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• W2007251619 abstract "The adenosine A2A receptor (A2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein αolf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ7 subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ7 protein controls the hierarchial assembly of a specific G-protein αolfβ2γ7 heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ7 subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A2AR G-protein αolfβ2γ7 interface as a possible therapeutic target for Parkinson disease. The adenosine A2A receptor (A2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein αolf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ7 subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ7 protein controls the hierarchial assembly of a specific G-protein αolfβ2γ7 heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ7 subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A2AR G-protein αolfβ2γ7 interface as a possible therapeutic target for Parkinson disease. G-protein-coupled receptors represent the single largest family of target proteins for drug development. Their actions require the participation of heterotrimeric guanine nucleotide binding proteins (G-proteins) whose roles in these diverse signaling pathways may be determined by their specific αβγ subunit combinations. The existence of 16 α, 5 β, and 12 γ subtypes creates the potential to generate a large number of distinct G-protein αβγ heterotrimers (1Hildebrandt J.D. Biochem. Pharmacol. 1997; 54: 325-339Crossref PubMed Scopus (127) Google Scholar, 2Robishaw J.D. Berlot C.H. Curr. Opin. Cell Biol. 2004; 16: 206-209Crossref PubMed Scopus (89) Google Scholar). Although their biochemical properties have been well studied (3Birnbaumer L. Biochim. Biophys. Acta. 2007; 768: 772-793Crossref Scopus (131) Google Scholar, 4Smrcka A.V. Cell Mol. Life Sci. 2008; 65: 2191-2214Crossref PubMed Scopus (276) Google Scholar), key questions regarding which G-protein αβγ heterotrimers actually exist in vivo and determining whether they perform specific signaling roles and biological functions remain to be answered. To address these questions, a gene-targeting approach has been used to delete the various α subunit genes in mice, leading to the identification of physiological functions for most of them (5Offermanns S. Oncogene. 2001; 20: 1635-1642Crossref PubMed Scopus (75) Google Scholar). By contrast, little attention has focused on the β and γ subunit genes. In particular, several features of the γ subunit genes suggest they may perform heterogeneous functions in vivo. Analogous to their α partners, the various γ subtypes show substantial structural diversity and exhibit pleiotropic patterns of expression (2Robishaw J.D. Berlot C.H. Curr. Opin. Cell Biol. 2004; 16: 206-209Crossref PubMed Scopus (89) Google Scholar). Accordingly, we have undertaken a gene targeting approach to systematically ablate the individual γ subtypes in mice (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7Schwindinger W.F. Giger K.E. Betz K.S. Stauffer A.M. Sunderlin E.M. Sim-Selley L.J. Selley D.E. Bronson S.K. Robishaw J.D. Mol. Cell Biol. 2004; 24: 7758-7768Crossref PubMed Scopus (74) Google Scholar), with the ultimate goal of elucidating their biological functions. Our recent work has demonstrated that knock-out of Gng7, encoding the γ7 subtype, produces a behavioral phenotype resulting in part from a localized defect in dopamine D1 receptor (D1R) 2The abbreviations used are: D1Rdopamine D1 receptorA2ARadenosine A2A receptorSPstriatopallidalSNstriatonigralGTPγSguanosine 5′-3-O-(thio)triphosphate. signaling in the brain (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Within the brain, the striatum collects and processes information from the cerebral cortex and thalamus affecting the control of voluntary movements (8Matamales M. Bertran-Gonzalez J. Salomon L. Degos B. Deniau J.M. Valjent E. Hervé D. Girault J.A. PLoS One. 2009; 4e4770Crossref PubMed Scopus (179) Google Scholar). Accounting for >90% of neurons within the striatum, the medium spiny neurons are comprised of two distinct subpopulations that are classified on the basis of their distinct circuitries (9Cauli O. Morelli M. Behav. Pharmacol. 