Introduction

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The nucleoside adenosine can influence most mammalian cell types via stimulation of four G protein-coupled receptors: subtypes A₁, A_2A, A_2B, and A₃ (Fredholm et al., 1994). The nature of the responses to adenosine and other agonists depends on the selective coupling of the activated receptor to distinct G proteins. The A₁ (Freissmuth et al., 1991; Munshi et al., 1991; Jockers et al., 1994) and A₃ (Zhou et al., 1992; Palmer et al., 1995) receptors are coupled with G_i proteins inhibiting adenylyl cyclase, whereas the A_2A and A_2B receptors (van Calker et al., 1979; Londos et al., 1980; Pierce et al., 1992) couple to G_s-like proteins and activate adenylyl cyclase. Three stimulatory G proteins have been biochemically characterized: G_s short, G_s long, and G_olf (Jones et al., 1990). They show 88% identity in their amino acid sequences (Jones and Reed, 1989). The G_s subunits have a widespread distribution, whereas the G_olf subunit is distributed in a more restricted manner (Herve et al., 1993). G_olf was first shown to be expressed in olfactory epithelium and was postulated to be exclusively involved in olfactory signaling (Jones and Reed, 1989). However, G_olf was later found also in other brain regions and the expression was particularly high in striatum (Drinnan et al., 1991; Herve et al., 1993). The striatal expression may be functionally important: recently developed transgenic mice deficient in G_olf are not only anosmic but are also hyperactive (Belluscio et al., 1998).

Thus, G_olf present in striatum probably plays a key role in the signal transduction mediated by G protein-coupled receptors in this area. Previous work has shown that G_olf is the main stimulatory G protein coupling dopamine D₁ receptors to adenylyl cyclase in the striatum (Herve et al., 1993). D₁ receptors are abundantly expressed in approximately 50% of the striatal neurons, mostly in those projecting to the substantia nigra and containing -aminobutyric acid, substance P, and dynorphin (Gerfen et al., 1990; Le Moine et al., 1991). Adenosine A_2A receptors are also highly expressed in the striatum (Jarvis and Williams, 1989; Parkinson and Fredholm, 1990). However, adenosine A_2A receptors are segregated from dopamine D₁ receptors and are selectively expressed in the striatopallidal neurons that also contain enkephalin and dopamine D₂ receptors (Schiffmann et al., 1991; Fink et al., 1992; Svenningsson et al., 1997). This is the other major subpopulation of projection neuron within the striatum. Because G_olf is highly expressed in the striatal neurons and A_2A receptors stimulate adenylyl cyclase and cAMP-dependent signal transduction in striatum (Fredholm, 1977; Svenningsson et al., 1998), it seemed important to examine whether A_2A receptors may couple to G_olf in the striatum. The aim of the present study was therefore to investigate the cellular localization of G_olf, G_s, and adenosine A_2A receptors in rat brain, and to investigate, using a photoaffinity labeling technique, whether adenosine A_2A receptors are functionally coupled to G_olf.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Chemicals. Aprotinin, m-aminoacetophenone, 1,4-dioxane, Triton X-100, sodium deoxycholate, phenymethylsulfonyl fluoride, protein A-Sepharose, tergitol Nonidet P-40, 4-morpholineethanesulfonic acid, N-ethyl-N'-(3-dimethylamino-propyl)carbodi-imide hydrochloride, and cAMP were purchased from Sigma (St. Louis, MO). CGS 21680 [2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine] was obtained from Research Biochemicals International (Natick, MA). [³H]cAMP was obtained from DuPont-NEN (Boston, MA). SCH 58261 [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine] was a gift from Dr. E. Ongini (Schering-Plough Research Institute, Milan, Italy). Digoxigenin-11-UTP and FuGENE 6 transfection reagent were purchased from Boehringer Mannheim (Mannheim, Germany). ³⁵S-UTP, [-³²P]GTP, and 4-morpholinepropanesulfonic acid were purchased from Amersham (Little Chalfont, England).

