©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Interaction with and Regulation of Multiple G-proteins by the Rat A Adenosine Receptor (*)

Timothy M. Palmer (1), Thomas W. Gettys (2)(§), Gary L. Stiles (1)(¶)

From the (1)Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 and the (2)Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interaction of the rat A adenosine receptor (AAR) 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-[-P]guanosine 5`-triphosphate (AA-[P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of G -subunits indicated that NECA stimulated incorporation of label into both G-2 and G-3. Additional experiments revealed an AAR stimulation of label into G and/or G -subunits, albeit to a lesser degree than that elicited by endogenous P purinergic receptors. No interaction with G could be detected. Sustained cellular exposure to NECA induced AAR desensitization and specific down-regulation of G-3 and G-protein -subunits without changing levels of G-2, G, or G -subunits. Therefore the AAR can interact with G-2, G-3, and, to some extent, G-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 AAR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the AAR could elicit the heterologous desensitization of signaling events mediated by G3.


INTRODUCTION

The multiple physiological effects of adenosine are mediated by activation of cell surface adenosine receptors (ARs)()(1) . Biochemical and molecular cloning studies have facilitated classification of these G-protein-coupled receptors into four subtypes, denoted as A, A, A, and A (2, 3). The AAR 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 A and AAR subtypes and, most intriguingly, its insensitivity to inhibition by alkylxanthine compounds(4) . The isolation of an AAR cDNA has implicated this receptor in mediating some physiological effects of adenosine. In particular, it has been demonstrated that the AAR 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 AAR, have also been designated as AARs, 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(7, 8) .

Agonist-induced desensitization, or refractoriness, is a universal feature of G-protein-coupled receptors, including the various AR subtypes(9) . 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(9, 10) . 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(9) .

Rapid, homologous functional desensitization of AAR-stimulated Ca mobilization has been reported in RBL-2H3 cells(5, 6) . However, the effects of chronic agonist exposure on AAR 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 (11) and in primary adipocyte cultures (12, 13) and suggests that chronic stimulation of the rat AAR results in the heterologous desensitization of other anti-lipolytic hormone responses due to the down-regulation of G proteins(13) . These changes are not the result of reduced gene transcription, as mRNA levels for each of the three G-subunits expressed in adipocytes are unaffected by agonist treatment(11) . Similar phenomena have since been described for several G-protein-coupled receptors, including those coupled to stimulation of adenylyl cyclase via G(14) and phospholipase C via G and G(15, 16, 17) . 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(18, 19) .

With these observations in mind, the goals of the present study were: (a) to determine the identity of the G-proteins with which the AAR interacts, and (b) to determine whether chronic AAR activation could modulate expression of these proteins.


EXPERIMENTAL PROCEDURES

Materials

Cell culture supplies were from Life Technologies, Inc. NECA was the generous gift of Dr. Ray Olsson (University of South Florida, Tampa, FL). I-AB-MECA was synthesized and purified to homogeneity by reverse phase high performance liquid chromatography as described previously(20) . 4-Azidoanilido-[-P]GTP (AA-[P]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.(21) . 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 Culture and Transfections

Cell lines stably expressing the pCMV5-rat AAR and pBC12BI-canine AAR constructs have been previously described and characterized(20, 22) . 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% CO. Cells were grown as monolayers in T-75 flasks and used just prior to reaching confluence.

Membrane Preparation

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.

Antibodies and Immunoblotting

The generation and specificities of all but one of the anti-peptide antisera used in this study have been demonstrated previously(23, 24, 25) . Antiserum 457 was generated against a decapeptide whose sequence is identical to that of the COOH termini of G and G(26) . Reactivity with G and G and a lack of cross-reactivity with inhibitory G-protein -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 G-1 and G-2, the restricted expression of the transducins and the lack of expression of G-1 in CHO cells (23, 25) means that this antiserum can be used as a specific tool for identification of G-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 G-2, 977 (1 in 4000 dilution of serum) for detection of G-3, 951 (1 in 8000 dilution of serum) for detection of G, 457 (1 in 1000 dilution of protein A affinity-purified IgG) for detection of G and G, and 987 (1 in 4000 dilution of serum) for detection of G-protein -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).

