cDNA constructs encoding human adenosine A_2A receptor (A_2A in pEYFP-N1, A_2A in pRluc-N1, and A_2A in pGFP-2-N1 vectors) or human dopamine D₂ receptor and CaM in pEYFP-N1 vector, were previously described (Navarro et al., 2009). NCS-1 in pEYFP-N1 vector was published previously (Zhao et al., 2001). NCS-1 in pTagRFP-N or pEGFP-N1 vectors was subcloned from the pNCS-1-YFP-N1 plasmid using XhoI and BamHI restriction sites. Caldendrin was subcloned from a caldendrin-EGFP-N1 plasmid (Dieterich et al., 2008) into a pEYFP-N1 vector using EcoRI and BamHI restriction sites. The identity of the newly cloned construct was confirmed by sequencing analysis. pEGFP-N1 (Clontech), pTagRFP-N (Evrogen, Moscow, Russia), and pcDNA3.1 (Invitrogen, Darmstadt, Germany) were used as a corresponding negative controls for co-immunoprecipitation experiments and in surface biotinylation studies.

The following primary antibodies were used: Anti-NCS-1 rabbit (Santa Cruz Biotechnology, Heidelberg, Germany), anti-GFP mouse (MMS-118R, HiSS Diagnostics, Freiburg, Germany), anti-Renilla Luciferase mouse (Millipore, Schwalbach, Germany), anti-phospho-ERK1/2 mouse (Sigma-Aldrich, Madrid, Spain), anti-ERK1/2 rabbit (Sigma-Aldrich, Madrid, Spain), and anti-phospho-AKT rabbit (SAB Signalway antibody, Madrid, Spain). The secondary antibodies were: goat anti-mouse immunoglobulins HRP conjugated (P0447, Dako, Hamburg, Germany), goat anti-rabbit IgG HRP conjugated (#7074, Cell Signaling, Frankfurt am Main, Germany), peroxidase-AffiniPure goat anti-mouse IgG, light chain^* specific antibody (Jackson ImmunoResearch) and Alexa Fluor 568 goat anti-rabbit IgG (A11031, A11036, Invitrogen, Darmstadt, Germany).

HEK293, HEK-293T cells, and COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 5% (v/v) heat inactivated Fetal Bovine Serum (FBS) (Invitrogen, Paisley, Scotland, UK). For immunocytochemistry experiments HEK-293 and COS-7 cells were plated on 18 mm coverslips, grown for 24 h and then transfected with Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. Twenty-four hours after transfection the cells were fixed with 4% paraformaldehyde (PFA) and processed for immunostaining as described below. For surface biotinylation experiments HEK293T cells were grown in 75 cm² culture flasks and transfected with Polyfect (Qiagen) according to the manufacturer's protocol. HEK-293T cells for BRET experiments were transfected with the plasmids encoding CaM, NCS-1, caldendrin, and adenosine A_2A receptor fusion proteins by PEI (PolyEthylenImine, Sigma, Steinheim, Germany) as previously described (Carriba et al., 2008).

Coverslips with transfected HEK-293 and COS-7 were fixed with 4% PFA for 10 min at 37°C, extensively washed with PBS and immunostained for endogenous NCS-1 as described before (Mikhaylova et al., 2009) with anti-NCS-1 rabbit antibody in a dilution of 1:300. COS-7 cells transfected with A_2A-Rluc construct were stained with anti-Renilla Luciferase mouse antibody in dilution 1:500. F-actin was stained with Alexa Fluor 568 phalloidin (Molecular probes, Life Technologies, Darmstadt, Germany) diluted in 1:1000 in PBS and incubated for 10 min at room temperature. Fluorescence images were obtained on a TCS SP5 II confocal laser scanning microscope (Leica, Germany) using a 63× oil objective and zoom factors in the range of 1–4×. A 405 laser line was used for visualizing DAPI staining, 488 for GFP/YFP, 568 for the Alexa 568, and TagRFP. Images were acquired as z-stacks with 0.3 μm z-step. Maximum projections of z-stack were created in the ImageJ program (ImageJ, NIH).