2005; 16: 63-77Crossref PubMed Scopus (139) Google Scholar). The striato-nigral (SN) neurons projecting to the substania nigra pars reticulata and entopeduncular nucleus constitute the direct tract, whereas the striato-pallidal (SP) neurons projecting to the lateral part of the globus pallidus comprise the indirect tract (8Matamales M. Bertran-Gonzalez J. Salomon L. Degos B. Deniau J.M. Valjent E. Hervé D. Girault J.A. PLoS One. 2009; 4e4770Crossref PubMed Scopus (179) Google Scholar). Typically, a coordinated balance between these two tracts produces normal movements, whereas a preponderance of one tract over the other is implicated in producing motor abnormalities associated with basal ganglia disorders (10Albin R.L. Young A.B. Penney J.B. Trends Neurosci. 1989; 12: 366-375Abstract Full Text PDF PubMed Scopus (4064) Google Scholar, 11Popoli P. Reggio R. Pèzzola A. Neuropsychopharmacology. 2000; 22: 522-529Crossref PubMed Scopus (42) Google Scholar). dopamine D1 receptor adenosine A2A receptor striatopallidal striatonigral guanosine 5′-3-O-(thio)triphosphate. In the SN neurons, the D1R acts through the G-protein αolf subunit to stimulate cAMP production (12Corvol J.C. Studler J.M. Schonn J.S. Girault J.A. Hervé D. J. Neurochem. 2001; 76: 1585-1588Crossref PubMed Scopus (176) Google Scholar). Based on recent analyses of Gng7−/− mice, this action is also dependent on the γ7 subtype (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Intriguingly, in the SP neurons, the adenosine A2A receptor (A2AR) also couples through the G-protein αolf subunit to enhance cAMP production even though this pathway produces the opposite behavioral effect (12Corvol J.C. Studler J.M. Schonn J.S. Girault J.A. Hervé D. J. Neurochem. 2001; 76: 1585-1588Crossref PubMed Scopus (176) Google Scholar). In the present study, we explored whether a specific G-protein αolfβ2γ7 subunit combination is required for this pathway in SP neurons. Using mice with targeted deletions of Gng7 or Gnal, lacking the G-protein γ7 (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) or αolf (13Belluscio L. Gold G.H. Nemes A. Axel R. Neuron. 1998; 20: 69-81Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar) subunits, respectively, we showed that levels of the αolf and β2 proteins were selectively and coordinately reduced in Gng7−/− mice, whereas levels of γ7 were largely unaffected in the Gnal−/− mice. Notably, these results indicate that assembly of the αolfβ2γ7 heterotrimer is an ordered process that is controlled by the amount of the γ7 subtype. Moreover, loss of the G-protein γ7 subunit led to defects in both D1R (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and A2A receptor activation of adenylyl cyclase without producing any gross alterations in locomotor behavior typical of Parkinson disease. Importantly, these findings contribute to a growing literature that suggests that blockade of A2AR signaling in the striatum may be an effective strategy for treating various neurological and addictive disorders. Disruption of Gng7, the gene encoding the G-protein γ7 subunit in mice, was described previously (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Gng7+/− mice were backcrossed to C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) for 5 generations, or separately to BALB/c mice (Jackson Laboratories) for 5 generations. Gng7+/− mice were intercrossed to produce the Gng7−/− mice and wild-type littermates used in these experiments. In this work, we will describe these mice as “on a C57BL/6 background,” or “on a BALB/c background.” However, after only 5 generations of backcrosses, there is still some contribution to their genetic makeup from the original ES cells (129SvEvBrd, Lexicon Genetics, Inc., The Woodlands, TX), from the dam that was bred with the chimera (C57BL/6 albino, Lexicon), and from the Cre recombinase expressing strain that we utilized (BALB/c-TgN(CMV-Cre)#Cgn, Jackson Laboratories). Hence, it was essential to use littermates to control for the possible influence of genetic background to observed responses. On the C57BL/6 background, 16 Gng7−/− mice (8 males and 8 females) and 16 wild-type littermates (8 males and 8 females) were studied. On the BALB/c background, 12 Gng7−/− mice (6 males and 6 females), and 14 wild-type littermates (4 males and 10 females) were studied. Genotypes were determined by PCR analysis of tail biopsy DNA as described previously (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Mice with a disrupted Gnal gene, that lack the G-protein αolf, were described previously (13Belluscio L. Gold G.H. Nemes A. Axel R. Neuron. 