Tissue Preparation. Adult male Sprague-Dawley rats (200-250 g) were briefly anesthetized with CO₂ and sacrificed immediately by decapitation. For the in situ hybridization experiments, the brains were dissected out and frozen on dry ice. Consecutive coronal sections (14 µm) were made through the rostral part of striatum. For the photolabeling experiments, striatum was dissected out and homogenized on ice in 50 mM Tris·HCl, pH 7.4 with an Ultra Turrax (3 ×10 s). The homogenate was centrifuged for 10 min (1,000g) after which the pellet was discarded. The supernatant was centrifuged for 50 min (30,000g) and washed once with 50 mM Tris·HCl, pH 7.4. After the second centrifugation, the pellet containing the membranes was resuspended in an incubation buffer (20 mM MgCl₂, 400 mM NaCl, 120 mM HEPES, and 0.4 mM EDTA) and stored at 80°C in aliquots until used.

Subcloning of cDNA Fragments for In Vitro Transcription Templates. cDNA fragments corresponding to the rat adenosine A_2A receptor, the rat  subunit G_s, and the rat  subunit G_olf were amplified with the use of polymerase chain reaction (PCR). The PCR products were subcloned into pBluescript II (Stratagene, La Jolla, CA). To verify the incorporation of the cDNA into pBluescript II, the subcloned cDNA fragments were sequenced using an automated DNA sequencer, ABI 373A (Applied Biosystems Inc., Foster City, CA).

Probe Synthesis and Labeling for In Situ Hybridization. ³⁵S- or digoxigenin-labeled antisense and sense cRNA probes were prepared by in vitro transcription from cDNA clones corresponding to fragments of rat adenosine A_2A receptor (Fink et al., 1992), rat G protein  subunit G_s (Jones and Reed, 1987), and rat G protein  subunit G_olf (Jones and Reed, 1989). The transcription was performed using MAXI-script in vitro transcription kit according to the manufacturer's protocol (Ambion Inc., Austin, TX).

Single In Situ Hybridization. The in situ hybridization experiments were performed as described previously by Le Moine and Bloch (1995). Cryostat sections were postfixed in 4% paraformaldehyde, dehydrated in graded alcohol, and were hybridized overnight at 55°C with 10⁶ cpm of ³⁵S-labeled probe in 50 µl of hybridization solution. After washing, the slides were dipped into Ilford K5 emulsion [diluted 1:3 in standard sodium citrate (SSC) buffer], exposed for 8 to 12 weeks, developed, and stained with cresyl violet.

Double In Situ Hybridization. In double in situ hybridization experiments, the adenosine A_2A receptor cRNA probe was labeled with ³⁵S-UTP, whereas the probes against G_s and G_olf mRNAs were labeled with digoxigenin-11-UTP. Cryostat sections were pretreated as described above. The sections were hybridized overnight at 55°C with a combination of ³⁵S- and digoxigenin-labeled probes (10⁶ cpm of ³⁵S-labeled probe and approximately 20 ng of digoxigenin-labeled probe in 50 µl of hybridization solution). The slides were washed in RNase A and various concentrations of SSC, but without dithiothreitol (DTT). At the end of the washes, the slides were put in 0.1× SSC at room temperature. The sections were rinsed twice for 5 min in buffer A (1 M NaCl/0.1 M Tris/2 mM MgCl₂, pH 7.5), and then for 30 min in buffer A containing 3% normal goat serum and 0.3% Triton X-100. After 5 h of incubation at room temperature with alkaline phosphatase-conjugated antidigoxigenin antiserum (Boehringer Mannheim; 1:1000 in buffer A/3% normal goat serum/0.3% Triton X-100), the sections were rinsed in buffer A (5 min, twice), then twice for 10 min in buffer B (1 M NaCl/0.1 M Tris/5 mM MgCl₂, pH 9.5), and twice for 10 min in 0.1 M buffer B (containing 0.1 M NaCl). The sections were then incubated overnight in the dark at room temperature in 0.1 M buffer B, pH 9.5, containing 0.34 mg/ml nitroblue tetrazolium and 0.18 mg/ml bromochloro-indolylphosphate. The sections were thereafter rinsed in 0.1 M buffer B, pH 9.5, then in 1× SSC, dried and dipped into Ilford K5 emulsion. After being exposed for 7 to 11 weeks, the sections were developed and mounted without counterstaining.