HEK-293T cells were grown in six-well plates till about 60% confluence and then were transiently co-transfected with a constant amount of cDNA encoding for Rluc fusion protein and with increasing amounts of cDNA, corresponding to the protein fused to YFP. Two hours before quantifying fluorescence intensity, the supplemented medium (DMEM) was replaced by HBSS in the presence (1.26 mM) or in the absence of Ca²⁺. Cells supplemented with Ca²⁺ were treated with 1 μm of ionomycin 10 min prior to the fluorescence measurements. To quantify protein-YFP expression, cells (20 μg protein) were distributed in 96-well microplates (black plates with a transparent bottom), and fluorescence was read in a Mithras LB 940 using an excitation filter at 400 nm. Protein-fluorescence expression was determined as fluorescence of the sample minus the fluorescence of cells expressing the BRET donor alone. For BRET measurements, cell suspensions (20 μg protein) were distributed in 96-well microplates (Corning 3600, white plates; Sigma) and 5 μM coelenterazine H (Molecular Probes, Eugene, OR) was added. After 1 min, the readings were collected using a Mithras LB 940 that allows the integration of the signals detected in the short-wavelength filter at 485 nm (440–500 nm) and the long-wavelength filter at 530 nm (510–590 nm). To quantify protein-Rluc luminescence readings were also collected after 10 min of adding coelenterazine H. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the donor construct expressed alone in the same experiment. BRET is expressed as mili BRET units (mBU) determined as net BRET × 1000. A hyperbolic saturation curve showing an increase in BRET signal as a function of the acceptor fusion protein expression (acceptor protein expression relative to the donor protein expression, YFP/Rluc) is indicative of a specific interaction between the receptor-Rluc and Ca²⁺ sensor-YFP fusion constructs (Ayoub and Pfleger, 2010). BRET curves were fitted by using a non-linear regression equation, assuming a single phase with GraphPad Prism software (San Diego, CA, USA) to obtain the BRET_max and BRET₅₀ values. The BRET₅₀ parameter represents the acceptor/donor ratio giving 50% of the BRET_max and was used to estimate the relative affinity of the interaction. BRET data are expressed as means ± S.E.M. of 4–6 different experiments grouped as a function of the amount of BRET acceptor. Statistical differences in BRET parameters were analyzed with bifactorial ANOVA followed by post-hoc Bonferroni's tests (p = 0.05).

Heterologous Co-IP was performed with extracts from HEK293T cells transiently expressing A_2A-YFP, D₂-YFP, GFP, and NCS-1-tagRFP. Endogenous NCS-1 was co-immunoprecipitated from HEK293T cells transfected only with A_2A-YFP or GFP plasmids. YFP-tagged receptors and GFP control were immunoprecipitated for 12 h at 4°C with anti-GFP mouse antibody coupled to magnetic beads. Purification of antibody bound complexes was done using the μMACS™ GFP Isolation Kit (Miltenyi Biotec GmbH, Germany) according to the protocols supplied by manufactures, except that six washing steps were introduced to remove the unspecific binding to the beads. Another set of experiments was performed for A_2A-YFP and GFP with overexpressed NCS-1-TagRFP. Extraction of proteins from HEK293T cells was done as described previously (Hradsky et al., 2011). NCS-1 was immunoprecipitated with anti-NCS-1 rabbit antibody coupled to Protein G sepharose (GE Healthcare) or corresponding rabbit IgG controls overnight at 4°C. In both cases, high Ca²⁺ and Ca²⁺-free conditions were achieved by addition of 0.5 mM of Ca²⁺ and 1 mM of Mg²⁺ or 2 mM EGTA and 1 mM Mg²⁺, respectively, to the cell extracts during immunoprecipitation as well as into the washing buffers. Beads with precipitated protein complexes were washed three times with corresponding extraction buffers and eluted with 2× SDS sample buffer. Eluted samples were checked on SDS-PAGE/WB using anti-NCS-1 and anti-GFP antibody. To measure the effect of Ca²⁺ on the efficiency of co-immunoprecipitation, the total amount of A_2A receptor co-purified at different conditions was quantified using the “Gel Analyzer” plug-in provided in the ImageJ software (NIH, USA). The maximal binding observed with anti-NCS-1 antibody in the presence of Ca²⁺ was taken as 100% for each experiment individually and % deviations from this condition were measured for the other groups. Confidence interval was calculated and data were represented as averages of 4–5 independent experiments ± standard error mean (S.E.M).

