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• W2053643150 abstract "Interaction of the rat A3 adenosine receptor (A3AR) with G-proteins has been assessed using a stably transfected Chinese hamster ovary cell system. The non-selective AR agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased the labeling of a 41-kDa membrane protein by 4-azidoanilido-[α-32P]guanosine 5′-triphosphate (AA-[32P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of Giα-subunits indicated that NECA stimulated incorporation of label into both Giα-2 and Giα-3. Additional experiments revealed an A3AR stimulation of label into Gq and/or G11α-subunits, albeit to a lesser degree than that elicited by endogenous P2U purinergic receptors. No interaction with Gs could be detected. Sustained cellular exposure to NECA induced A3AR desensitization and specific down-regulation of Giα-3 and G-protein β-subunits without changing levels of Giα-2, Gsα, or Gq+11α-subunits. Therefore the A3AR can interact with Giα-2, Giα-3, and, to some extent, Gq-like proteins, but sustained agonist exposure down-regulates only one of the G-proteins with which it interacts. This is the first description of the differing specificities of A3AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the A3AR could elicit the heterologous desensitization of signaling events mediated by Gi3. Interaction of the rat A3 adenosine receptor (A3AR) with G-proteins has been assessed using a stably transfected Chinese hamster ovary cell system. The non-selective AR agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased the labeling of a 41-kDa membrane protein by 4-azidoanilido-[α-32P]guanosine 5′-triphosphate (AA-[32P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of Giα-subunits indicated that NECA stimulated incorporation of label into both Giα-2 and Giα-3. Additional experiments revealed an A3AR stimulation of label into Gq and/or G11α-subunits, albeit to a lesser degree than that elicited by endogenous P2U purinergic receptors. No interaction with Gs could be detected. Sustained cellular exposure to NECA induced A3AR desensitization and specific down-regulation of Giα-3 and G-protein β-subunits without changing levels of Giα-2, Gsα, or Gq+11α-subunits. Therefore the A3AR can interact with Giα-2, Giα-3, and, to some extent, Gq-like proteins, but sustained agonist exposure down-regulates only one of the G-proteins with which it interacts. This is the first description of the differing specificities of A3AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the A3AR could elicit the heterologous desensitization of signaling events mediated by Gi3. The multiple physiological effects of adenosine are mediated by activation of cell surface adenosine receptors (ARs) 1The abbreviations used are: ARadenosine receptorG-proteinguanine nucleotide-binding regulatory proteinAA-[32P]GTP4-azidoanilido-[α-32P]guanosine 5′-triphosphate125I-AB-MECA125I-4-aminoben-zyl-5′-N-methylcarboxamidoadenosineNECA5′-N-ethylcarboxamidoadenosinePVDFpolyvinylidene difluorideCHOChinese hamster ovaryPAGEpolyacrylamide gel electrophoresisPTxpertussis toxin. 1The abbreviations used are: ARadenosine receptorG-proteinguanine nucleotide-binding regulatory proteinAA-[32P]GTP4-azidoanilido-[α-32P]guanosine 5′-triphosphate125I-AB-MECA125I-4-aminoben-zyl-5′-N-methylcarboxamidoadenosineNECA5′-N-ethylcarboxamidoadenosinePVDFpolyvinylidene difluorideCHOChinese hamster ovaryPAGEpolyacrylamide gel electrophoresisPTxpertussis toxin.1(1Olsson R.A. Pearson D. Physiol. Rev. 1990; 70: 761-845Crossref PubMed Scopus (718) Google Scholar). Biochemical and molecular cloning studies have facilitated classification of these G-protein-coupled receptors into four subtypes, denoted as A1, A2a, A2b, and A3 (2, 3). The A3AR cDNA clone was initially isolated from a rat brain cDNA library and was classed as a distinct AR subtype due to its distinct agonist potency series compared with A1 and A2AR subtypes and, most intriguingly, its insensitivity to inhibition by alkylxanthine compounds(4Zhou Q.-Y. Li C. Olah M.E. Johnson R.A. Stiles G.L. Civelli O. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7432-7436Crossref PubMed Scopus (639) Google Scholar). The isolation of an A3AR cDNA has implicated this receptor in mediating some physiological effects of adenosine. In particular, it has been demonstrated that the A3AR is the AR responsible for enhancing antigen-stimulated secretion in a rat mast cell line, RBL-2H3 (5, 6). Subsequently isolated AR cDNAs from sheep and human sources, which exhibit a 70% amino acid identity with the rat A3AR, have also been designated as A3ARs, although differences in their pharmacological properties versus the rat protein make it unclear as to whether these proteins are species homologues or constitute a distinct AR subtype(7Linden J. Taylor H.E. Robeva A.S. Tucker A.L. Stehle J.H. Rivkees S.A. Fink J.S. Reppert S.M. Mol. Pharmacol. 