Preparation of Radioactive m-Acetylanilido-GTP. The preparation of radioactive m-acetylanilido-GTP (m-AcAGTP) was performed as described by Zor et al. (1995). Briefly, [-³²P]GTP (3000 Ci/mmol) was freeze-dried and redissolved in 50 µl of 0.125 M 4-morpholineethanesulfonic acid buffer, pH 6.5. N-Ethyl-N'-(3-dimethylamino-propyl)carbodi-imide hydrochloride (10 µmol) was then dissolved in this solution and m-aminoacetophenone (20 µmol) in 20 µl of 1,4-dioxane was added. After 5 h at room temperature, the mixture was freeze-dried and redissolved in 10 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0. Insoluble m-aminoacetophenone was removed by centrifugation. The product was stored at 18°C until used.

Photoaffinity Labeling of Striatal Membranes with [-³²P]m-AcAGTP. First we optimized the photoaffinity reaction with regard to Mg²⁺, NaCl, and GDP concentrations. The reaction mixture that gave the largest ratio of incorporated [-³²P]m-AcAGTP between stimulated and unstimulated membranes was selected for further use. The optimal reaction mixture contained, in a total volume of 60 µl, 50 µg of membranes, 5 mM MgCl₂, 100 mM NaCl, 0.1 mM EDTA, 0.6 mM ATP, 100 µM GDP, and the adenosine A_2A agonist CGS 21680 when indicated. After 3 min incubation at 37°C, 1.5 µCi of [-³²P]m-AcAGTP was added and the samples were incubated for additional 5 min at 37°C. The reaction was terminated by dilution with 400 µl of ice-cold incubation buffer (5 mM MgCl₂, 100 mM NaCl, 30 mM HEPES, 0.1 mM EDTA, and 2 mM DTT). Excess photoaffinity label was removed by centrifugation at 4°C (12,000g for 5 min) and the pellet was resuspended in 60 µl of incubation buffer. The membranes were pipetted into dimples made in aluminum foil pressed against an empty Eppendorf tube rack. The aluminum foil was wrapped over a glass dish filled with ice. UV irradiation (l = 320 nm) was applied for 20 min from a distance of 2 cm from the samples. Photolabeled membranes were pelleted and solubilized in 2% SDS for 10 min at room temperature before SDS/polyacrylamide gel electrophoresis (PAGE; 12%) and autoradiography or immunoprecipitation.

Immunoprecipitation of  G_olf Protein. The solubilized, photolabeled membranes were diluted 1:1 with immunoprecipitation buffer (10 mM Tris·HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.5% SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 µg/ml aprotinin, and 0.2 mM phenylmethylsulfonyl fluoride) and centrifuged at 12,000g for 10 min at 4°C. The pellet from this centrifugation was discarded and the supernatant was mixed with 3 µl of nondiluted, subtype-specific G protein  subunit G_olf antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which does not cross-react with the G protein  subunit G_s, and was incubated for 1 h at 4°C under constant rotation. Then 60 µl of protein A-Sepharose (4 mg) in immunoprecipitation buffer was added to each sample and incubated overnight at 4°C under constant rotation. Thereafter, the Sepharose beads were pelleted (1 min at 12,000g, 4°C) and washed twice with 1 ml of washing buffer A (50 mM Tris·HCl, 600 mM NaCl, 0.5% SDS, and 1% tergitol Nonidet P-40, pH 7.4) and twice with washing buffer B (100 mM Tris·HCl, 300 mM NaCl, and 10 mM EDTA, pH 7.4). The washed Protein A-Sepharose was then resuspended in 100 µl of Laemmli buffer and heated at 80°C for 5 min and then centrifuged as above. Fifty microliters of the supernatant was then subjected to SDS/PAGE (12%), as described by Laemmli (1970). The gel was dried in a gel drier and exposed to X-ray film.

