signal transduction by changing ligand binding affinity, activation of G proteins, or internalization and desensitization. For example, the adenosine A1 (A1R) and P2Y1 (P2Y1R) receptors form a heterodimer with each other in cotransfected HEK (human embryonic kidney)

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293T cells and in native rat brain (Yoshioka, Hosoda, Kuroda, & Nakata, 2002; Yoshioka, Saitoh, & Nakata, 2001, 2002). Upon heterodimerization, the P2Y1R agonist was capable of inducing signaling via Gi/o protein coupling to A1R (Yoshioka et al., 2001). Another example is the adenosine A2AR and dopamine D2R heterodimer, where both receptors are suggested to be involved in Parkinson’s disease. Longterm stimulation with D2R agonist led to the internalization of A2AR/ D2R oligomers, and opposing effects were observed in cells treated with D2R antagonist or with the A2AR agonist (Vidi, Chemel, Hu, & Watts, 2008). 2. Requirement for membrane trafficking: Some GPCRs have been reported to need heterodimerization for proper membrane trafficking. The GABAB receptor is composed of two subunits (GABAB1 and GABAB2) and is nonfunctional when the monomers are expressed individually but functional when they are coexpressed (Jones et al., 1998; Kaupmann et al., 1998). This phenomenon also occurs with M71 olfactory receptors that require the formation of heterodimers with A2AR, P2Y1R, and P2Y2R for functional expression at the cell membrane (Bush et al., 2007). 3. Altering ligand specificity: An intriguing example of the significance of GPCR heterodimerization is changes in ligand specificity. There are three subtypes of taste receptor T1R, that is, T1R1, T1R2, and T1R3. The T1R2/T1R3 heterodimer recognizes diverse natural and synthetic sweeteners. By contrast, the human T1R1/T1R3 heterodimer responds to the umami taste stimulus L-glutamate, and this response is enhanced by 50 -ribonucleotides, a hallmark of umami taste (Palmer, 2007). Adenosine and nucleotides exert various physiological effects via purinergic receptors. ATP is hydrolyzed extracellularly into ADP, AMP, and adenosine. ATP and ADP bind to P2 receptors, whereas adenosine binds to the adenosine receptor (Ralevic & Burnstock, 1998). This intriguing relationship between these ligands suggests some interaction between adenosine and P2 receptors. Adenosine receptors are further subclassified into A1, A2A, A2B, and A3, all of which are GPCRs. The P2 receptors are further subclassified into ligand-gated ion channel-type P2X1–7 receptors and G protein-coupled P2Y1, 2, 4, 6, 11, 12, 13, 14 receptors. P2Y receptors respond to a wider range of agonists including purines, pyrimidines, and UDP glucose. Evidence showing the existence of homo- and hetero-oligomerization between purinergic receptors and other GPCRs has been accumulating.

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According to our data and GRIPDB, homodimerization occurs in the A1R, A2AR, and P2Y2Rs (Kamiya, Saitoh, Yoshioka, & Nakata, 2003; Suzuki et al., 2009; Yoshioka, Saitoh, et al., 2002). Heterodimerization is observed in various combinations including A1R/P2Y1R (Yoshioka et al., 2001); A1R/P2Y2R (Suzuki, Namba, Tsuga, & Nakata, 2006); A2AR/P2Y1R/ P2Y12R (Nakata, Suzuki, Namba, & Oyanagi, 2010); A1R/thromboxane A2 receptor TPa (Mizuno, Suzuki, Hirasawa, & Nakahata, 2011); A1R/ D1R (Gine´s et al., 2000); A2AR/D2R (Hillion et al., 2002); A1R/A2AR (Ciruela et al., 2006); P2Y4R/P2Y6R (D’Ambrosi, Iafrate, Saba, Rosa, & Volonte´, 2007); M71/A2AR, P2Y1R, and P2Y2R (Bush et al., 2007); A1R/metabotropic glutamate receptor 1 (Ciruela et al., 2001); and A2AR/metabotropic glutamate receptor 5 (Ferre et al., 2002). There are many biochemical techniques to measure protein–protein interactions for soluble proteins, whereas different specific strategies are required for membrane-bound GPCRs. In this chapter, we describe some of the useful procedures to investigate GPCR oligomerization, especially hetero-oligomerization between purinergic receptors.