1993; 44: 524-532PubMed Google Scholar, 8Salvatore C.A. Jacobson M.A. Taylor H.E. Linden J. Johnson R.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10365-10369Crossref PubMed Scopus (393) Google Scholar). adenosine receptor guanine nucleotide-binding regulatory protein 4-azidoanilido-[α-32P]guanosine 5′-triphosphate 125I-4-aminoben-zyl-5′-N-methylcarboxamidoadenosine 5′-N-ethylcarboxamidoadenosine polyvinylidene difluoride Chinese hamster ovary polyacrylamide gel electrophoresis pertussis toxin. adenosine receptor guanine nucleotide-binding regulatory protein 4-azidoanilido-[α-32P]guanosine 5′-triphosphate 125I-4-aminoben-zyl-5′-N-methylcarboxamidoadenosine 5′-N-ethylcarboxamidoadenosine polyvinylidene difluoride Chinese hamster ovary polyacrylamide gel electrophoresis pertussis toxin. Agonist-induced desensitization, or refractoriness, is a universal feature of G-protein-coupled receptors, including the various AR subtypes(9Ramkumar V. Stiles G.L. Sibley D.R. Houslay M.D. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, New York1994: 217-231Google Scholar). Several studies have indicated that, for many receptors, desensitization can be divided into two temporally and mechanistically distinct phases: short term agonist exposure can induce receptor phosphorylation, which in turn can impair receptor/G-protein interaction(9Ramkumar V. Stiles G.L. Sibley D.R. Houslay M.D. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, New York1994: 217-231Google Scholar, 10Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1077) Google Scholar). Longer agonist treatment times can result in down-regulation of the receptor and/or its associated G-protein, as well as the up-regulation of components controlling opposing signaling pathways(9Ramkumar V. Stiles G.L. Sibley D.R. Houslay M.D. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. John Wiley & Sons, New York1994: 217-231Google Scholar). Rapid, homologous functional desensitization of A3AR-stimulated Ca2+ mobilization has been reported in RBL-2H3 cells(5Ali H. Cunha-Melo J.R. Saul W.F. Beaven M.A. J. Biol. Chem. 1990; 265: 745-753Abstract Full Text PDF PubMed Google Scholar, 6Ramkumar V. Stiles G.L. Beaven M.A. Ali H. J. Biol. Chem. 1993; 268: 16887-16890Abstract Full Text PDF PubMed Google Scholar). However, the effects of chronic agonist exposure on A3AR signaling have not been investigated. In this regard, it has been suggested that chronic agonist exposure can result in the specific down-regulation of the G-protein with which a receptor preferentially couples. This phenomenon has been studied extensively in rat adipocytes both in the intact animal (11Longabaugh J.P. Didsbury J. Spiegel A. Stiles G.L. Mol. Pharmacol. 1989; 36: 681-688PubMed Google Scholar) and in primary adipocyte cultures (12Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar, 13Green A. Milligan G. Dobias S. J. Biol. Chem. 1992; 267: 3223-3229Abstract Full Text PDF PubMed Google Scholar) and suggests that chronic stimulation of the rat A1AR results in the heterologous desensitization of other anti-lipolytic hormone responses due to the down-regulation of Gi proteins(13Green A. Milligan G. Dobias S. J. Biol. Chem. 1992; 267: 3223-3229Abstract Full Text PDF PubMed Google Scholar). These changes are not the result of reduced gene transcription, as mRNA levels for each of the three Giα-subunits expressed in adipocytes are unaffected by agonist treatment(11Longabaugh J.P. Didsbury J. Spiegel A. Stiles G.L. Mol. Pharmacol. 1989; 36: 681-688PubMed Google Scholar). Similar phenomena have since been described for several G-protein-coupled receptors, including those coupled to stimulation of adenylyl cyclase via Gs(14McKenzie F.R. Milligan G. J. Biol. Chem. 1990; 265: 17084-17093Abstract Full Text PDF PubMed Google Scholar) and phospholipase C via Gq and G11(15Mullaney I. Dodd M.W. Buckley N. Milligan G. Biochem. J. 1992; 289: 125-131Crossref Scopus (62) Google Scholar, 16Shah E.H. Milligan G. Mol. Pharmacol. 1994; 46: 1-7PubMed Google Scholar, 17Kim G.-D. Carr I.C. Anderson L.A. Zabavnik J. Eidne K.A. Milligan G. J. Biol. Chem. 1994; 269: 19933-19940Abstract Full Text PDF PubMed Google Scholar). In the few cases examined, it has been proposed that agonist occupation of the appropriate receptor reduces the half-life of the G-protein with which it interacts, thereby resulting in down-regulation(18Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar, 19Hadcock J.R. Ros M. Watkins D.C. Malbon C.C. J. Biol. Chem. 1990; 265: 14784-14790Abstract Full Text PDF PubMed Google Scholar). With these observations in mind, the goals of the present study were: (a) to determine the identity of the G-proteins with which the A3AR interacts, and (b) to determine whether chronic A3AR activation could modulate expression of these proteins. Cell culture supplies were from Life Technologies, Inc. NECA was the generous gift of Dr. Ray Olsson (University of South Florida, Tampa, FL). 