1998; 20: 69-81Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). These were backcrossed for up to 9 generations with C57BL/6 mice to obtain homozygous (Gnal−/−) and their control littermates (Gnal+/+). For comparing homozygous mutant and wild-type mice, 6-week-old male and female mice were used for experiments. Mice were segregated by sex and group housed in plastic microisolator cages in ventilated racks (Thoren Caging Systems, Inc., Hazelton, PA). Mice were given ad lib access to water and Mouse Diet 9F (Purina Mills, LLC, St. Louis, MO). Environmental factors included temperature and humidity control and a 12-hour light/dark cycle. The animal facility is maintained as virus antibody-free and parasite-free. The Geisinger Clinic Institutional Animal Care and Use Committee approved animal research protocols. Striatal tissues were homogenized in Buffer A (10 mm Tris, pH 7.4, 1 mm EDTA, 1 mm DTT, 0.3 mm AEBSF, 30 μm leupeptin, 1 μm pepstatin A) with 10% sucrose using a Brinkmann Homogenizer (Brinkmann Instruments Co, Westbury, NY). Membranes were then isolated by centrifugation (65 min at 100,000 × g) onto a cushion of Buffer A with 44.5% (w/v) sucrose. The membranes at the interface were transferred to a new tube and twice washed with Buffer A and collected by centrifugation (30 min at 100,000 × g). Protein concentrations were determined with Coomassie Plus (Thermo Fisher Scientific, Inc., Rockford, IL). Adenylyl cyclase activity was determined by incubating membrane protein (20 μg) at 30 °C for 15 min in 0.1 ml of buffer containing 50 mm HEPES (pH 7.4), 1 mm EGTA, 5 mm MgCl2, 0.1 mm ATP, 1 × 106 cpm of [α-32P]ATP, 10 μm rolipram, 1 unit/ml adenosine deaminase, 5 mm creatine phosphate, 50 units/ml creatine phosphokinase, and various agonists as indicated in the text. Reactions were terminated by addition of 0.1 ml of 2% SDS, 40 mm ATP, 1.4 mm cAMP, 10,000 cpm of [3H]cAMP, and heating to 100 °C for 3 min. [32P]cAMP was isolated by chromatography on Dowex and Alumina columns, using [3H]cAMP as a recovery marker, and quantified by liquid scintillation counting. Radioligand binding to A2AR in striatal membranes prepared from Gng7−/− mice and wild-type littermates was performed using either the radiolabeled agonist [125I]2-[2-(4-amino-3-iodo-phenyl)ethylamino]adenosine (125I-APE) or the radiolabeled antagonist 125I-ZM241385 as described previously (14Luthin D.R. Olsson R.A. Thompson R.D. Sawmiller D.R. Linden J. Mol. Pharmacol. 1995; 47: 307-313PubMed Google Scholar, 15Murphree L.J. Marshall M.A. Rieger J.M. MacDonald T.L. Linden J. Mol. Pharmacol. 2002; 61: 455-462Crossref PubMed Scopus (86) Google Scholar). Binding was performed using striatal membranes prepared from wild type or Gng7−/− mice in buffer containing 10 mm HEPES pH 7.4, 1 mm EDTA, 5 mm MgCl2, and 1 unit/ml adenosine deaminase. The agonist, 125I-APE binds to two affinity states of the A2AR, a high affinity state corresponding to receptor-G-protein complexes, and a low affinity state corresponding to receptors uncoupled from G-proteins (15Murphree L.J. Marshall M.A. Rieger J.M. MacDonald T.L. Linden J. Mol. Pharmacol. 2002; 61: 455-462Crossref PubMed Scopus (86) Google Scholar). Because GTPγS added to membranes uncouples receptors from G-proteins, 50 μm GPTγS was added to some membranes to measure agonist binding to largely uncoupled receptors. In the absence of added GTPγS, 125I-APE binds preferentially to G-protein coupled receptors. 125I-APE also binds to uncoupled receptors, but may under measure total receptor number because a fraction of the radioligand may dissociate for the low affinity site during washing of filters. Hence, the total number of receptors (Bmax) was more accurately detected as the number of specific binding sites for the high affinity antagonist, 125I-ZM241385. Nonspecific radioligand binding was measured in the presence of 50 μm N-ethylcarboxamidoadenosine (NECA). To examine the expression of G-protein subunits in mouse striatum, Western blot analysis was performed on cholate-solublized membranes that were prepared as described previously (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Antisera for Gαs was used at a 1:500 dilution, for Gαolf (16Hervé D. Le Moine C. Corvol J.C. Belluscio L. Ledent C. Fienberg A.A. Jaber M. Studler J.M. Girault J.A. J. Neurosci. 2001; 21: 4390-4399Crossref PubMed Google Scholar) at 1:2000, and for Ras (BD Biosciences, Palo Alto, CA) at a 1:2000 dilution. Antisera for β1 (1:500), β2 (1:500), γ2, γ3, γ5 (1:100), and γ7 were described previously (17Foster K.A. McDermott P.J. Robishaw J.D. Am. J. Physiol. 