HEK293T cells overexpressing A_2A-YFP alone or together with NCS-1-TagRFP and GFP control co-transfected with NCS-1-TagRFP were grown for 48 h in supplemented DMEM. Then growth medium was removed, cells were washed twice with HBSS and serum-free DMEM was added for another 2 h. Cells were labeled with Sulfo-NHS-SS-Biotin (Pierce) for 15 min at 4°C according to the manufacturers manual. Unbound biotin was sequestered and cells were harvested by scrapping, proteins were extracted with 1× TBS containing 1% Triton-X-100 over 1 h at 4°C. Equal amounts of extract were bound to Streptavidin beads (Life Technologies) and biotinylated proteins were eluted with 2× SDS and subjected to SDS-PAGE. A_2A-YFP protein bands were detected with anti-GFP mouse antibody. NCS-1-TagRFP was visualized with anti-NCS-1 rabbit antibody on the same membrane. The efficiency of surface biotinylation for A_2A–YFP in the presence or absence of NCS-1-TagRFP (n = 4) was quantified by measuring the optical densities of GFP signal as described above. Obtained values were compared between groups with and without overexpressed NCS-1 (the later one is taken as 100%). Statistical comparison was done with bifactorial ANOVA followed by post-hoc Bonferroni's tests.

A detailed protocol for activation of adenosine A_2A receptors in HEK293T cells by addition of the A_2A receptor agonist CGS21680 (100 nM) has been described previously (Navarro et al., 2009). Briefly, HEK293T cells expressing A_2A and NCS-1 or the corresponding controls were grown in 25 cm² flasks to 50% confluence and cultured in serum-free medium overnight before the experiment. Two hours before the experiment, the cells medium was changed to HBSS buffer containing 1.26 mM Ca²⁺ and cells were treated or not with 1 μM ionomycin for 5 min before the addition of CGS21680. After cell lysis and estimation of protein concentration equal amounts of each sample (10 μg) were subjected to SDS-PAGE. To determine the level of ERK1/2 and pAKT phosphorylation, the membranes were then probed with a mouse anti-phospho-ERK1/2 antibody or phospho-AKT antibody. Total-ERK1/2 antibody was used as a loading control. The levels of phosphorylated ERK1/2 and phosphorylated AKT were normalized for differences in loading using the total ERK1/2 protein bands. Quantitative analysis of detected bands was performed by Odyssey V3.0 software. Statistical comparison was done with bifactorial ANOVA followed by post-hoc Bonferroni's tests (^*p = 0.05).

The BRET technique can be successfully used as a method to test a protein—protein interaction in living cells (De and Gambhir, 2005; Carriba et al., 2008). Particularly, previously reported direct binding between the carboxy terminus of the A_2A receptor and CaM (Woods et al., 2008) was verified and extensively characterized using BRET (Navarro et al., 2009). This method is based on the fact that a luminescent donor (Rluc) oxidates the substrate coelenterazine H emitting bioluminescence that can be transferred to a fluorescent acceptor (YFP) when the distance between the donor and the acceptor is about 4.4 nm, (Dacres et al., 2010)—a typical distance between proteins interacting in a macro complex. The advantage of the BRET technique over classical techniques like co-immunoprecipitation is that it is performed in living cells and the protein concentrations of each protein can be carefully controlled with the interaction monitored over a range of protein concentrations and ratios. We have chosen BRET to test if the A_2A receptor is capable of forming a complex with other members of the CaM superfamily, namely NCS-1 and caldendrin. We compared the BRET efficiency between HEK293T cells co-expressing A_2A fused to Renilla luciferase (Rluc) and CaM, NCS-1 or caldendrin fused to YFP (Figures 1A–C). In agreement with our previous findings, the saturation curve obtained upon increasing CaM-YFP expression, indicated a specific interaction between CaM and A_2A (BRET_max 60 ± 7 mBU and BRET₅₀ 41 ± 6). BRET occurred already at resting conditions but BRET_max was increased (p = 0.05) after 10 min of stimulation with 1 μM of ionomycin (BRET_max 78 ± 7 and BRET₅₀ 48 ± 8/Figure 1A). Interestingly, a clear and saturable BRET signal was also observed when increasing amounts of NCS-1-YFP were co-expressed with a constant amount of A_2A-Rluc (BRET_max 43 ± 4 mBU and BRET₅₀ 11 ± 5, Figure 1B) again demonstrating a specific interaction. Elevation of intracellular Ca²⁺ concentration by preincubation of HEK293T cells with ionomycin (1 μM) for 10 min, increased (p = 0.05) both BRET_max (57 ± 6 mBU) and BRET₅₀ (33 ± 9/Figure 1B). These results can be interpreted in two ways, neither of which is exclusive of the other. In one, Ca²⁺ led to conformational changes in the A_2A-NCS-1 complex that reduces the distance between Rluc and YFP fused to the C-terminal domain of the two interacting fusion proteins. In the other, Ca²⁺ increases complex formation by increasing the affinity between the two proteins.