125I-AB-MECA was synthesized and purified to homogeneity by reverse phase high performance liquid chromatography as described previously(20Olah M.E. Gallo-Rodriguez C. Jacobson K.A. Stiles G.L. Mol. Pharmacol. 1994; 45: 978-982PubMed Google Scholar). 4-Azidoanilido-[α-32P]GTP (AA-[32P]GTP) was the generous gift of Dr. John Raymond (Duke University and VA Medical Centers, Durham, NC) and was prepared by the method of Offermans et al.(21Offermans S. Schultz G. Rosenthal W. Methods Enzymol. 1991; 195: 286-301Crossref PubMed Scopus (59) Google Scholar). Protein A-Sepharose was from Pharmacia Biotech Inc. PVDF membranes and horseradish peroxidase-conjugated recombinant protein A were from Pierce. UTP was from Sigma. Sources of other materials have been described elsewhere (20, 22). Cell lines stably expressing the pCMV5-rat A3AR and pBC12BI-canine A2aAR constructs have been previously described and characterized(20Olah M.E. Gallo-Rodriguez C. Jacobson K.A. Stiles G.L. Mol. Pharmacol. 1994; 45: 978-982PubMed Google Scholar, 22Palmer T.M. Gettys T.W. Jacobson K.J. Stiles G.L. Mol. Pharmacol. 1994; 45: 1082-1094PubMed Google Scholar). Cells were maintained in Ham's F-12 medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a 37°C humidified atmosphere containing 5% CO2. Cells were grown as monolayers in T-75 flasks and used just prior to reaching confluence. Cells from one T-75 flask were washed, scraped into 5 ml of lysis buffer (5 mM Hepes, 2 mM EDTA, pH 7.5, containing 10 μg/ml soybean trypsin inhibitor, 10 μg/ml leupeptin, 5 μg/ml pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride) and disrupted by Dounce homogenization on ice (20 strokes). After centrifugation at 48,000 × g for 10 min, the crude membrane pellet was resuspended in lysis buffer to a concentration of approximately 1 mg of protein/ml and aliquoted for storage at −80°C. The generation and specificities of all but one of the anti-peptide antisera used in this study have been demonstrated previously(23Raymond J.R. Olsen C.L. Gettys T.W. Biochemistry. 1993; 32: 11064-11073Crossref PubMed Scopus (136) Google Scholar, 24Gettys T.W. Fields T.A. Raymond J.R. Biochemistry. 1994; 33 (1994): 4283-4290Crossref PubMed Scopus (99) Google Scholar, 25Gettys T.W. Sheriff-Carter K. Moomaw J. Taylor I.L. Raymond J.R. Anal. Biochem. 1994; 220: 82-91Crossref PubMed Scopus (44) Google Scholar). Antiserum 457 was generated against a decapeptide whose sequence is identical to that of the COOH termini of Gqα and G11α(26Strathmann M. Wilkie T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7407-7409Crossref PubMed Scopus (120) Google Scholar). Reactivity with Gqα and G11α and a lack of cross-reactivity with inhibitory G-protein α-subunits have been assessed using recombinant proteins in immunoblotting studies (data not shown). Additionally, although antiserum 982 was raised against the carboxyl-terminal decapeptide sequence common to rod and cone transducins, as well as Giα-1 and Giα-2, the restricted expression of the transducins and the lack of expression of Giα-1 in CHO cells (23Raymond J.R. Olsen C.L. Gettys T.W. Biochemistry. 1993; 32: 11064-11073Crossref PubMed Scopus (136) Google Scholar, 25Gettys T.W. Sheriff-Carter K. Moomaw J. Taylor I.L. Raymond J.R. Anal. Biochem. 1994; 220: 82-91Crossref PubMed Scopus (44) Google Scholar) means that this antiserum can be used as a specific tool for identification of Giα-2 in this system. The following primary antibodies were used for immunoblotting at the concentrations indicated in parentheses: 982 (1 in 4000 dilution of serum) for detection of Giα-2, 977 (1 in 4000 dilution of serum) for detection of Giα-3, 951 (1 in 8000 dilution of serum) for detection of Gsα, 457 (1 in 1000 dilution of protein A affinity-purified IgG) for detection of Gqα and G11α, and 987 (1 in 4000 dilution of serum) for detection of G-protein β-subunits. After solubilization in electrophoresis sample buffer, equivalent amounts of membrane protein (typically 75 μg/sample) were resolved by SDS-PAGE using 10% (w/v) polyacrylamide resolving gels. Resolved proteins were transferred to PVDF membranes and nonspecific protein binding sites blocked by a 60-min incubation at room temperature in blocking buffer (5% (w/v) skim milk in phosphate-buffered saline containing 0.2% (v/v) Triton X-100 and 0.02% (w/v) thimerosal). Membranes were then incubated with the appropriate dilution of primary antiserum in fresh blocking buffer for either 2 h at room temperature or overnight at 4°C. After removal of antiserum and extensive washing with three changes of blocking buffer, the membrane was incubated for 60 min at room temperature with a 1 in 5000 dilution of horseradish peroxidase-conjugated