2. MEASUREMENT OF GPCR DIMERIZATION 2.1. Coimmunoprecipitation Coimmunoprecipitation of detergent-solubilized receptors is the most common and classical technique for research into possible GPCR dimerization. The key points of this strategy are the selection of antibodies for two different GPCR targets and membrane solubilization conditions. Therefore, exploration of the optimum antibodies and optimum condition for receptor solubilization may be required. We explored the coimmunoprecipitation between HA-tagged A1R and Myc-tagged P2Y2R using cotransfected HEK293T cells (Suzuki et al., 2006). In addition, we performed immunoprecipitation of nontagged A1R and Myc-P2Y2R using solubilized membranes from nontagged A1R/MycP2Y2R-cotransfected HEK293T cells. We detected nontagged A1R in the complex precipitated with anti-Myc (Fig. 13.1). This result indicates that a complex between nontagged A1R and Myc-P2Y2R was formed in the membranes of the cotransfected cells, and coimmunoprecipitates of HA-A1R/Myc-P2Y2R (Suzuki et al., 2006) were not due to nonspecific interactions between both tags.

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Figure 13.1 Coimmunoprecipitation of cells coexpressing A1R/Myc-P2Y2R. Anti-Myc antibody was used to coimmunoprecipitate A1R with Myc-P2Y2R WT (seventh lane from left). The first to fourth lanes represent expression of receptor proteins and reactivity of the antibodies used. Membrane extract, solubilized membrane extract; IP, immunoprecipitation; sup, supernatant following immunoprecipitation; IB, immunoblotting.

2.1.1 Protocol On day 1, detach HEK293T cells grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum using trypsin–EDTA solution and seed into 100-mm dishes at a density of 3.5  106 cells/dish. The following day, transfect cells with a mixture of 5 mg of cDNA plasmids, nontagged A1R, and Myc-tagged P2Y2R at the N-terminus and 15 ml of FuGENE HD (Roche Applied Bioscience, Manheim, Germany). On day 4, collect cells (1  107 cells), wash cells twice with DPBS (Dulbecco’s phosphate-buffered saline) (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4), and sonicate in hypotonic lysis buffer (50 mM Tris-acetate, pH 7.4) containing protease inhibitor cocktail (Roche Applied Bioscience). After centrifugation at 17,400  g for 30 min, solubilize cell membranes by incubating with 300 ml of Tx buffer (50 mM Tris-acetate, pH 7.4, 300 mM NaCl, and 1% Triton X-100) containing protease inhibitor cocktail and incubate for 1 h at 4  C. After centrifugation, preclear the supernatant containing the solubilized membranes by incubating, for 30 min at 4  C, with 30 ml of 50% Protein G-Sepharose™ 4 Fast Flow (GE Healthcare, Piscataway, NJ) in Tx buffer, followed by centrifugation at 17,400  g for 10 s. Then collect the supernatant and mix with 1 mg of anti-Myc 9E10 antibody (Roche Applied Bioscience) and incubate

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for 1 h at 4  C, followed by incubation for 2 h with 30 ml of 50% Protein G-Sepharose™ 4 Fast Flow (GE Healthcare). Wash the immune complex twice with 300 ml of Tx buffer, and elute the bound proteins with 30 ml of SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) sample buffer. Then separate the solubilized proteins using SDS-PAGE and transfer electrophoretically onto a nitrocellulose membrane. After blocking with 5% skimmed milk in 20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20, probe for the receptors using rabbit polyclonal anti-A1R antibody (Sigma-Aldrich, St. Louis, MO), followed by horseradish peroxidase-conjugated anti-rabbit IgG antibody (MP Biomedicals, Irvine, CA).