Next we investigated if A_2A and caldendrin can form heteromers. A non-specific (linear) and low BRET signal was obtained in this case, a result consistent with two proteins not interacting (Figure 1C). An increase in intracellular Ca²⁺ levels had no effect on BRET efficiency either. Taken together these results suggest that caldendrin might not be an interaction partner of the A_2A receptor and that not all EF-hand proteins produce a BRET signal indicative of an interaction.

To get more insight in this, we performed co-immunoprecipitation experiments with transfected HEK293T cells. Interestingly, we found that NCS-1 is expressed endogenously in this cell line (Figures 2A,B). Immunostaining with anti-NCS-1 antibody showed an extranuclear punctate pattern as expected for a N-terminal myristoylated protein that exhibits membrane localization. The co-localization between endogenous NCS-1 and overexpressed A_2A-YFP was mostly restricted to the cell periphery (Figure 2A). We then performed co-immunoprecipitation experiments with HEK293T cells co-transfected with A_2A-YFP and NCS-1-TagRFP or the corresponding control contructs (Figure 2C). Dopamine D₂ receptor (D₂-YFP) was included as a positive control since its interaction with NCS-1 is well established (Kabbani et al., 2002; Lian et al., 2011). The mouse anti-GFP antibody led to immunoprecipitation of receptor-YFP in complex with overexpressed (Figure 2C) or endogenous (Figure 2D) NCS-1. In the case of overexpression, the binding of NCS-1 to D₂ and A_2A receptors already occurred in the presence of EGTA (Figure 2C). Immunoprecipitation of overexpressed and endogenous NCS-1 with rabbit anti-NCS-1 bound to Protein G sepharose have shown that A_2A-YFP was efficiently co-purified with NCS-1-TagRFP (Figure 2E). The corresponding controls with unspecific rabbit IgG showed again some unspecific binding of N-myristoylated NCS-1 to the beads but it was significantly lower as compared to immunoprecipitations done with the anti-NCS-1 antibody (Figures 2E,F). Interestingly, analogous to the BRET experiments the presence Ca²⁺ had a positive effect on the interaction (Figures 2D,E and F) and mostly a homomeric A_2A-YFP receptor formed a complex with NCS-1 (Figure 2E).

NCS-1 is involved in regulation of vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane and associates with the Golgi membranes via its N-terminal myristoyl tail where it regulates activity of phosphatidylinositol 4-kinase IIIβ (PI-4KIIIβ), thus providing a Ca²⁺-dependent control for the regulated local synthesis of phosphatidylinositol 4-phosphate (PI(4)P) and for the exit of vesicles from TGN (Zhao et al., 2001; Haynes et al., 2005; Mikhaylova et al., 2009). At the plasma membrane NCS-1 is involved in transduction of Ca²⁺ signaling and regulating the activity of different kinases and surface receptors (for review see Mikhaylova et al., 2011). A_2A receptor traverses the secretory trafficking pathway from the ER to the Golgi complex on the way to the plasma membrane. Therefore, we next asked how the presence of NCS-1 would affect the expression of A_2A receptor at the plasma membrane and where in the cell the complex of A_2A receptor and NCS-1 might occur. To address the first question, we performed a quantitative surface biotinylation assay to compare the surface expression of A_2A receptor with and without co-transfection of NCS-1. Two major bands corresponding to the monomer and the dimer of A_2A receptor were biotinylated and purified with streptavidin beads (Figure 3A). Additional bands appearing in streptavidin-bound fraction but not in whole-cell extract might represent a differentially modified (for example glycosilated, phosphorylated, etc.) plasma membrane receptor fraction. Although there we observed a slight increase in immunoreactivity of biotinylated A_2A when co-expressed with NCS-1, we have not found any significant effect on surface receptor expression in non-stimulated HEK293T cells (Figure 3B). Next, for the co-localization study we have chosen COS-7 cells because they are significantly larger than HEK293T cells. After 24 h of overexpression, significant amounts of A_2A-YFP fluorescence was still associated with the ER and the Golgi membranes. Considerable overlap of A_2A-YFP and NCS-1-TagRFP fluorescence, however, was mostly seen close to the plasma membrane compartment (Figure 3C). Therefore, in another set of experiments we included phalloidin-568 staining to visualize a dense cortical actin cytoskeleton along the cell membranes. Additionally, since the excitation/emission spectrum of TagRFP is largely overlapping with Alexa Fluor 568, we replaced the fusion constructs by NCS-1-YFP and A_2A-Rluc (Figure 3D). Again we could see a co-localization of A_2A and NCS-1 fluorescence overlapping with F-actin at the edges of the cell membrane as well as in small clusters in the cytosol (Figure 3D). These clusters could represent post-Golgi transport carriers, endosomes or some other vesicular compartments.