protein A in a high detergent skim milk solution. The series of washes described above was then repeated and followed by two further washes in phosphate-buffered saline alone. Reactive proteins were visualized by an enhanced chemiluminescence protocol in accordance with the manufacturer's instructions (Renaissance, DuPont NEN). Quantitation of immunoblots was by densitometric scanning of autoradiographs using a Bio-Rad model 620 densitometer with analysis by the 1-D Analyst software package. Preliminary experiments demonstrated that the amounts of membrane protein and primary antibody dilutions employed produced signals within the linear response range of our detection methods (data not shown). These were performed as described previously (24Gettys T.W. Fields T.A. Raymond J.R. Biochemistry. 1994; 33 (1994): 4283-4290Crossref PubMed Scopus (99) Google Scholar) except that freshly isolated crude cell membranes (prepared as described above) were used and adenosine deaminase was added to a concentration of 1 unit/ml. Quantitation was by densitometric scanning of autoradiographs. Immunoprecipitations using the indicated anti-G-protein subunit antisera described above were performed essentially as described previously(23Raymond J.R. Olsen C.L. Gettys T.W. Biochemistry. 1993; 32: 11064-11073Crossref PubMed Scopus (136) Google Scholar, 24Gettys T.W. Fields T.A. Raymond J.R. Biochemistry. 1994; 33 (1994): 4283-4290Crossref PubMed Scopus (99) Google Scholar). Binding studies using the high affinity A3AR radioligand 125I-AB-MECA and adenylyl cyclase assays were performed and analyzed as described previously(20Olah M.E. Gallo-Rodriguez C. Jacobson K.A. Stiles G.L. Mol. Pharmacol. 1994; 45: 978-982PubMed Google Scholar). To examine A3AR interaction with and regulation of G-proteins, a transfected CHO cell system was chosen, thereby allowing the use of non-transfected CHO cells as an appropriate negative control. The level of expression of the recombinant A3AR in this system was determined using the high affinity A3AR agonist radioligand 125I-AB-MECA in saturation binding experiments (Fig. 1A). This ligand bound to a single saturable high affinity site in membranes from transfected cells, with Kd and Bmax values of 1.4 ± 0.4 nM and 3.7 ± 0.4 pmol/mg membrane protein, respectively (three experiments). Moreover, the expressed A3AR was functional as the non-selective AR agonist NECA could elicit a dose-dependent inhibition of forskolin-stimulated adenylyl cyclase activity in membranes from transfected cells (Fig. 1B). Neither specific binding of 125I-AB-MECA nor inhibition of forskolin-stimulated adenylyl cyclase activity was observed in non-transfected CHO cells (data not shown). It has been documented that the abilities of recombinant A3ARs in transfected CHO cells to inhibit cAMP accumulation and endogenous A3ARs in RBL-2H3 cells to stimulate phospholipase C are abolished by pretreatment with pertussis toxin(4Zhou Q.-Y. Li C. Olah M.E. Johnson R.A. Stiles G.L. Civelli O. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7432-7436Crossref PubMed Scopus (639) Google Scholar, 5Ali H. Cunha-Melo J.R. Saul W.F. Beaven M.A. J. Biol. Chem. 1990; 265: 745-753Abstract Full Text PDF PubMed Google Scholar). To determine the nature of the A3AR/Gi-protein interaction, a photolabile GTP analogue (AA-[32P]GTP) was used to assess A3AR-stimulated G-protein activation(22Palmer T.M. Gettys T.W. Jacobson K.J. Stiles G.L. Mol. Pharmacol. 1994; 45: 1082-1094PubMed Google Scholar, 23Raymond J.R. Olsen C.L. Gettys T.W. Biochemistry. 1993; 32: 11064-11073Crossref PubMed Scopus (136) Google Scholar). Using this approach, it was determined that NECA could stimulate an increase in the labeling of a 41-kDa protein in membranes from A3AR cDNA-transfected but not in non-transfected CHO cells; this effect was dose-dependent, with half-maximal effects occurring at a NECA concentration of 0.2 μM (Fig. 2A). The -fold increase in labeling over basal at a saturating dose of NECA was between 3- and 6-fold (four experiments). The identity of the Gi protein(s) activated by the A3AR was deduced by immunoprecipitating each of Giα-2 and Giα-3 from crude membranes after AA-[32P]GTP labeling using subtype-specific antibodies (Fig. 2B). This demonstrated that NECA increased incorporation of label into each of the precipitated proteins, thereby demonstrating that the A3AR is capable of interacting with both Giα-2 and Giα-3 in this system. It has been recently demonstrated that receptors thought previously to couple exclusively to Gi proteins can also interact with other signaling systems. One example of this is the α2A adrenergic receptor, which, as well as coupling to multiple Gi proteins, can also activate Gs and Gq(27Kurose H. Regan J.W. Caron M.G. Lefkowitz R.J. Biochemistry. 1991; 30: 3335-3341Crossref PubMed Scopus (82) Google Scholar, 28Eason M.G. Jacinto M.T. Liggett S.B. Mol. Pharmacol. 