Subcloning of the Rat G_olf cDNA. The rat G  subunit G_olf cDNA was amplified with PCR using a plasmid (Bluescript) containing the rat G_olf cDNA sequence (a gift from Dr. A.G. Gilman, Southwestern Medical Center, Dallas, TX). The PCR product was subcloned into the vector pCI-neo (Promega, Scandinavian Diagnostic Services, Falkenberg, Sweden). To verify the incorporation of the cDNA into pCI-neo, the subcloned cDNA fragments were sequenced using an automated DNA sequencer, ABI 373A (Applied Biosystems Inc.).

Transient Transfection of G_olf cDNA into Chinese Hamster Ovary (CHO) Cells Expressing Human Adenosine A_2A Receptors. CHO-K1 cells (American Type Culture Collection, Rockville, MD) stably expressing human adenosine A_2A receptors (Kull et al., 1999) were grown adherent and maintained in -minimum essential medium without nucleosides, containing 10% fetal calf serum, penicillin (50 U/ml), streptomycin (50 µg/ml), L-glutamine (2 mM), and geneticin (Life Technologies, Täby, Sweden; 500 µg/ml) at 37°C in 5% CO₂/95% air. Transient transfection of G_olf cDNA or control plasmid was performed using FuGENE 6 transfection reagent (Boehringer Mannheim) according to the instruction manual.

Measurement of cAMP Accumulation. Thirty-six hours after transfection, both G_olf cDNA-transfected cells and control cells were sown into 12-well plates (200,000 cells per well) and allowed to grow for 36 h. Cells were then washed twice with HEPES-buffered (20 mM) -minimal essential medium, pH 7.4. The cells were incubated at 37° for 10 min in 0.9 ml of HEPES-buffered medium. The adenosine A_2A agonist CGS 21680 was added in 0.1 ml of medium and the cells were incubated for another 10 min. The reactions were terminated by the addition of perchloric acid to a final concentration of 0.4 M. After 1 h at 4°C, the acidified cell suspensions were transferred to tubes and neutralized with 4 M KOH/1 M Tris·HCl. The cAMP content in the samples was determined using a competitive radioligand-binding assay (Nordstedt and Fredholm, 1990). Radioactivity was measured in an LKB/Pharmacia scintillation counter with 3 ml of ReadySafe (Beckman, Bromma, Sweden) scintillation fluid.

Data Analysis. In the double in situ hybridization experiments with probes against adenosine A_2A receptor mRNA and G_olf or G_s mRNAs in the striatum, three categories of neurons were counted: those that only exhibited a radioactive signal (i.e., at least two times the background), those that showed only a nonradioactive signal, and those that showed both signals. Quantification was made in the lateral and medial parts of striatum and in the core and shell regions of nucleus accumbens. All neurons within the examined areas were counted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

Regional Distribution of G_olf, G_s, and A_2A Receptors in Rat Forebrain. To study the abundance and distribution of mRNAs for G_olf, G_s, and A_2A receptors, we performed in situ hybridization on coronal sections from the rat brain (Fig. 1). Adenosine A_2A receptor mRNA was, as shown previously (Svenningsson et al., 1997), abundant in the caudate putamen, the nucleus accumbens, and the olfactory tubercle (Fig. 1a). G_olf mRNA was also found in the caudate putamen, the nucleus accumbens, and the olfactory tubercle. In addition, G_olf mRNA, but not adenosine A_2A receptor mRNA, was detected in e.g., the pyramidal cell layer of piriform cortex and in the Islands of Calleja (Fig. 1b). These neurons in the Islands of Calleja are known to be interconnected with the olfactory tubercle (Fallon, 1983) and to express dopamine D₁ and D₃ receptors (Le Moine and Bloch, 1996; Svenningsson et al., 1997). G_s mRNA was most abundant in areas not expressing adenosine A_2A receptor mRNA, such as pyramidal cells of the piriform cortex, cerebral cortex, claustrum, endopiriform nucleus, and the diagonal band of Broca. In addition, G_s mRNA was found throughout septum, whereas mRNA encoding A_2A receptors was found only in occasional cells in lateral septum. Moderate levels of G_s mRNA were detected in nucleus accumbens, whereas caudate putamen only exhibited very low expression (Fig. 1c).