2.2. Bioluminescence resonance energy transfer2 The coimmunoprecipitation strategies described above are simple and do not require special equipment. However, the above techniques may seem limited because the solubilization of hydrophobic GPCRs can lead to aggregation that could be mistaken for oligomerization. Furthermore, the solubilization process with detergents can inhibit the association between GPCRs. Biophysical assays based on light resonance energy transfer (RET) are therefore quite valuable for overcoming these issues. Bioluminescence resonance energy transfer (BRET) is a biophysical technique that represents a powerful tool with which to measure protein–protein interaction in living cells (Pfleger & Eidne, 2006). BRET is a natural process involving nonradiative energy transfer between donor and suitable acceptor fluorophores after oxidation of substrate. An appropriate distance between ˚ , which is consistent with the disfluorophores causing BRET is 10–100 A tance where two molecules can interact with each other. BRET2 is an advanced BRET technology, which uses Renilla luciferase (Rluc) as the donor, codon-humanized form of wild-type GFP (green fluorescent protein) (GFP2) as the acceptor, and DeepBlueC (Coelenterazine 400A) as the substrate. The use of this substrate results in a 395-nm Rluc emission peak and an emission maximum at 510 nm for GFP2, which offers greater separation of the emission spectra of the donor and acceptor moieties. We employed this technique to measure homodimerization of A1R (Suzuki et al., 2009 and Fig. 13.2A and B) and P2Y2R (Fig. 13.2A and C) and heterodimerization of A1R/P2Y1R (Yoshioka, Saitoh, et al., 2002), A2AR/D2R (Kamiya et al., 2003), and A1R/P2Y2R (Fig. 13.2A). Although a powerful strategy, BRET studies can be misleading if not conducted using the appropriate controls. Nonspecific BRET can result from the overexpression of noninteracting proteins when acceptor

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Figure 13.2 Single-point BRET2 (A) and competitive BRET2 (B and C) analysis. (A) HEK293T cells were cotransfected with an equal concentration of indicated plasmids. For nonspecific control, constant amounts of Rluc-N3 and GFP2-N3 vectors were cotransfected (Rluc/GFP2). BRET signals were measured using cells cultured for 48 h after transfection. The BRET2 ratio was plotted as a function of the expression ratio of receptor-GFP2 over receptor-Rluc. Experiments were performed three times using six replicates. *P < 0.05 versus negative control by Dunnett's test. HEK293T cells were cotransfected with a fixed concentration of (B) HA-A1R-Rluc and HA-A1R-GFP2 plasmids and increasing amounts of HA-A1R not fused to donor or acceptor BRET2 probes. HEK293T cells were also cotransfected with a fixed concentration of (C) Myc-P2Y2R-Rluc and Myc-P2Y2R-GFP2 plasmids and Myc-P2Y2R not fused to donor or acceptor BRET2 probes. Data are expressed as mean S.E.M. of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 (Dunnett's test).

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molecules are in close proximity to donor molecules. Thus, it is important to select proper negative controls that can be compared at similar expression levels. According to the guidelines for RET experiments (Marullo & Bouvier, 2007), three BRET methods have been recommended including competitive BRET experiments, as mentioned below. Results of single-point BRET2 and competitive BRET2 analysis are shown in Fig. 13.2A–C. For single-point BRET2, HEK293T cells were cotransfected with equal amounts of two kinds of plasmids, A1R-Rluc/A1R-GFP2, P2Y2 R-Rluc/P2Y2R-GFP2, A1R-Rluc/P2Y2R-GFP2, and soluble-free Rluc/ GFP2 for negative control. All three combinations resulted in significantly high BRET2 signal in the negative control (Fig. 13.2A), indicating formation of A1R homodimer, P2Y2R homodimer, and A1R/P2Y2R heterodimer in living cells. For competitive BRET2 analysis, fixed amounts of A1R-Rluc/A1RGFP2 or P2Y2R-Rluc/P2Y2R-GFP2 and increasing amounts of nonlabeled A1R or nonlabeled P2Y2R were coexpressed in HEK293T cells. As shown in Fig. 13.2B and C, BRET signal was decreased as the concentration of nonlabeled A1R or P2Y2R increased. In addition, BRET2 of P2Y2R-Rluc/ P2Y2R-GFP2 was inhibited as cotransfection of nonlabeled A1R was increased (data not shown). These data confirmed the specific formation of A1R homodimer, P2Y2R homodimer, and A1R/P2Y2R heterodimer. 2.2.1 Protocol 2.2.1.1 Plasmid construction and transient transfection