1994; 45: 696-702PubMed Google Scholar, 29Conklin B.R. Chabre O. Wong Y.H. Federman A.D. Bourne H.R. J. Biol. Chem. 1992; 267: 31-34Abstract Full Text PDF PubMed Google Scholar). Recent experiments performed on recombinant G-protein α-subunits have demonstrated that Gsα-subunits have a much lower affinity for AA-[32P]GTP than Gi proteins (30). Therefore, despite the fact that no agonist-stimulated photoincorporation into a 48-kDa band was observed (the size of Gsα-subunits in CHO cells as determined by immunoblotting; Ref. 22), we could not immediately eliminate the possibility that the A3AR was capable of coupling to Gs. Therefore we assayed cell membranes for effects of A3AR activation on GTP-stimulated, i.e. “basal,” adenylyl cyclase activity. In non-transfected CHO cells, 50 μM NECA produced a 6 ± 4% activation of adenylyl cyclase activity above that of GTP alone. In contrast, the addition of 50 μM NECA to membranes from A3AR-expressing CHO cells inhibited basal activity by 57 ± 7% (three experiments). Under the same conditions, the addition of 50 μM NECA to membranes from CHO cells expressing 0.26-0.30 pmol/mg A2aAR, a prototypical Gs-coupled receptor(22Palmer T.M. Gettys T.W. Jacobson K.J. Stiles G.L. Mol. Pharmacol. 1994; 45: 1082-1094PubMed Google Scholar), elicited a 19.7 ± 0.5-fold increase over GTP-stimulated activity (10 experiments). To determine whether A3AR activation of Gi was masking an interaction between the A3AR and Gs, assays were also performed after treatment of CHO cells with 20 ng/ml PTx for 24 h, an incubation sufficient to ADP-ribosylate the total cellular pool of Gi as determined by subsequent [32P]ADP-ribosylation experiments on isolated membranes (22). Under these conditions, we observed a 79 ± 12% loss of the ability of 50 μM NECA to inhibit forskolin-stimulated adenylyl cyclase activity (three experiments) consistent with the inactivation of almost all functional Gi. However, this treatment did not unmask an activation of adenylyl cyclase activity: in membranes from PTx-treated cells, the addition of 50 μM NECA to membranes merely attenuated inhibition of GTP-stimulated activity from 57 ± 7% to 17 ± 12% (three experiments). To assess potential A3AR interaction with Gq and G11, membranes were photolabeled with AA-[32P]GTP followed by immunoprecipitation with antibody 457 (anti-Gq+11). Preliminary immunoblotting experiments with 457 demonstrated that under these conditions 457 specifically immunoprecipitates a 42-kDa band that co-migrates with Gq+11α-subunits on SDS-PAGE, and that these immunoprecipitates are devoid of detectable Giα-2 and Giα-3 proteins, as determined by immunoblotting of the immunoprecipitates with antisera 982 and 977, respectively (data not shown). As a positive control for the photolabeling experiments, we made use of the endogenous P2U purinergic receptor expressed by CHO-K1 cells. Agonist occupation of this receptor raises intracellular Ca2+ concentrations in a manner that is resistant to modulation by PTx treatment, indicating that the response is mediated by G-proteins belonging to the Gq family(31Iredale P.A. Hill S.J. Br. J. Pharmacol. 1993; 110: 1305-1310Crossref PubMed Scopus (106) Google Scholar). Analysis of total membranes after photolabeling indicated that under conditions where the addition of 10 μM NECA produced a 2-3-fold increase in the incorporation of label into the 41-kDa band (which presumably consists of Giα-2 and Giα-3; Fig. 2), no such increase was noted after the addition of 100 μM UTP, a P2U purinergic receptor agonist (Fig. 3A). However, after membrane solubilization and immunoprecipitation with antiserum 457, a single 42-kDa labeled band is observed (Fig. 3B), which presumably consists of a mixture of Gqα and G11α, both of which are expressed in CHO cells(32Mullaney I.M. Mitchell F.M. Milligan G. FEBS Lett. 1993; 324: 241-245Crossref PubMed Scopus (44) Google Scholar). The agonist-modulated labeling of the immunoprecipitated 42-kDa band is quite distinct from that observed for the 41-kDa band in total membranes (Fig. 3, A and B). The addition of UTP increases the labeling of the 42-kDa band by some 2-2.5-fold, whereas 10 μM NECA increases labeling by 1.3-1.5-fold (ranges of values from two experiments). Therefore agonist-occupied A3ARs are capable of increasing the labeling of Gq-like proteins in membranes from transfected CHO cells, albeit to a lesser degree than that elicited by endogenous activated P2U purinergic receptors. To determine whether the activated A3AR could regulate the expression of the G-proteins with which it interacts, cells were treated with 10 μM NECA for up to 24 h and membranes prepared for comparative immunoblotting experiments. These demonstrated that while the expression of Giα-2 and Gq+11α-subunits are unaffected by prolonged agonist occupancy of