G-protein Photolabeling

These were performed as described previously (24) 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(23, 24) .

Radioligand Binding and Adenylyl Cyclase Assays

Binding studies using the high affinity AAR radioligand I-AB-MECA and adenylyl cyclase assays were performed and analyzed as described previously(20) .


RESULTS

Expression of the AAR in CHO Cells

To examine AAR 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 AAR in this system was determined using the high affinity AAR agonist radioligand I-AB-MECA in saturation binding experiments (Fig. 1A). This ligand bound to a single saturable high affinity site in membranes from transfected cells, with K and B values of 1.4 ± 0.4 nM and 3.7 ± 0.4 pmol/mg membrane protein, respectively (three experiments). Moreover, the expressed AAR 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 I-AB-MECA nor inhibition of forskolin-stimulated adenylyl cyclase activity was observed in non-transfected CHO cells (data not shown).


Figure 1: Stable expression of the rat AAR in CHO cells. A, saturation isotherm for I-AB-MECA binding to membranes from AAR-transfected CHO cells. Nonspecific binding was defined by the inclusion of 10 µM NECA in the assay. This is one of three experiments, composite data from which are given under ``Results.'' B, NECA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in membranes from AAR-transfected CHO cells. At a concentration of 5 µM, forskolin elevated adenylyl cyclase activity by some 10-12-fold above basal. Each point is the mean of three separate experiments. Curve-fitting analysis gave an IC for NECA of 4.7 ± 1.4 µM and a maximal inhibition of 42 ± 2% of the forskolin-stimulated activity.



AAR-stimulated G-protein Labeling

It has been documented that the abilities of recombinant AARs in transfected CHO cells to inhibit cAMP accumulation and endogenous AARs in RBL-2H3 cells to stimulate phospholipase C are abolished by pretreatment with pertussis toxin(4, 5) . To determine the nature of the AAR/G-protein interaction, a photolabile GTP analogue (AA-[P]GTP) was used to assess AAR-stimulated G-protein activation(22, 23) . Using this approach, it was determined that NECA could stimulate an increase in the labeling of a 41-kDa protein in membranes from AAR 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 G protein(s) activated by the AAR was deduced by immunoprecipitating each of G-2 and G-3 from crude membranes after AA-[P]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 AAR is capable of interacting with both G-2 and G-3 in this system.


Figure 2: AAR-stimulated photoincorporation of AA-[P]GTP into G-proteins. A, dose-dependent incorporation of AA-[P]GTP into a 41-kDa membrane protein by NECA in membranes from AAR-transfected CHO cells. Membranes were preincubated for 10 min at 30 °C with differing concentrations of NECA prior to the addition of AA-[P]GTP and incubation for another 10 min. After UV irradiation, 10% of each sample was resolved by SDS-PAGE and labeled proteins visualized by autoradiography. Quantitation was by densitometric scanning. B, after incubation with or without 10 µM NECA, membranes were photolabeled as described above. The final membrane pellets were then solubilized in detergent buffer, divided into two aliquots, and each aliquot immunoprecipitated with antibodies specific for either G-2 or G-3 and protein A-Sepharose. Immunoprecipitates were then analyzed by SDS-PAGE and autoradiography.



Assessment of AAR Interaction with G

It has been recently demonstrated that receptors thought previously to couple exclusively to G proteins can also interact with other signaling systems. One example of this is the adrenergic receptor, which, as well as coupling to multiple G proteins, can also activate G and G(27, 28, 29) .

Recent experiments performed on recombinant G-protein -subunits have demonstrated that G -subunits have a much lower affinity for AA-[P]GTP than G proteins (30). Therefore, despite the fact that no agonist-stimulated photoincorporation into a 48-kDa band was observed (the size of G -subunits in CHO cells as determined by immunoblotting; Ref. 22), we could not immediately eliminate the possibility that the AAR was capable of coupling to G. Therefore we assayed cell membranes for effects of AAR 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 AAR-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 AAR, a prototypical G-coupled receptor(22) , elicited a 19.7 ± 0.5-fold increase over GTP-stimulated activity (10 experiments). To determine whether AAR activation of G was masking an interaction between the AAR and G, 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 G as determined by subsequent [P]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 G. 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).