1990; 259: H432-H441Crossref PubMed Google Scholar, 18Cali J.J. Balcueva E.A. Rybalkin I. Robishaw J.D. J. Biol. Chem. 1992; 267: 24023-24027Abstract Full Text PDF PubMed Google Scholar, 19Wang Q. Mullah B.K. Robishaw J.D. J. Biol. Chem. 1999; 274: 17365-17371Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and were used at a 1:200 dilution, except as indicated. His-tagged G-protein β and γ subunits (CytoSignal Research Products, Irvine, CA) and His-tagged αolf (12Corvol J.C. Studler J.M. Schonn J.S. Girault J.A. Hervé D. J. Neurochem. 2001; 76: 1585-1588Crossref PubMed Scopus (176) Google Scholar) were used as standards for quantitative immunoblotting. To examine levels of mRNA for αolf, β2, γ7, and DARPP-32, RNA was prepared from striatum of six Gng7−/− mice and six wild-type littermates at the N24 backcross to C57BL/6 and real-time RT-PCR was conducted as described previously (20Schwindinger W.F. Borrell B.M. Waldman L.C. Robishaw J.D. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009; 297: R1494-R1502Crossref PubMed Scopus (26) Google Scholar). Primers were as follows: Gnal (CCT TCC TAC TTG CCT GAC CGC; TGA CGA TAG TGC TTT TCC CGG), Gng7 (GCT GGG ATC GAA CGC ATC AAG; CAG GAA GAT CCC GGC ATT CAC), Gnb2 (TCA TAG GTC ACG AGT CGG ACA TCA; ATG GCA TCC CAG ATG TTG CAG TTG), Ppp1r1b (CAC CAC CCA AAG TCG AAG AGA; CGA AGC TCC CCT AAC TCA TCCT), and to correct for variation in cDNA yield Eef1a1 (GGA ATG GTG ACA ACA TGC TG; CGT TGA AGC CTA CAT TGT CC). Locomotor activity was quantified in CLAMS cages (Columbus Instruments, Columbus, OH). The cages consist of clear plastic boxes (20 cm × 10 cm × 12.5 cm) fitted with three rows of 8 photoelectric sensors (x, y, and z directions). The mice were placed in the CLAMS cages at 11 am and remained in the cages for 4 h. During this time the mice had ad lib access to water. Every minute the numbers of individual (total) and consecutive (ambulatory) photobeam breaks for each of 3 sensor arrays, and the number of contacts with the sipper tube were recorded. For caffeine trials, mice were removed from the cages after 1 h and given an intraperitoneal injection of saline or caffeine (5 ml/kg). Mice that had been habituated to the CLAMS cages and to the injection procedure with saline were used in drug trials. The locomotor response to drug is expressed as an increment over the response to saline. Locomotor activities were studied at age 10.0 ± 0.8 weeks for C57BL/6 background mice and age 8.5 ± 0.3 weeks for BALB/c background mice. The response to caffeine was studied at age 23.6 ± 3.0 weeks for C57BL/6 background mice and age 14.6 ± 0.3 weeks for BALB/c background mice. Sample statistics and Student's t-tests were computed using Excel (Microsoft). Data are presented as means ± S.E. of the mean. Locomotor activity was compared by repeated measures multivariate analysis of variance (MANOVA), using JMP (SAS Institute Inc., Carey, NC). The A2AR is primarily responsible for the psychostimulant action of caffeine (21Ledent C. Vaugeois J.M. Schiffmann S.N. Pedrazzini T. El Yacoubi M. Vanderhaeghen J.J. Costentin J. Heath J.K. Vassart G. Parmentier M. Nature. 1997; 388: 674-678Crossref PubMed Scopus (788) Google Scholar, 22Huang Z.L. Qu W.M. Eguchi N. Chen J.F. Schwarzschild M.A. Fredholm B.B. Urade Y. Hayaishi O. Nat. Neurosci. 2005; 8: 858-859Crossref PubMed Scopus (459) Google Scholar). Because blockade of this receptor has been shown to reverse the hypolocomotor phenotype resulting from dopamine deficiency (23Salamone J.D. Ishiwari K. Betz A.J. Farrar A.M. Mingote S.M. Font L. Hockemeyer J. Müller C.E. Correa M. Parkinsonism Relat. Disord. 2008; 14: S130-S134Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and dysfunctional dopamine signaling (24Aoyama S. Kase H. Borrelli E. J. Neurosci. 2000; 20: 5848-5852Crossref PubMed Google Scholar), the A2AR signaling components are increasingly recognized as valid therapeutic targets for treating Parkinson disease and for reducing the side effects of levodopa therapy (25Bara-Jimenez W. Sherzai A. Dimitrova T. Favit A. Bibbiani F. Gillespie M. Morris M.J. Mouradian M.M. Chase T.N. Neurology. 2003; 61: 293-296Crossref PubMed Scopus (319) Google Scholar, 26Schwarzschild M.A. Agnati L. Fuxe K. Chen J.F. Morelli M. Trends Neurosci. 