View larger version (72K): [in this window] [in a new window]   Fig. 1.   Distribution of adenosine A2A, Golf, and Gs in rat brain. Darkfield autoradiograms showing the expression of (a) adenosine A2A receptor mRNA, (b) Golf mRNA, and (c) Gs mRNA in coronal sections of the rat forebrain. Regions expressing the genes appear white. Scale bars, 1 mm.

Cellular Localization of G_olf, G_s, and A_2A Receptor mRNA within the Striatum. There are three major subcategories of striatal neurons: medium-sized (~20 µm in diameter) neurons (which are further subdivided into spiny and aspiny neurons) and large-sized (~40 µm in diameter) neurons. Of the striatal neurons, 95% are medium-sized spiny neurons and the remaining 5% are medium-sized aspiny neurons and large-sized neurons. Because no staining for detecting spines was used in the present study, we could only distinguish between neurons based on their size_.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Colocalization of adenosine A2A receptors with Golf and Gs. Emulsion autoradiograms from double in situ hybridization experiments showing (a) the colocalization of adenosine A2A receptor mRNA (silver grains) with Golf mRNA (dark cells) in caudate putamen (arrows indicate colocalization) and (b) the absence of colocalization of adenosine A2A receptor mRNA (silver grains) with Gs mRNA (dark cell) in caudate putamen (arrow indicates a single labeled neuron for Gs mRNA). Scale bars, 10 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Histograms showing the numbers of neurons in the lateral and medial caudate-putamen and in the core and shell of nucleus accumbens that are labeled (+) or not labeled () with probes for adenosine A2A receptor mRNA, Golf mRNA and Gs mRNA. All neurons within the examined areas were counted.

Photolabeling of G Proteins in Striatal Membranes. To investigate whether G_olf is activated on stimulation with an A_2A receptor agonist, we used a photolabeling technique (Zor et al., 1995). We first established that G proteins present in the membranes can be photolabeled by [-³²P]m-AcAGTP and that activation of G proteins leads to an increase in this photolabeling. We incubated striatal membranes with [-³²P]m-AcAGTP with and without a high concentration (10 µM) of the adenosine A_2A agonist CGS 21680 and with different concentrations (3-100 µM) of GDP. The labeling was then visualized by SDS/PAGE followed by X-ray autoradiography. The crude photolabeled membranes gave a broad band with an apparent molecular mass of 40 to 50 kDa (Fig. 4a). In a parallel experiment, the G_olf subunits were immunoprecipitated with a specific antibody directed against G_olf, and separated with the use of SDS-PAGE. The incorporated [-³²P]m-AcAGTP label was determined by autoradiography (Fig. 4b). The immunoprecipitate gave a single band with an apparent molecular mass of 44 to 45 kDa, a size identical with the molecular mass of the protein visualized in immunoblot experiments (data not shown). By comparing the intensity of [-³²P]m-AcAGTP labeling in the crude membranes, it was not possible to distinguish between control conditions and in the presence of agonist at any of the GDP concentrations. However, by comparing the intensity of labeling after immunoprecipitation, it was clear that the labeling was enhanced in the stimulated samples. At low (3-10 µM) GDP concentrations, the basal labeling was strongest, but the ratio of agonist-stimulated to basal photolabeling of the G_olf protein was largest at high (30 to 100 µM) GDP concentrations (Fig. 4, b and c).