AAR Interaction with G-like Proteins

To assess potential AAR interaction with G and G, membranes were photolabeled with AA-[P]GTP followed by immunoprecipitation with antibody 457 (anti-G). Preliminary immunoblotting experiments with 457 demonstrated that under these conditions 457 specifically immunoprecipitates a 42-kDa band that co-migrates with G -subunits on SDS-PAGE, and that these immunoprecipitates are devoid of detectable G-2 and G-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 P purinergic receptor expressed by CHO-K1 cells. Agonist occupation of this receptor raises intracellular Ca concentrations in a manner that is resistant to modulation by PTx treatment, indicating that the response is mediated by G-proteins belonging to the G family(31) .

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 G-2 and G-3; Fig. 2), no such increase was noted after the addition of 100 µM UTP, a P 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 G and G, both of which are expressed in CHO cells(32) . 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 AARs are capable of increasing the labeling of G-like proteins in membranes from transfected CHO cells, albeit to a lesser degree than that elicited by endogenous activated P purinergic receptors.


Figure 3: AAR and P purinergic receptor-stimulated incorporation of AA-[P]GTP into G -subunits. A, membranes were preincubated for 10 min at 30 °C with the indicated agonists prior to the addition of AA-[P]GTP and incubation for another 10 min. After UV irradiation, 10% of each sample was resolved by SDS-PAGE and labeled proteins visualized by autoradiography. B, membranes were photolabeled as described above prior to solubilization and immunoprecipitation with antiserum 457 (anti-G -subunits) and protein A-Sepharose. Immunoprecipitates were then analyzed by SDS-PAGE and autoradiography.



AAR Regulation of G-protein Expression

To determine whether the activated AAR 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 G-2 and G -subunits are unaffected by prolonged agonist occupancy of AARs, G-3 undergoes a profound down-regulation (Fig. 4, A and B; ). This effect was absolutely dependent on the expression of the AAR since parallel treatment of non-transfected CHO cells failed to alter levels of G-3 (Fig. 4C). Additionally this figure also demonstrates that constitutive expression of the AAR did not significantly alter the levels of expression of G-3 in transfected versus non-transfected CHO cells (Fig. 4C). Expression levels of G were not greatly affected by agonist treatment (). However, levels of the -subunits common to all G-proteins decreased by approximately 60% ( Fig. 5and ); as for G-3, this effect could not be observed in non-transfected CHO cells (data not shown). Under these conditions, the AAR underwent a functional desensitization, as manifested by an increase in the IC value for NECA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity (Fig. 6).


Figure 4: Selective agonist-mediated down-regulation of G-3 in AAR-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 membranes for immunoblotting with antisera 982, specific for G-2 (panelA), or 977, specific for G-3 (panelB). C, non-transfected and AAR-transfected CHO cells were treated with or without 10 µM NECA for 24 h prior to membrane preparation and immunoblotting with antiserum 977 (anti-G-3).




Figure 5: Agonist-mediated down-regulation of G-protein -subunits in AAR-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--subunits) as described under ``Experimental Procedures.''




Figure 6: Agonist-mediated desensitization of AAR function. After treatment with or without 10 µM NECA for 24 h, membranes were prepared for assay of adenylyl cyclase activity as described in Fig. 1. In this experiment, the IC 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.



The effects on G-protein subunit expression of treating transfected cells with increasing concentrations of NECA are shown in Fig. 7. The EC values for loss of G-3 and -subunits are similar (60 nM and 80 nM, respectively), suggesting an equivalent dependence of each process on agonist occupation of AARs. 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 G-3 undergoes a steady down-regulation observable at 4 h and maximal by 16 h exposure (Fig. 8, A and C), -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 -subunit down-regulation more closely parallels that of G-3 (Fig. 8, B and C).


Figure 7: Dose dependence values of G-protein subunit down-regulation to increasing concentrations of NECA. 75 µg of membrane protein from AAR-expressing CHO cells treated with the indicated concentrations of NECA for 24 h were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting with anti-G-3 antiserum 977 (panelA) or anti--subunit antiserum 987 (panelB). PanelC is a quantitative analysis of three separate dose dependence experiments performed for each of these proteins.