For BRET2 experiments, construct plasmids that have Rluc and GFP2 at the C-terminus of the cDNA of target GPCRs, without stop codon. We constructed HA-A1R-GFP2, HA-A1R-Rluc, Myc-P2Y2R-Rluc, and MycP2Y2R-GFP2 by amplification of the HA-tagged rat A1R or Myc-tagged rat P2Y2R coding sequence, without stop codon, using sense and antisense primers containing distinct restriction enzyme sites at the 50 and 30 ends, respectively (Mizuno et al., 2011; Suzuki et al., 2006). The fragments were then subcloned in-frame into the appropriate sites of the pGFP2-N3 and pRluc-N3 expression vectors (Perkin Elmer Life Sciences, Boston, MA). For single-point BRET experiments, cotransfect HEK293T cells (5  106 cells per 35-mm dish) with a fixed amount (e.g., 1 mg) of Rluc-tagged and GFP2-tagged plasmids. For competitive BRET experiments, cotransfect HEK293T cells (5  105 per 35-mm dish) with a fixed amount (e.g., 1 mg) of Rluc-tagged and GFP2-tagged plasmids and increasing concentrations of unlabeled receptor plasmids (e.g., 0, 0.4, 0.8, 1.2, 1.6, 2.0 mg). Transfection was done using 3 ml of FuGENE HD (Roche Applied Bioscience). Nontransfected cells or cells transfected with only Rluc-tagged plasmids can be used for control.

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2.2.1.2 BRET2 measurement

Forty-eight hours after transfection, harvest cells and suspend in assay buffer (DPBS containing 0.1 mg/ml CaCl2, 0.1 mg/ml MgCl2, and 1 mg/ml Dglucose). Next, distribute the cells into white-walled 96-well plates (OptiPlate; Perkin Elmer Life Sciences) at a density of 1  106 cells per well, and incubate for 20 min at 37  C. Then add DeepBlueC (Perkin Elmer Life Sciences) or Coelenterazine 400A (Biotium Inc., CA) to cells at a final concentration of 5 mM, and immediately determine BRET signal using a Fusion a universal microplate analyzer (Perkin Elmer Life Sciences) for the detection of Rluc at 410 nm and GFP2 at 515 nm. The BRET ratio was calculated as emission at 515 nm over emission at 410 nm. Correct the values by subtracting the background signal detected using nontransfected cells.

2.3. Immunoelectron microscopy Immunoelectron microscopy is another tool for the more direct assessment of GPCR heterodimerization. In this chapter, we introduce this method to examine heterodimerization of transfected and endogenous purinergic receptors using HEK293T and intact rat brain, respectively. To assess the hetero-oligomerization of endogenous GPCRs in brain tissue, three experimental steps are typically required. The first step is to obtain immunoelectron microscopic data acquired from preembedding methods using transfected culture cells. The second step is to acquire data using transfected cells with postembedding methods. The last step is to acquire data from tissues using postembedding methods. As shown in Fig. 13.3A and B, immunogold particles were localized individually or in clusters, indicating that both transfected HA-A1R and Myc-P2Y2R form monomers and homo-oligomers in HEK293T cells. When Myc-P2Y2R-transfected HEK293T cells were incubated with both anti-HA and anti-Myc antibodies, we detected single particles scattered all over the cells (Fig. 13.3A). A similar observation was also observed in HA-A1R-transfected HEK293T cells incubated with both anti-HA and anti-Myc antibodies (Fig. 13.3B). By contrast, HEK293T cells cotransfected with both HA-A1R and Myc-P2Y2R displayed clusters of different-sized particles, mainly at the cell surface (Fig. 13.3C), suggesting the formation of hetero-oligomers. Similarly, we observed clusters of different-sized gold particles at cytoplasmic membranes in cell bodies in the rat cerebellum, indicating the presence of heteromeric complexes of endogenous A1R and P2Y2R in the rat cerebellum (Fig. 13.3D).

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A

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Figure 13.3 Immunogold electron microscopy (postembedding) method to detect A1R and P2Y2R in transfected HEK293T cells using nanogold particles. (A) Localization of Myc-P2Y2R (small particles) detected with anti-Myc and nonspecific immunoreaction with anti-HA (large particles) in Myc-P2Y2R-transfected HEK293T cells. (B) Localization of HA-A1R detected with anti-HA and nonspecific immunoreaction with anti-Myc in HA-A1R-transfected HEK293T cells. (C) Anti-HA and anti-Myc immunolocalization in HEK293T cells cotransfected with HA-A1R and Myc-P2Y2R. (D) Visualization of A1R and P2Y2R in rat brain. Localization of A1R (small particles) and P2Y2R (large particles) on the cell surface of Purkinje cells detected with both anti-A1R and anti-P2Y2R. Arrows indicate two adjacent receptors on the cell membrane. CM, cell membrane; CP, cytoplasm. Bars represent 100 nm.