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Agonist-stimulated photoaffinity labeling of Golf is influenced by GDP. Membranes from striatum were photolabeled with [-32P]m-AcAGTP at various GDP concentrations in the absence () or presence (+) of CGS 21680 (10 µM). Autoradiogram showing solubilized labeled membranes directly subjected to SDS-PAGE (a) or one representative autoradiogram of three independent experiments showing immunoprecipitated membranes with the subtype-specific G protein  subunit Golf antibody subjected to SDS-PAGE (b). c, histograms showing the quantification of 10 µM CGS 21680-induced increased labeling of immunoprecipitated Golf subunits in percent of control at various GDP concentrations (n = 3) (Error bars, S.E.M.).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Concentration dependent increase of Golf photolabeling by CGS 21680. Membranes from striatum were stimulated in the presence of 100 µM GDP with increasing concentrations of CGS 21680 (1 nM to 10 µM). Golf was immunoprecipitated with the subtype-specific G protein  subunit Golf antibody and subjected to SDS-PAGE. The autoradiogram (a) showing the 45-kDa region is representative of three independent experiments. b, dose-response curve of CGS 21680 induced incorporation of [32P]m-AcAGTP as percent of maximal incorporation (n = 3) (Error bars, S.E.M.).

cAMP Accumulation Experiments. The adenosine A_2A agonist CGS 21680 induced a concentration-dependent increase in cAMP accumulation both in the control cells and in the G_olf cDNA transfected CHO cells (Fig. 6). There was no significant change in the potency of CGS 21680: 19 nM (13-27) for G_olf transfected cells and 29 nM (16-52) for control cells. However, there was a significant increase (t test P = .0082) in the efficacy of the agonist. The plateau was 2.8 (2.6-3.0) pmol/50 µl for G_olf transfected cells and 2.2 (1.9-2.4) pmol/50 µl for control cells (95% confidence intervals within parentheses).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Dose-response curves of CGS 21680 induced cAMP accumulation in CHO cells stably expressing () adenosine A2A receptors and CHO cells transiently transfected () with Golf cDNA and expressing adenosine A2A receptors. Results are mean of two experiments with duplicates and are presented as picomoles of cAMP per 50 µl (Error bars, S.E.M.). The plateau is significantly increased in Golf transfected cells compared with nontransfected cells (t test P = .0082).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

It has been taken for granted that adenosine A_2A receptors are coupled to the stimulatory G protein subunit G_s. The present data suggest that this may not be the complete truth, at least not in dopamine-rich areas of the brain.

Some previous reports suggest that G_s plays a minor role in striatal neurons, which contain the highest levels of A_2A receptors in the brain. Although the highest levels of activated adenylyl cyclase, measured using [³H]forskolin binding, are found in the cautate putamen (Worley et al., 1986), this region possesses the lowest levels of G_s mRNA in brain (Largent et al., 1988) (see also Figs. 1c, 2b, and 3). It can also be mentioned that Albright and McCune-Albright syndromes, which are associated with mutations of the G_s gene, do not exhibit any extrapyramidal neurological symptoms (Weinstein et al., 1990; Schwindinger et al., 1992).

Herve et al. (1993) have shown that selective lesioning of striatonigral neurons, using a retrograde neurotoxin, markedly decreases the levels of dopamine D₁ receptors and G_olf in striatum. Moreover, 6-hydroxydopamine lesioning of the dopaminergic axons in neonatal rats induces hypersensitivity to dopamine receptor agonists in adulthood without any change in D₁ receptor bindingbut there is an increase in G_olf expression. There is also an increase in G_s protein, but mainly in glial cells (Penit-Soria et al., 1997). Furthermore, mice deficient in G_olf are hyperactive (Belluscio et al., 1998). These results could indicate that G_olf might, in striatum, play some of the role(s) otherwise attributed to G_s.