Figure 8: Time courses of G-protein subunit down-regulation. 75 µg of membrane protein from AAR-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-G-3 antiserum 977 (panelA) or anti--subunit antiserum 987 (panelB). PanelC is a quantitative analysis of three separate time course experiments performed for each of these proteins.




DISCUSSION

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(11, 12, 13, 33) . 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 G by cholera toxin-catalyzed ADP-ribosylation induces a rapid loss of total cellular levels of G that is independent of the initial increase in cAMP levels(34, 35) . In S49 cyc lymphoma cells transfected with an epitope-tagged G 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 G from the membrane to the cytosol(36) .

With these observations in mind, the present study was undertaken. The AAR 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 G proteins. In the transfected CHO cell system we have shown, using sequential G-protein photolabeling and immunoprecipitation with specific antibodies, that the AAR is capable of interacting with, and presumably activating, both G-2 and G-3 in isolated membranes. Interestingly, these are the same two pertussis toxin substrates that are expressed in RBL-2H3 cells (37) and suggest that the endogenously expressed AAR in these cells is at least capable of interacting with these proteins to produce its downstream effects. Presumably, the pertussis toxin-sensitive AAR-stimulated increase in inositol 1,4,5-trisphosphate production in RBL-2H3 cells is due to increased levels of dissociated G-derived -subunits activating phosphatidylinositol-specific phospholipase C- isoforms, an interaction that has been demonstrated both in intact cells (38) and with purified components(39) . However, we were also able to reveal an interaction of the AAR with G-like proteins expressed in CHO cells, and this would suggest that, at least in some instances, AAR stimulation of phospholipase C may have a PTx-insensitive component. The generality and significance of AAR/G interaction in systems expressing AARs endogenously remain to be determined. No stimulatory effect of AAR activation on adenylyl cyclase activity could be determined, suggesting that the AAR does not interact significantly with G.

Chronic agonist exposure of AAR-expressing CHO cells induces the specific down-regulation of G-3 and not G-2 or G -subunits, despite the ability of the receptor to activate each of these proteins. This effect required agonist occupation of the AAR, as no down-regulation was observed in native CHO cells, which express undetectable levels of functional AARs. The loss of G-3 was associated with a similarly profound reduction in the levels of -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 EC value for NECA stimulation of AA-[P]GTP incorporation into G proteins was also similar to the EC values for G-protein subunit down-regulation, suggesting a possible link between G-protein activation and down-regulation. Under these conditions, AAR-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 AAR 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 AAR. Nevertheless, such a profound down-regulation of G-3 would be expected to impair downstream signaling events elicited by any receptor with which it could interact.

The profound loss of -subunits was a surprising finding from these studies and is perplexing considering the quantity of G-3 expressed in these cells. Quantitative immunoblotting studies in CHO-K1 cells using E. coli-derived recombinant proteins as standards have shown that G-2 is present at an 8-fold molar excess over G-3 (4.8 versus 0.6 pmol/mg membrane protein)(23, 25) . Transfection of CHO cells with the AAR 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 G-3 represents a loss of approximately 0.4 pmol of G-3/mg of membrane protein. Assuming that 1 mol of -subunits is down-regulated with 1 mol of G-protein -subunit, the loss of 0.4 pmol/mg -subunits would be undetectable since it represents a small proportion of the total pool of -subunits. Therefore, additional -subunits, not associated with the loss of G-3, must be down-regulated in order to account for the 60% loss measured by immunoblotting (). This indicates that additional processes distinct from those involved in G-3 down-regulation are involved in regulating -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 G-3 over G-2 and G -subunits despite their ability to each couple with activated AARs may be due to any of several reasons. While we are not able to quantitate the amount of G -subunits expressed in CHO cell membranes, it is possible that large molar excesses of G-2 or G -subunits over G-3 mask an equivalent down-regulation such that it is not detectable: for example, a molar reduction in levels of G-2 equivalent to that of G-3 (0.4 pmol/mg membrane protein) would represent less than 10% of the total pool of G-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 -adrenergic receptor has suggested that increasing the receptor:G-protein ratio enhances observation of agonist-mediated down-regulation of G -subunits(40) . 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 AAR to activate and regulate specific G proteins in a transfected CHO cell system. The AAR is capable of activating both G-2 and G-3, the two pertussis toxin substrates expressed in CHO cells, as well as G -subunits. Moreover, sustained exposure of AAR-expressing cells to agonist results in the selective down-regulation of G-3 and G-protein -subunits but not of G-2, G, or G -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 AAR is capable of interacting with multiple G-proteins in a given cell type, agonist treatment specifically induces the down-regulation of G-3, thereby providing a potential mechanism for heterologous desensitization of hormone signaling events mediated by this G-protein.