2006; 29: 647-654Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). In SP neurons, the G-protein αolf subunit has been shown to positively couple this receptor to stimulation of cAMP production (27Kull B. Svenningsson P. Fredholm B.B. Mol. Pharmacol. 2000; 58: 771-777Crossref PubMed Scopus (170) Google Scholar, 12Corvol J.C. Studler J.M. Schonn J.S. Girault J.A. Hervé D. J. Neurochem. 2001; 76: 1585-1588Crossref PubMed Scopus (176) Google Scholar). However, little information is available on the obligatory β and γ components involved in this context. By applying biochemical and behavioral approaches to a novel mouse model, we found that the G-protein γ7 subunit is specifically required for both A2AR signaling and psychostimulant response to caffeine. Identifying a mechanistic basis for this requirement, we show that the γ7 protein drives the preferential assembly of a G-protein αolfβ2γ7 heterotrimer in the striatum that is involved in a key signaling pathway controlling locomotion and reward. Regulation of cAMP production in medium spiny neurons represents a primary target of many neurotransmitters and psychoactive drugs that affect short- and long-term locomotor responses (28Borgkvist A. Fisone G. Neurosci. Biobehav. Rev. 2007; 31: 79-88Crossref PubMed Scopus (53) Google Scholar). Virtually all medium spiny neurons, including SN and SP neurons, express substantial levels of γ7 mRNA (29Watson J.B. Coulter 2nd, P.M. Margulies J.E. de Lecea L. Danielson P.E. Erlander M.G. Sutcliffe J.G. J. Neurosci. Res. 1994; 39: 108-116Crossref PubMed Scopus (52) Google Scholar, 30Doyle J.P. Dougherty J.D. Heiman M. Schmidt E.F. Stevens T.R. Ma G. Bupp S. Shrestha P. Shah R.D. Doughty M.L. Gong S. Greengard P. Heintz N. Cell. 2008; 135: 749-762Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 31Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. Suárez-Fariñas M. Schwarz C. Stephan D.A. Surmeier D.J. Greengard P. Heintz N. Cell. 2008; 135: 738-748Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). Because adenylyl cyclase signaling by the D1R was shown to be dependent on γ7 expression in SN neurons (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), we explored whether adenylyl cyclase signaling by the A2AR is similarly dependent on expression of γ7 in SP neurons. As a biochemical assay, we used the selective A2AR agonist, CGS-21680, to stimulate adenylyl cyclase activity in striatal membranes prepared from Gng7−/− mice. By comparison to their wild type littermates, adenylyl cyclase activity in response to 10 μm or 25 μm CGS-21680 was reduced by 30–40% in striatal membranes from Gng7−/− mice (Fig. 1A). Furthermore, cAMP production in response to 100 μm forskolin was also reduced by ∼35% in striatal membranes from Gng7−/− mice (Fig. 1B). Because forskolin-stimulated adenylyl cyclase activity was reduced, we could not assess whether the impaired response to the A2AR agonist was due to a defect in G-protein coupling and/or adenylyl cyclase activation. Therefore, as a second biochemical assay, we used high affinity agonist binding to directly measure the actual interaction between the A2AR and the G-protein. Initial saturation binding experiments were performed with the A2AR agonist, 125I-APE, on pooled samples of striatal membranes from either Gng7−/− mice or their wild-type littermates (12 mice in each group). In the wild-type sample, the addition of GTPγS dramatically reduced agonist binding by greater than 75%, indicating a significant portion of the A2AR was associated with G-protein (Fig. 2A). In contrast, in the knock-out sample, addition of GTPγS produced little reduction in A2AR agonist binding (Fig. 2B), suggesting most A2AR was no longer coupled to G-protein. Finally, there was no significant difference in the binding of the A2AR antagonist, 125I-ZM241385, between the samples (Fig. 2C), indicating the total number of A2AR was comparable between the two genotypes. Taken together, these results indicate a striking reduction in the fraction of the A2AR that was coupled to G-protein in striatal membranes from Gng7−/− mice. Subsequent binding studies were performed on striatal membranes from individual mice representing each genotype. To calculate the fraction of the A2AR pool that was coupled to G-protein, we determined the ratio of specific GTPγS-sensitive 125I-APE agonist binding sites relative to 125I-ZM421385 antagonist binding sites in striatal membranes from both genotypes. By comparison to their wild-type littermates, the fraction of the A2AR pool