A3ARs, Giα-3 undergoes a profound down-regulation (Fig. 4, A and B;). This effect was absolutely dependent on the expression of the A3AR since parallel treatment of non-transfected CHO cells failed to alter levels of Giα-3 (Fig. 4C). Additionally this figure also demonstrates that constitutive expression of the A3AR did not significantly alter the levels of expression of Giα-3 in transfected versus non-transfected CHO cells (Fig. 4C). Expression levels of Gsα were not greatly affected by agonist treatment (Table I). However, levels of the β-subunits common to all G-proteins decreased by approximately 60% (Fig. 5 and); as for Giα-3, this effect could not be observed in non-transfected CHO cells (data not shown). Under these conditions, the A3AR underwent a functional desensitization, as manifested by an increase in the IC50 value for NECA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity (Fig. 6).Table I:Agonist regulation of G-protein expression in CHO cells expressing the rat A3AR Open table in a new tab Figure 5:Agonist-mediated down-regulation of G-protein β-subunits in A3AR-transfected CHO cells. 75 μg of crude membrane protein from cells treated in the absence (Control) or presence (Treated) of 10 μM NECA for 24 h were resolved by SDS-PAGE and transferred to PVDF for immunoblotting with antisera 987 (anti-β-subunits) as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6:Agonist-mediated desensitization of A3AR function. After treatment with or without 10 μM NECA for 24 h, membranes were prepared for assay of adenylyl cyclase activity as described inFig. 1. In this experiment, the IC50 value for NECA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity increased from 2.0 ± 0.4 μM (Control) to 73 ± 20 μM (Treated). This is one of multiple experiments, which produced quantitatively similar data.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects on G-protein subunit expression of treating transfected cells with increasing concentrations of NECA are shown in Fig. 7. The EC50 values for loss of Giα-3 and β-subunits are similar (∼60 nM and 80 nM, respectively), suggesting an equivalent dependence of each process on agonist occupation of A3ARs. However, treatment of transfected CHO cells with 10 μM NECA for various times demonstrates that their respective time courses of down-regulation are distinct (Fig. 8). While Giα-3 undergoes a steady down-regulation observable at 4 h and maximal by 16 h exposure (Fig. 8, A and C), β-subunit down-regulation is biphasic; a rapid initial reduction in expression, observable at 2 h, stabilizes until after 8 h, when the subsequent rate of β-subunit down-regulation more closely parallels that of Giα-3 (Fig. 8, B and C).Figure 8:Time courses of G-protein subunit down-regulation. 75 μg of membrane protein from A3AR-expressing CHO cells treated with 10 μM NECA for the indicated times were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting with anti-Giα-3 antiserum 977 (panel A) or anti-β-subunit antiserum 987 (panel B). Panel C is a quantitative analysis of three separate time course experiments performed for each of these proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The adaptive responses of cells to sustained hormone exposure operate at several different levels. While rapid covalent modifications of receptors by various kinases are most likely responsible for initiating short term desensitization, prolonged agonist treatment invokes distinct processes. A common phenomenon exhibited by many G-protein-coupled receptors is that of down-regulation, defined as a loss in the total number of receptors expressed by the cell. Such receptor-specific events are presumably responsible for the homologous desensitization exhibited by many receptors, but cannot explain heterologous desensitization phenomena, whereby exposure to a given agonist can desensitize responses elicited by other hormone receptors. One potential mechanism of heterologous desensitization is that of regulation of G-protein function and expression, since changes at this level via one receptor would be expected to affect the signaling capacity of any other receptor that utilizes the particular G-protein to activate its appropriate effector. Evidence in support of this hypothesis has accumulated over recent years, due to the availability of antisera capable of discriminating among the multiple G-protein subunits expressed in many cell types(11Longabaugh J.P. Didsbury J. Spiegel A. Stiles G.L. Mol. Pharmacol. 