  
Table: Agonist regulation of G-protein expression in CHO cells expressing the rat AAR

AAR-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, G and G 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%.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Grant DK42486.

Supported by National Institutes of Health NHLBI SCOR Grant in Ischemic Disease P50HL17670 and in part by NHLBI Grant RO1HL35134. To whom reprint requests should be addressed: Duke University Medical Center, Box 3444, Durham, NC 27710.

The abbreviations used are: AR, adenosine receptor; G-protein, guanine nucleotide-binding regulatory protein; AA-[P]GTP, 4-azidoanilido-[-P]guanosine 5`-triphosphate; I-AB-MECA,I-4-aminoben-zyl-5`-N-methylcarboxamidoadenosine; NECA, 5`-N-ethylcarboxamidoadenosine; PVDF, polyvinylidene difluoride; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; PTx, pertussis toxin.


ACKNOWLEDGEMENTS

We thank Dr. Mark Olah for donating the AAR-transfected cell line used in this study, Dr. John Raymond for generously supplying AA-[P]GTP, and Linda Scherich for preparation of the manuscript.


REFERENCES
  1. Olsson, R. A., and Pearson, D. (1990) Physiol. Rev.70, 761-845 [Free Full Text]
  2. Tucker, A. L., and Linden, J. (1993) Cardiovasc. Res.27, 62-67 [Medline] [Order article via Infotrieve]
  3. Palmer, T. M., and Stiles, G. L. (1994) Adv. Enzymol.69, 83-120 [Medline] [Order article via Infotrieve]
  4. Zhou, Q.-Y., Li, C., Olah, M. E., Johnson, R. A., Stiles, G. L., and Civelli, O. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 7432-7436 [Abstract]
  5. Ali, H., Cunha-Melo, J. R., Saul, W. F., and Beaven, M. A. (1990) J. Biol. Chem.265, 745-753 [Abstract/Free Full Text]
  6. Ramkumar, V., Stiles, G. L., Beaven, M. A., and Ali, H. (1993) J. Biol. Chem.268, 16887-16890 [Abstract/Free Full Text]
  7. Linden, J., Taylor, H. E., Robeva, A. S., Tucker, A. L., Stehle, J. H., Rivkees, S. A., Fink, J. S., and Reppert, S. M. (1993) Mol. Pharmacol.44, 524-532 [Abstract]
  8. Salvatore, C. A., Jacobson, M. A., Taylor, H. E., Linden, J., and Johnson, R. G. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 10365-10369 [Abstract]
  9. Ramkumar, V., and Stiles, G. L. (1994) Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification (Sibley, D. R., and Houslay, M. D., eds) pp. 217-231, John Wiley & Sons, New York
  10. Hausdorff, W. P., Caron, M. G., and Lefkowitz, R. J. (1990) FASEB J.4, 2881-2889 [Abstract]
  11. Longabaugh, J. P., Didsbury, J., Spiegel, A., and Stiles, G. L. (1989) Mol. Pharmacol.36, 681-688 [Abstract]
  12. Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem.265, 5206-5210 [Abstract/Free Full Text]
  13. Green, A., Milligan, G., and Dobias, S. (1992) J. Biol. Chem.267, 3223-3229 [Abstract/Free Full Text]
  14. McKenzie, F. R., and Milligan, G. (1990) J. Biol. Chem.265, 17084-17093 [Abstract/Free Full Text]
  15. Mullaney, I., Dodd, M. W., Buckley, N., and Milligan, G. (1992) Biochem. J.289, 125-131
  16. Shah, E. H., and Milligan, G. (1994) Mol. Pharmacol.46, 1-7 [Abstract]