Here we confirm that mRNAs for the adenosine A_2A receptor (Svenningsson et al., 1997) and for G_olf (Drinnan et al., 1991; Herve et al., 1993) were most abundant in the caudate putamen, the nucleus accumbens, and the olfactory tubercle, whereas these areas express little G_s (Largent et al., 1988). We also show directly that adenosine A_2A receptors and G_olf are colocalized. In situ hybridization experiments showed that approximately 50% of the medium-sized neuron-like cells exhibited a strong signal for adenosine A_2A receptor mRNA and a large majority (95%) of these neurons coexpressed G_olf mRNA. G_olf mRNA was also abundant in A_2A receptor mRNA negative neurons. Thus, the relative enrichment of A_2A receptors and G_olf mRNA in the striatum is likely to reflect the preferential localization in the striatopallidal neurons (A_2A receptors) and in striatonigral and striatopallidal neurons (G_olf). The labeling for G_s mRNA in striatum was weak and seemed to be preferentially located in glial cells, as described previously (Feinstein et al., 1992). In the present study, only 3 to 5% of the neurons in caudate putamen and 13 to 21% of the neurons in nucleus accumbens expressed both A_2A receptor mRNA and G_s mRNA.

Using a photoaffinity labeling method, we show that the adenosine A_2A receptor is not only colocalized with G_olf in striatum, but also functionally coupled to it. Photoaffinity labeling is a method used for identification of distinct molecular components in biochemical processes. The simplest procedure for labeling G proteins is by UV irradiation of [-³²P]GTP (Basu and Modak, 1987). However, this method has low sensitivity. The most widely used photoaffinity label for G proteins is 4-azidoanilido-GTP and was developed by Pfeuffer (1977). However, this compound has some disadvantages: it is unstable and its synthesis, purification, and application must be performed in complete darkness. The photoaffinity label we have used, m-acetylanilido-GTP, developed by Zor and coworkers (1995), has several advantages: 1) no need for purification; 2) quantitative conversion of GTP into m-AcAGTP; 3) excellent stability in solution; and 4) resistant to hydrolysis and remains bound to the G protein during centrifugal washing. Using this photolabeling method, we could demonstrate that the adenosine A_2A agonist CGS 21680 induced an increased incorporation of [-³²P]m-AcAGTP in immunoprecipitated G_olf subunits. The magnitude of the increase was GDP-concentration-dependent, with the largest increase seen at a high GDP concentration (100 µM). CGS 21680 increased labeling in a dose-dependent manner with a calculated EC₅₀ value of 16 nM (4.7-54). This increase was receptor mediated because it was blocked by the A_2A receptor antagonist SCH 58261 (data not shown). The CGS 21680 potency is in agreement with the potency in the cAMP assay.

In an attempt to determine whether A_2A receptor-activated G_olf subunits could activate adenylyl cyclase, we transiently transfected G_olf into CHO cells stably expressing A_2A receptors (Kull et al., 1999). In the G_olf transfected cells, there was a significant increase in the maximal agonist stimulated adenylyl cyclase activity without any change in the potency of CGS 21680. A change in the maximal effect was expected from the previous demonstration of increased constitutive accumulation of cAMP and the observation that agonist-stimulated cAMP levels were proportional to the amount of G_s expression (Yang et al., 1997).

	Conclusion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

The present study shows that adenosine A_2A receptor mRNA was coexpressed with G_olf mRNA in striatal medium-sized neurons to a much higher extent than with G_s mRNA. Using a photolabeling technique, we show that activation of adenosine A_2A receptors in striatal membranes led to activation of G_olf. Moreover, transfection of G_olf cDNA into cells that express human adenosine A_2A receptors led to an increase of the maximal agonist stimulated cAMP level. When taken together, these results provide strong anatomical and biochemical evidence that adenosine A_2A receptors stimulate adenylyl cyclase and cAMP-dependent signal transduction in striatum by activating G_olf rather than G_s. These findings have the more general implication that a given receptor may couple to different G proteins in different locations.