1989; 36: 681-688PubMed Google Scholar, 12Green A. Johnson J.L. Milligan G. J. Biol. Chem. 1990; 265: 5206-5210Abstract Full Text PDF PubMed Google Scholar, 13Green A. Milligan G. Dobias S. J. Biol. Chem. 1992; 267: 3223-3229Abstract Full Text PDF PubMed Google Scholar, 33Milligan G. Trends Pharmacol. Sci. 1993; 14: 413-418Abstract Full Text PDF PubMed Scopus (110) Google Scholar). In particular, many studies have demonstrated that chronic cellular exposure to specific agonists results in a rapid down-regulation of the G-protein which that receptor might be expected to activate upon agonist occupancy; these effects are generally independent of second messenger generation despite the requirement for receptor activation (33). Additionally, receptor-independent, constitutive activation of Gs by cholera toxin-catalyzed ADP-ribosylation induces a rapid loss of total cellular levels of Gs that is independent of the initial increase in cAMP levels(34Milligan G. Unson C.G. Wakelam M.J.O. Biochem. J. 1989; 262: 643-649Crossref PubMed Scopus (52) Google Scholar, 35Chang F.-H. Bourne H.R. J. Biol. Chem. 1989; 264: 5352-5357Abstract Full Text PDF PubMed Google Scholar). In S49 cyc− lymphoma cells transfected with an epitope-tagged Gsα construct, the cholera toxin-stimulated down-regulation is due to an enhanced degradation of the protein, as manifested by a reduced biological half-life, and is associated with a shift in the subcellular distribution of Gsα from the membrane to the cytosol(36Levis M.J. Bourne H.R. J. Cell Biol. 1992; 119: 1297-1307Crossref PubMed Scopus (179) Google Scholar). With these observations in mind, the present study was undertaken. The A3AR has been shown to increase phosphatidylinositol-specific phospholipase C activity in RBL-2H3 cells (5, 6) and inhibit adenylyl cyclase activity in transfected CHO cells (4). Both of these effects are abolished by pretreatment with pertussis toxin, suggesting a role for Gi proteins. In the transfected CHO cell system we have shown, using sequential G-protein photolabeling and immunoprecipitation with specific antibodies, that the A3AR is capable of interacting with, and presumably activating, both Giα-2 and Giα-3 in isolated membranes. Interestingly, these are the same two pertussis toxin substrates that are expressed in RBL-2H3 cells (37Hide M. Ali H. Price S.R. Moss J. Beaven M.A. Mol. Pharmacol. 1991; 40: 473-479PubMed Google Scholar) and suggest that the endogenously expressed A3AR in these cells is at least capable of interacting with these proteins to produce its downstream effects. Presumably, the pertussis toxin-sensitive A3AR-stimulated increase in inositol 1,4,5-trisphosphate production in RBL-2H3 cells is due to increased levels of dissociated Gi-derived βγ-subunits activating phosphatidylinositol-specific phospholipase C-β isoforms, an interaction that has been demonstrated both in intact cells (38Hawes B.E. Luttrell L.M. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 15776-15785Abstract Full Text PDF PubMed Google Scholar) and with purified components(39Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). However, we were also able to reveal an interaction of the A3AR with Gq-like proteins expressed in CHO cells, and this would suggest that, at least in some instances, A3AR stimulation of phospholipase C may have a PTx-insensitive component. The generality and significance of A3AR/Gq/11 interaction in systems expressing A3ARs endogenously remain to be determined. No stimulatory effect of A3AR activation on adenylyl cyclase activity could be determined, suggesting that the A3AR does not interact significantly with Gs. Chronic agonist exposure of A3AR-expressing CHO cells induces the specific down-regulation of Giα-3 and not Giα-2 or Gq+11α-subunits, despite the ability of the receptor to activate each of these proteins. This effect required agonist occupation of the A3AR, as no down-regulation was observed in native CHO cells, which express undetectable levels of functional A3ARs. The loss of Giα-3 was associated with a similarly profound reduction in the levels of β-subunits common to all G-proteins. The reductions in expression of these proteins exhibited similar dose dependence values to increasing concentrations of NECA. Additionally, the EC50 value for NECA stimulation of AA-[32P]GTP incorporation into Gi proteins was also similar to the EC50 values for G-protein subunit down-regulation, suggesting a possible link between G-protein activation and down-regulation. Under these conditions, A3AR-mediated inhibition of adenylyl cyclase underwent a functional desensitization. We cannot determine at the present time the contribution of G-protein down-regulation to the overall process of A3AR desensitization; as we have previously demonstrated, receptor desensitization is a complex phenomenon involving several distinct processes whose importance to the overall effect varies upon length of agonist exposure. In particular, receptor down-regulation may play a role, but we cannot test this due to the unavailability of a radiolabeled antagonist ligand for the A3AR. Nevertheless, such a profound down-regulation of Giα-3 would be expected to impair downstream signaling events elicited by any receptor with which it could interact. The profound loss of β-subunits was a surprising finding from these studies and is perplexing considering the quantity of Giα-3 expressed in these cells. Quantitative immunoblotting studies in CHO-K1 cells using E. coli-derived recombinant proteins as standards have shown that Giα-2 is present at an 8-fold molar excess over Giα-3 (4.8 versus 0.6 pmol/mg membrane protein)(23Raymond J.R. Olsen C.L. Gettys T.W. Biochemistry. 