that was coupled to G-protein was markedly reduced in striatal membranes from Gng7−/− mice (p < 0.001) (Fig. 2D). Collectively, these results confirm that A2AR signaling shows a specific requirement for G-protein γ7 expression and that its loss is associated with an impaired ability of this receptor to couple to G-protein. One mechanism that could account for the observed defects in both G-protein coupling and adenylyl cyclase activation is a coordinate reduction in the cellular amount of the G-protein αs or αolf subunit. These two structurally related isoforms are both able to stimulate cAMP production (12Corvol J.C. Studler J.M. Schonn J.S. Girault J.A. Hervé D. J. Neurochem. 2001; 76: 1585-1588Crossref PubMed Scopus (176) Google Scholar) and are both expressed in the striatum (32Hervé D. Lévi-Strauss M. Marey-Semper I. Verney C. Tassin J.P. Glowinski J. Girault J.A. J. Neurosci. 1993; 13: 2237-2248Crossref PubMed Google Scholar). Previously, we showed that loss of the γ7 subunit coordinately reduced levels of the αolf protein in the striatum (6Schwindinger W.F. Betz K.S. Giger K.E. Sabol A. Bronson S.K. Robishaw J.D. J. Biol. Chem. 2003; 278: 6575-6579Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To confirm and extend this finding, we performed immunoblot analysis on micropunch samples from dorsal striatum (caudate) and ventral striatum (nucleus accumbens) of Gng7−/− mice on two different genetic backgrounds (i.e. C57BL/6 and BALB/c). By comparison to their wild-type littermates, αolf protein levels were strikingly reduced by >85% in both dorsal and ventral striatal membranes from knock-out animals on a C57BL/6 genetic background (Fig. 3A). The effect was remarkably specific in that αs protein levels (i.e. 45- and 52-kDA forms) were not affected in the dorsal striatum and were reduced by only 20% in the ventral striatum of knock-out mice (Fig. 3B). Attesting to the generality of this finding, immunoblot analysis of the corresponding regions of knock-out mice on a BALB/c genetic background yielded similar results (supplemental Fig. S1). Taken together, these results demonstrate that the cellular level of the αolf but not the αs subunit is dependent on expression of the γ7 subunit. Next, we investigated how loss of the γ7 protein impacts the levels of particular β protein(s) in the striatum. Because targeting of the βγ dimer to the plasma membrane is dependent upon post-translational lipid modifications of the γ subunit (33Iñiguez-Lluhi J.A. Simon M.I. Robishaw J.D. Gilman A.G. J. Biol. Chem. 1992; 267: 23409-23417Abstract Full Text PDF PubMed Google Scholar, 34Simonds W.F. Butrynski J.E. Gautam N. Unson C.G. Spiegel A.M. J. Biol. Chem. 1991; 266: 5363-5366Abstract Full Text PDF PubMed Google Scholar, 35Muntz K.H. Sternweis P.C. Gilman A.G. Mumby S.M. Mol. Biol. Cell. 1992; 3: 49-61Crossref PubMed Scopus (118) Google Scholar), we reasoned that loss of the γ7 protein could affect the level of a specific β subtype in striatal membranes from Gng7−/− mice. By comparison to their wild-type littermates, β2 protein levels were selectively reduced by 31% with no significant changes in the amounts of β1 and β4 proteins (Fig. 3C). Taken together, these findings show both coordinate and selective suppression of the αolf, β2, and γ7 subunits at the protein level. To assess whether the αolf subunit plays a reciprocal role in this process, we used the Gnal−/− mouse model (13Belluscio L. Gold G.H. Nemes A. Axel R. Neuron. 1998; 20: 69-81Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar) to determine whether loss of the αolf protein causes a corresponding suppression of the γ7 protein. By comparison to their wild type littermates, γ7 protein levels were not significantly different in striatal membranes from Gnal−/− mice (Fig. 4), indicating that the expression of the γ7 but not the αolf protein drives the assembly of a specific G-protein heterotrimer in the striatum. One mechanism that could account for suppression of the αolf protein is a reduced level of the corresponding mRNA transcript. To test this possibility, we performed real time RT-PCR analysis on striatal tissue from Gng7−/− mice and their wild-type littermates on the C57BL/6 background. Despite the loss of αolf protein (Fig. 4), the level of αolf mRNA was not significantly reduced in the striatum of Gng7−/− mice (Fig. 5A). Moreover, the level of β2 mRNA was not significantly different in wild type and Gng7−/− str" @default.