In our earlier experiments, under the same experimental conditions, oligomerization of A1R and P2Y2R in rat brain tissue occurred in hippocampal pyramidal cells, cerebellum, and pyramidal cells in the frontal cortex (Namba et al., 2010). Hetero-oligomers of A1R/P2Y2R were detected in significant numbers at the cell surface. From these studies, it can be speculated that the A1R/P2Y2R hetero-oligomer responsible for unique signal transduction (Suzuki et al., 2006) also occurs in the rat brain. 2.3.1 Protocol 2.3.1.1 Preembedding method of transfected HEK293T cells

Forty-eight hours after transfection of HA-A1R and/or Myc-P2Y2R plasmids, briefly wash the cells with 0.1 M phosphate-buffered saline (PBS) and fix with 4% paraformaldehyde and permeabilize with 0.25% Triton X-100 (PBST) for 15 min. Incubate the cells with anti-HA rat antibody (1 mg/ml)

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and anti-Myc mouse antibody (1 mg/ml) for 3 h at 4  C. After washing with PBS, incubate the cells with 10-nm gold particle-conjugated goat anti-rat IgG antibody (rat IgG-10, 1:1000; BBI International, Lakewood, CO) and 5-nm gold particle-conjugated goat anti-mouse IgG antibody (mouse IgG-5, 1:1000; BBI International) for 4 h at 4  C. After washing, fix the cells with 2.5% glutaraldehyde in 0.15 M sodium cacodylate (pH 7.4) for 2 h. Postfix the cells with 1% osmium tetroxide for 4 h at room temperature. After washing twice with 50% ethanol, stain the cells with 1% uranyl acetate in 50% ethanol for 30 min. Then dehydrate the cells on culture dishes with 50%, 70%, and 95% ethanol for 10 min each and in 100% ethanol for 2  15 min. Further dehydrate the cells in 1:1 mixture of ethanol and propylene oxide for 1 min. Following this step, rinse the cells on culture dishes with 100% propylene oxide, immediately add 1:1 mixture of propylene oxide and Epon 812 resin (NISSIN EM, Tokyo, Japan) and leave for 8 min. Embed the cells on the culture dishes with straight Epon 812 resin for 2  5 h. Flat-embed the culture dishes with another straight Epon 812 resin, and bake at 60  C for 48 h. Freeze the dishes in liquid nitrogen for 1 min, and subsequently submerge the dishes in room temperature water. Then, crack the frozen dishes to remove the dish from the resin completely. Cut the polymerized resin into 1.5  1.5-mm blocks using a razor blade on a hot plate, and fix to the tip of the foundation capsule, using instant glue, with the cellular surface lying face up. Cut the block into thin sections (40 nm) using a diamond knife in an ultramicrotome. Observe the specimens with an H7500 electron microscope (Hitachi, Japan). 2.3.1.2 Postembedding method of transfected HEK293T cells and brain tissue

Before transfection, prepare a polyethyleneimine-coated plain microscope slide (25  75  1 mm). Perform fixation and dehydration as described above. Remove the ethanol with the 100% propylene oxide for 10 min. Incubate the slide in 1:1 mixture of propylene oxide and LR-white resin (NISSIN EM) for 30 min. Embed the cells on the slide with straight LRwhite for 2  5 h. Put the polymerized LR-white capsule on cells embedded with straight LR-white, and bake at 60  C for 48 h. Detach the polymerized LR-white from the slide. Cut the block into thin sections using a diamond knife in an ultramicrotome. Cut the dissected brain tissue into 1.0-mm3 blocks that are then incubated with lead(II) acetate (Sigma-Aldrich) buffer for 1 h at room temperature. Following this, dehydrate the brain tissue with 50%, 70%, and 95% ethanol for 30 min each and in 100% ethanol for 2  2 h. Further dehydrate in 1:1 mixture of ethanol and propylene oxide for 2 h.