Homodimers of adenosine A_2A receptor are the predominant form of the receptor at the cell membrane (Canals et al., 2004). Moreover, based on the co-immunprecipitation data, where mostly the homomeric form of A_2A was co-purified with NCS-1, we questioned if NCS-1 could interact with the A_2A homodimer. We performed BRET saturation curves in HEK293T cells expressing an A_2A-Rluc and increasing amounts of A_2A-YFP in the presence or absence of NCS-1 (Figure 4A). The presence of NCS-1 modified the BRET_max (p = 0.01) and BRET₅₀ (p = 0.05) corresponding to the formation of a A_2A homodimer (BRET_max 196 ± 10 mBU and BRET₅₀ 63 ± 7 in the absence of NCS-1; BRET_max 131 ± 9 mBU and BRET₅₀ 90 ± 10 in the presence of NCS-1). This result demonstrates that NCS-1 interacts with A_2A receptor homodimers and induces changes in their quaternary structure in a manner that suggests a greater distance between donor and acceptor, a change in orientation, and/or a diminished number of heteromers. Another possibility that we cannot exclude would be a change in the density of adenosine A_2A receptors at the plasma membrane upon overexpression of NCS-1. However, we have not found significant increases in the amount of A_2A receptor associated with the plasma membrane using surface biotinylation assay (Figure 3A). BRET experiments were always carried out at constant amounts of receptors between compared groups. Since we performed our study in intact HEK293T cells, a small change in plasma membrane density of A_2A receptors would presumably be compensated for by a change in density in other intracellular membranes. Thus, the changes measured in the BRET assay are most likely due to differences in receptor-receptor interaction. An elevation in Ca²⁺ levels by treating cells with ionomycin (1 μM) did not modify A_2A–A_2A homomerization but modified the BRET₅₀ (p = 0.05) in the presence of NCS-1 (BRET_max 148 ± 10 mBU and BRET₅₀ 48 ± 7), indicating a change in the receptor association between NCS-1 and the A_2A heteromer in the presence of Ca²⁺ (Figure 4A).

The adenosine A_2A receptor is a G_s coupled receptor that regulates cAMP production. The activation of the A_2A receptor regulates a number of protein and lipid kinases, including MAPK 1-3 (ERK1/2) phosphorylation and activity (Wyatt et al., 2002; Navarro et al., 2009) and V-akt murine thymoma viral oncogene homolog 1 (AKT, also called PKB/Mori et al., 2004). To test the effect of NCS-1 overexpression on A_2A receptor function we analyzed MAPK and AKT signaling pathways. 2-[p-(2-carboxyethyl)phenylethylamino]-50 ethylcarboxamidoadenosine (CGS 21680) is a selective agonist of A_2A type of adenosine receptors. CGS21680-induced ERK1/2 and AKT phosphorylation was assessed in transiently transfected HEK293T cells at basal conditions or at elevated calcium levels. Agonist stimulation of A_2A in NCS-1-YFP transfected cells induced a significant increase in ERK1/2 phosphorylation at basal conditions and this effect was reversed when cells were pre-treated with Ca²⁺/Ionomycin (Figure 4B). As a negative control, overexpression of caldendrin-YFP, that showed no interaction with A_2A receptors, had no effect on ERK1/2 activity at any condition tested, demonstrating further the specificity of the NCS-1-induced effects (Figure 4B). Finally, NCS-1 promoted the CGS21680-induced AKT phosphorylation at resting Ca²⁺ levels and again this effect was blocked when intracellular Ca²⁺ concentrations were increased with ionomycin (Figure 4C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.