1993; 32: 11064-11073Crossref PubMed Scopus (136) Google Scholar, 25Gettys T.W. Sheriff-Carter K. Moomaw J. Taylor I.L. Raymond J.R. Anal. Biochem. 1994; 220: 82-91Crossref PubMed Scopus (44) Google Scholar). Transfection of CHO cells with the A3AR cDNA does not alter the steady-state levels of these proteins compared with non-transfected cells (Fig. 4C and data not shown). Therefore the 70% reduction in levels of Giα-3 represents a loss of approximately 0.4 pmol of Giα-3/mg of membrane protein. Assuming that 1 mol of β-subunits is down-regulated with 1 mol of G-protein α-subunit, the loss of 0.4 pmol/mg β-subunits would be undetectable since it represents a small proportion of the total pool of β-subunits. Therefore, additional β-subunits, not associated with the loss of Giα-3, must be down-regulated in order to account for the 60% loss measured by immunoblotting (Table I). This indicates that additional processes distinct from those involved in Giα-3 down-regulation are involved in regulating β-subunit expression. Such a scenario is also suggested by the distinct time courses displayed for down-regulation of each of these proteins (Fig. 8). The selective loss of Giα-3 over Giα-2 and Gq+11α-subunits despite their ability to each couple with activated A3ARs may be due to any of several reasons. While we are not able to quantitate the amount of Gq+11α-subunits expressed in CHO cell membranes, it is possible that large molar excesses of Giα-2 or Gq+11α-subunits over Giα-3 mask an equivalent down-regulation such that it is not detectable: for example, a molar reduction in levels of Giα-2 equivalent to that of Giα-3 (0.4 pmol/mg membrane protein) would represent less than 10% of the total pool of Giα-2 and would probably not be detected by immunoblotting. Also, the importance of receptor/G-protein stoichiometry in determining extent of down-regulation has not been thoroughly examined, although a recent study on cell lines expressing different levels of β2-adrenergic receptor has suggested that increasing the receptor:G-protein ratio enhances observation of agonist-mediated down-regulation of Gsα-subunits(40Adie E.J. Milligan G. Biochem. J. 1994; 300: 709-715Crossref PubMed Scopus (34) Google Scholar). Therefore, if a receptor couples to more than one G-protein with equal efficiency, it is most likely that the least abundant G-protein would be more susceptible to down-regulation. In conclusion, we have assessed the ability of the A3AR to activate and regulate specific Gi proteins in a transfected CHO cell system. The A3AR is capable of activating both Giα-2 and Giα-3, the two pertussis toxin substrates expressed in CHO cells, as well as Gq+11α-subunits. Moreover, sustained exposure of A3AR-expressing cells to agonist results in the selective down-regulation of Giα-3 and G-protein β-subunits but not of Giα-2, Gsα, or Gq+11α-subunits. However, the extent to which both of these proteins are down-regulated and the differing time courses exhibited suggest that distinct processes may be involved in regulating the levels of these proteins in response to receptor activation. Whatever these mechanisms are, we have clearly shown that while the A3AR is capable of interacting with multiple G-proteins in a given cell type, agonist treatment specifically induces the down-regulation of Giα-3, thereby providing a potential mechanism for heterologous desensitization of hormone signaling events mediated by this G-protein. A3AR-transfected CHO cells were treated with or without 10 μM NECA for 24 hours at 37°C prior to membrane preparation and comparative immunoblotting using the antisera described under “Experimental Procedures.” Under these conditions, Gqα and G11α co-migrate and therefore the signal observed on immunoblots represents a composite signal for these proteins. Data are presented as means ± standard error from the number of experiments in parentheses, with the signal obtained for untreated control membranes set at 100%. We thank Dr. Mark Olah for donating the A3AR-transfected cell line used in this study, Dr. John Raymond for generously supplying AA-[32P]GTP, and Linda Scherich for preparation of the manuscript." @default.
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• W2053643150 title "Differential Interaction with and Regulation of Multiple G-proteins by the Rat A3 Adenosine Receptor" @default.
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