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• W2007251619 title "Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum" @default.
• W2007251619 cites W1494195183 @default.
• W2007251619 cites W1521659744 @default.
• W2007251619 cites W1527306897 @default.
• W2007251619 cites W1530195638 @default.
• W2007251619 cites W1604834232 @default.
• W2007251619 cites W1844553425 @default.
• W2007251619 cites W1904392094 @default.
• W2007251619 cites W1907647691 @default.
• W2007251619 cites W1911782737 @default.
• W2007251619 cites W1967383331 @default.
• W2007251619 cites W1976310120 @default.
• W2007251619 cites W1977221298 @default.
• W2007251619 cites W1978559289 @default.
• W2007251619 cites W1982729847 @default.
• W2007251619 cites W1983203888 @default.
• W2007251619 cites W1987380827 @default.
• W2007251619 cites W1990269363 @default.
• W2007251619 cites W1997117846 @default.
• W2007251619 cites W2013190001 @default.
• W2007251619 cites W2017179120 @default.
• W2007251619 cites W2018348038 @default.
• W2007251619 cites W2018384224 @default.
• W2007251619 cites W2019347876 @default.
• W2007251619 cites W2025837534 @default.
• W2007251619 cites W2027092355 @default.
• W2007251619 cites W2027182286 @default.
• W2007251619 cites W2028129201 @default.
• W2007251619 cites W2033922593 @default.
• W2007251619 cites W2038370141 @default.
• W2007251619 cites W2040473331 @default.
• W2007251619 cites W2046384382 @default.
• W2007251619 cites W2051274411 @default.
• W2007251619 cites W2052280808 @default.
• W2007251619 cites W2052582754 @default.
• W2007251619 cites W2060096092 @default.
• W2007251619 cites W2060588252 @default.
• W2007251619 cites W2063225331 @default.
• W2007251619 cites W2065066076 @default.
• W2007251619 cites W2065138156 @default.
• W2007251619 cites W2072237956 @default.
• W2007251619 cites W2079001140 @default.
• W2007251619 cites W2083747704 @default.
• W2007251619 cites W2084058724 @default.
• W2007251619 cites W2085426620 @default.
• W2007251619 cites W2085460988 @default.
• W2007251619 cites W2088615332 @default.
• W2007251619 cites W2089015327 @default.
• W2007251619 cites W2089627071 @default.
• W2007251619 cites W2090614971 @default.
• W2007251619 cites W2092666379 @default.
• W2007251619 cites W2095061120 @default.
• W2007251619 cites W2105737054 @default.
• W2007251619 cites W2107495585 @default.
• W2007251619 cites W2113557771 @default.
• W2007251619 cites W2115155255 @default.
• W2007251619 cites W2118774378 @default.
• W2007251619 cites W2123456748 @default.
• W2007251619 cites W2123491442 @default.
• W2007251619 cites W2135578299 @default.
• W2007251619 cites W2138787681 @default.
• W2007251619 cites W2142803792 @default.
• W2007251619 cites W2143544378 @default.
• W2007251619 cites W2150626351 @default.
• W2007251619 cites W2154914042 @default.
• W2007251619 cites W2160427817 @default.
• W2007251619 cites W2163162724 @default.
• W2007251619 cites W2164683872 @default.
• W2007251619 cites W2463767959 @default.
• W2007251619 cites W61768019 @default.
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