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Exchange with 100% propylene oxide for 2  2 h, and add 1:1 mixture of propylene oxide and LR-white to the brain tissue for 2 h. Embed the brain tissues with straight LR-white for 2  5 h. Place specimen at the bottom of a gelatin capsule and fill with LR-white. Polymerize in a 60  C oven for 48 h. Cut out the specimen in polymerized resin and place the tip in a polymerized LR-white capsule. Mount the ultra-thin sections (40 nm) on 200-mesh nickel grids (NISSIN EM) and incubate in PBST containing 1% bovine serum albumin for 10 min. After immunoreactions, rinse sections for 2  2 min by placing grids on large droplets of PBST. Stain sections with uranyl acetate for 10 min and rinse in PBS for 2  2 min.

3. RECEPTOR PHARMACOLOGY Hetero-oligomerization of GPCRs can affect various aspects of receptors, including altering ligand binding specificity, signal transduction, and cellular trafficking. We therefore explored purinergic receptor activity in cotransfected HEK293T cells by ligand binding assay, adenylate cyclase assay, and intracellular Ca2þ assay.

3.1. Ligand binding assay We revealed that A1R ligand binding was attenuated by P2Y2R ligands in the A1R/P2Y2R coexpressing HEK293T cells (Suzuki et al., 2006). To analyze what caused the inhibition of A1R agonist binding by P2Y2R activation in the A1R/P2Y2R-coexpressing cells, we examined [3H]CCPA (2-chloroN6-cyclopentyladenosine), a selective A1R agonist, saturation binding in the presence or absence of 100 mM UTP, a P2Y2R agonist (Fig. 13.4). In the absence of UTP, the Kd and Bmax values were 0.23  0.03 nM and 0.84  0.04 pmol/mg protein, respectively. Conversely, in the presence of UTP, these values were estimated to be 1.61  0.11 nM and 0.34  0.02 pmol/mg protein, respectively. These Kd and Bmax values are significantly different from the original values. Therefore, inhibition of A1R agonist binding by the P2Y2R agonist UTP is due to a decrease in both the affinity and the number of binding sites of the A1R in the membranes from A1R/P2Y2R-coexpressing cells. 3.1.1 Protocol Perform transfection into HEK293T as above using FuGENE HD (Roche Applied Bioscience) and harvest these cells 48 h later for use in the ligand binding assay. Collect transfected cells (approx. 107 cells) and wash twice with DPBS. Disrupt the cells by sonication in hypotonic lysis buffer

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Figure 13.4 [3H]CCPA saturation binding in the presence or absence of 100 mM UTP in membranes from cells coexpressing HA-A1R and Myc-P2Y2R. Saturation binding of [3H] CCPA to HEK293T cell membranes coexpressing HA-A1R and Myc-P2Y2R was determined in the presence (circle) and absence (triangle) of 100 mM UTP. Results are shown as the mean of three independent experiments performed in duplicate.

containing protease inhibitor cocktail. Centrifuge at 17,400  g for 30 min and suspend the pellet using the same buffer. Repeat this three times. Incubate the membranes (15–30 mg) for 1.5 h at 25  C with various concentrations of radiolabeled ligand [3H]CCPA (42.6 Ci/mmol; PerkinElmer Life Sciences, Boston, MA), 50 mM Tris-acetate (pH 7.4), 1 mM EDTA, 5 mM MgCl2, and 4 units/ml of adenosine deaminase (ADA; Sigma-Aldrich). Nonspecific binding can be determined by examining the binding in the presence of xanthine amine congener (10 mM), an adenosine receptor antagonist. After filtration through GF/B glass fiber filters, quantify radioactive ligand bound to the receptors by counting the radioactivity of the filters. Determine KD and Bmax values by nonlinear regression analysis using one-site binding model (GraphPad Prism 4; GraphPad, San Diego, CA).

3.2. cAMP assay We examined whether heteromerization affects cellular signaling transduction. For this purpose, we studied A1R agonist-induced inhibition of adenylyl cyclase, a key index of A1R function (Fig. 13.5). As expected, CPA (N6-cyclopentyladenosine), a selective A1R agonist, decreased forskolin (FSK)-evoked cAMP generation in cells expressing HA-A1R alone and in cells coexpressing HA-A1R and Myc-P2Y2R (Fig. 13.5A and B). Interestingly, we found that UTP inhibited the CPA-mediated signaling in the HA-A1R/Myc-P2Y2R-coexpressing cells (Fig. 13.5B, black bar).