  17. Kim, G.-D., Carr, I. C., Anderson, L. A., Zabavnik, J., Eidne, K. A., and Milligan, G. (1994) J. Biol. Chem.269, 19933-19940 [Abstract/Free Full Text]
  18. Mitchell, F. M., Buckley, N. J., and Milligan, G. (1993) Biochem. J.293, 495-499 [Medline] [Order article via Infotrieve]
  19. Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem.265, 14784-14790 [Abstract/Free Full Text]
  20. Olah, M. E., Gallo-Rodriguez, C., Jacobson, K. A., and Stiles, G. L. (1994) Mol. Pharmacol.45, 978-982 [Abstract]
  21. Offermans, S., Schultz, G., and Rosenthal, W. (1991) Methods Enzymol.195, 286-301 [Medline] [Order article via Infotrieve]
  22. Palmer, T. M., Gettys, T. W., Jacobson, K. J., and Stiles, G. L. (1994) Mol. Pharmacol.45, 1082-1094 [Abstract]
  23. Raymond, J. R., Olsen, C. L., and Gettys, T. W. (1993) Biochemistry32, 11064-11073 [Medline] [Order article via Infotrieve]
  24. Gettys, T. W., Fields, T. A., and Raymond, J. R. (1994) Biochemistry33, 4283-4290 (1994) [Medline] [Order article via Infotrieve]
  25. Gettys, T. W., Sheriff-Carter, K., Moomaw, J., Taylor, I. L., and Raymond, J. R. (1994) Anal. Biochem.220, 82-91 [CrossRef][Medline] [Order article via Infotrieve]
  26. Strathmann, M., Wilkie, T., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A.86, 7407-7409 [Abstract]
  27. Kurose, H., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1991) Biochemistry30, 3335-3341 [Medline] [Order article via Infotrieve]
  28. Eason, M. G., Jacinto, M. T., and Liggett, S. B. (1994) Mol. Pharmacol.45, 696-702 [Abstract]
  29. Conklin, B. R., Chabre, O., Wong, Y. H., Federman, A. D., and Bourne, H. R. (1992) J. Biol. Chem.267, 31-34 [Abstract/Free Full Text]
  30. Fields, T. A., Linder, M. E., and Casey, P. J. (1994) Biochemistry33, 6877-6883 [Medline] [Order article via Infotrieve]
  31. Iredale, P. A., and Hill, S. J. (1993) Br. J. Pharmacol.110, 1305-1310 [Abstract]
  32. Mullaney, I. M., Mitchell, F. M., and Milligan, G. (1993) FEBS Lett.324, 241-245 [CrossRef][Medline] [Order article via Infotrieve]
  33. Milligan, G. (1993) Trends Pharmacol. Sci.14, 413-418 [CrossRef][Medline] [Order article via Infotrieve]
  34. Milligan, G., Unson, C. G., and Wakelam, M. J. O. (1989) Biochem. J.262, 643-649 [Medline] [Order article via Infotrieve]
  35. Chang, F.-H., and Bourne, H. R. (1989) J. Biol. Chem.264, 5352-5357 [Abstract/Free Full Text]
  36. Levis, M. J., and Bourne, H. R. (1992) J. Cell Biol.119, 1297-1307 [Abstract]
  37. Hide, M., Ali, H., Price, S. R., Moss, J., and Beaven, M. A. (1991) Mol. Pharmacol.40, 473-479 [Abstract]
  38. Hawes, B. E., Luttrell, L. M., Exum, S. T., and Lefkowitz, R. J. (1994) J. Biol. Chem.269, 15776-15785 [Abstract/Free Full Text]
  39. Hepler, J. R., Kozasa, T., Smrcka, A. V., Simon, M. I., Rhee, S. G., Sternweis, P. C., and Gilman, A. G. (1993) J. Biol. Chem.268, 14367-14375 [Abstract/Free Full Text]
  40. Adie, E. J., and Milligan, G. (1994) Biochem. J.300, 709-715 [Medline] [Order article via Infotrieve]

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