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Figure 13.5 Effects of A1R and P2Y2R agonists and antagonists on FSK-stimulated cAMP accumulation in cells expressing HA-A1R alone (A) and coexpressing HA-A1R and MycP2Y2R (B). The results were normalized by the cAMP concentration in control cells (without ligands). Results shown are the means of more than two independent experiments performed in quadruplicate. Values were compared by Dunnett's test. n.s., no significant difference (P > 0.05) where indicated. *P < 0.05, ***P < 0.001 versus where indicated, between groups. Part of this figure was reprinted from Suzuki et al. (2006), with permission from Elsevier.

This result is consistent with the inhibitory effect of UTP on [3H]CCPA binding to the membranes from cells coexpressing A1R and P2Y2R (Fig. 13.4, open circles). The antagonist-like effect of UTP on A1Rmediated signaling in cells expressing A1R alone was negligible (Fig. 13.5A, black bars). UTP alone did not significantly affect FSK-induced cAMP production in cells expressing HA-A1R alone and in cells coexpressing HA-A1R and Myc-P2Y2R (Fig. 13.5A and B). 3.2.1 Protocol Transfect HEK293T cells plated on collagen-coated 24-well plates at 1  105 cells/well as described above and culture for another 24 h. Next, treat the cells for 1 h at 37  C with 1 unit/ml ADA and incubate for 15 min at 37  C with an equal volume of serum-free DMEM containing 100 mM of Ro-20-1724 and 1 unit/ml ADA with or without receptor antagonist. Subsequently, incubate the cells with receptor agonist and FSK (10 mM final concentration) for Gi/o-coupled receptor for a further 10 min. Terminate the reaction by adding ice-cold 0.1N HCl for 10 min,

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followed by neutralization with 1N NaOH. Extract the intracellular cAMP with 65% ethanol for 10 min on ice. Determine the intracellular cAMP concentration using a cAMP Biotrak Enzyme Immunoassay System (GE Healthcare).

3.3. Ca2þ assay Measurement of intracellular Ca2þ concentration is needed for analysis of signal transduction of Gq/11-coupled GPCRs. Changes in intracellular free Ca2þ concentration ([Ca2þ]i) were monitored by the intensity of Fura-2 fluorescence at 37  C using a spectrofluorometer. Fura-2-acetoxymethyl ester (Fura-2-AM) is a membrane-permeable derivative of the ratiometric calcium indicator Fura-2, used to measure cellular calcium concentrations by fluorescence. When added to cells, Fura-2-AM crosses cell membranes, and once inside the cell, the acetoxymethyl groups are removed by cellular esterases. Removal of the acetoxymethyl esters regenerates “Fura-2,” the pentacarboxylate calcium indicator. Measurement of Ca2þ-induced fluorescence at both 340 and 380 nm allows for calculation of calcium concentration-based 340/380 ratios. We focused on three purinergic receptor subtypes, A2AR, P2Y1R, and P2Y12R, which are expressed in platelets and are thought to play individual roles in platelet aggregation (Jin & Kunapuli, 1998). Formation of a heteromeric complex composed of these three receptors has been demonstrated using coimmunoprecipitation techniques in HEK293T cells coexpressing these three receptors (Nakata et al., 2010). For further exploration, we examined the possibility of functional interaction between these purinergic receptors (Suzuki, Obara, Moriya, Nakata, & Nakahata, 2011). We analyzed the effect of ligands for A2AR and P2Y12R on the mobilization of intracellular Ca2þ as a key index of P2Y1R-mediated signaling. These observations were performed using HEK293T cells transfected with A2AR and P2Y12R, which endogenously express P2Y1R (Schachter, Sromek, Nicholas, & Harden, 1997). As expected, stimulation of the P2Y1R with 2MeSADP (2-methylthio ADP), an agonist of P2Y1R and P2Y12R, caused an increase in Ca2þ release (Fig. 13.6A), which was absolutely blocked by pretreatment with MRS2179, a P2Y1R-specific antagonist (Fig. 13.6B). These findings indicated that normal P2Y1R signaling via Gq/11 in HEK293T cells existed. Pretreatment with CGS21680, an A2AR specific agonist, did not affect Ca2þ mobilization induced by 2MeSADP (Fig. 13.6C). By contrast, treatment with ZM241385 and ARC69931MX, which are specific antagonists for

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Figure 13.6 Mobilization of Ca2þ evoked by 2MeSADP prestimulated with various ligands. Fura 2-loaded HEK293T cells transfected with A2AR and P2Y12R were preincubated for 1 min with either vehicle as control (A), 10 mM MRS2179 (B), 10 mM CGS21680 (C), 10 mM ZM241385 (D), or 10 mM ARC69931MX (E) as indicated. Cells were then stimulated with 1 mM 2MeSADP (arrow), and fluorescence intensity was measured. Traces shown are representative of at least three separate experiments.

A2AR and P2Y12R, respectively, caused decreased Ca2þ release (Fig. 13.6D and E). These results indicate negative regulation of P2Y1R function by A2AR- and P2Y12R-specific antagonists. 3.3.1 Protocol Wash the transfected cells twice with Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, 20 mM HEPES, and 5.6 mM glucose, pH 7.4). After centrifugation at 250  g for 2 min, treat the cells with Fura-2-AM (1 mM; Dojindo Laboratories, Kumamoto, Japan) at 37  C for 15 min. Then centrifuge the cells at 250  g for 2 min and wash twice with Tyrode’s buffer. Suspend the cells in modified Tyrode’s buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 0.18 mM CaCl2, 20 mM HEPES, and 5.6 mM glucose, pH 7.4) at a density of 2  105 cells/ml. Use 1.5 ml of the cell suspension for Fura-2 assays at 37  C, with gentle stirring, in a spectrofluorometer with excitation at 340 and 380 nm and emission at 510 nm. Calibration of the signal should be performed for each sample by adding 0.15% Triton X-100 to obtain maximal fluorescence and then 2.5 mM EGTA to obtain minimal fluorescence. The ratio of fluorescence at 340 nm to that at 380 nm is a measure of [Ca2þ]i, assuming a KD of 244 nM Ca2þ for Fura-2-AM.

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4. CONCLUSION As mentioned above, purinergic receptors frequently form heterodimers, which exert specific alterations in signal transduction. It is likely that the physiological effects of adenine nucleotides and nucleosides rely on a membrane network of appropriate receptors and enzymes rather than the presence of isolated proteins. Depending on the partners included in the network, a previously unforeseen number of different responses can be expected to arise in response to ATP, ADP, and adenosine. These results suggest that a large number of combinations for direct interaction may occur to form a membrane network of receptors and enzymes for adenine nucleosides and nucleotides. Purinergic receptors are known to be widely expressed in various cells. Frequently, the output by the addition of nucleotides or nucleosides to cells is not always what is expected from independent molecularly defined cloned purinergic receptor types (Burnstock & Knight, 2004). Although other mechanisms may exist, it is quite feasible that such functional modification can be produced by the direct interaction between purinergic receptors of other colocalized subtypes. As reviewed in this chapter, purinergic receptors tend to form heterodimers with other GPCRs, and therefore, if purinergic receptors are located in close proximity on the cell membrane, it is natural to speculate that the resulting heterodimers between purinergic receptors produce various changes or modification in the purinergic signaling. GPCRs are known to be proteins that are difficult to crystallize. Efforts to elucidate the crystal structures of GPCRs bound to diffusible ligands have recently yielded structures for five rhodopsin-like GPCRs: the human b2 adrenergic receptor (Rosenbaum et al., 2011), turkey b1 adrenergic receptor (Warne et al., 2011), human A2A adenosine receptor (Xu et al., 2011), human CXCR4 chemokine receptor (Wu et al., 2010), and the D3 dopamine receptor (Chien et al., 2010). GPCR hetero-oligomerization may increase the diversity of GPCR-responsive signals by altering receptor pharmacology, and such oligomers thus represent a potential set of novel therapeutic targets. Therefore, agents that promote or disrupt oligomerization, or that selectively induce heterodimerization, may be of clinical and research value. Future clarification of the crystal structure of heterodimerized GPCRs will dramatically facilitate new drug discovery.

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