Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on October 3, 2006; DOI: 10.1124/jpet.106.113589
0022-3565/07/3201-218-228$20.00
JPET 320:218-228, 2007
CELLULAR AND MOLECULAR
A Comparison of the Antagonist Affinities for the Gi- and Gs-Coupled States of the Human Adenosine A1-Receptor
Jillian G. Baker, and
Stephen J. Hill
Institute of Cell Signalling, Medical School, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
Received for publication
September 7, 2006
Accepted
October 2, 2006.
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Abstract
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The antagonist affinity for a given receptor is traditionally considered to be constant, reflecting the chemical nature of the specific ligand-receptor interaction. However, recent observations with all three 
-adrenoceptors have cast doubt on this basic pharmacological principle. The extent to which this finding applies to other G protein-coupled receptors and their interaction with different G proteins is unknown. Therefore, we studied the influence of different agonists on antagonist affinity measurements for Gi- and Gs-coupled conformations of the adenosine A1-receptor in Chinese hamster ovary cells stably expressing the human adenosine A1-receptor and a cAMP-response element (CRE)-secreted placental alkaline phosphatase reporter gene. Gi-coupled inhibition of [3H]cAMP accumulation via the A1-receptor was observed at low concentrations of agonist; however, a small increase in [3H]cAMP accumulation was also seen at higher agonist concentrations. This biphasic response was more evident for A1-stimulated CRE-gene transcription. The inhibitory component was abolished by pretreatment with pertussis toxin, whereas the stimulatory component was augmented, suggesting that the responses were due to an A1-Gi-coupled inhibition followed by an A1-Gs-coupled stimulation. However, the antagonist affinity values measured at the Gi-coupled and Gs-coupled conformations of the receptor were the same in both functional responses and whole-cell binding. Thus, in marked contrast to the -adrenoceptors, the A1-receptor conforms to the long-held principle of pharmacology that antagonist affinity measurements are constant regardless of the response being measured and the competing agonist used to stimulate that response. This was true even when the receptor was shown, in the same assay, to exist in two different conformational states coupled to two different G proteins.
The ability of an antagonist to bind to a given receptor is traditionally considered to be constant, reflecting the nature of the specific chemical interaction between a ligand and its receptor (Kenakin et al., 1995
). Consequently, antagonist affinity measurements at a given species homolog of a particular receptor should remain constant regardless of the method used to measure it, provided that the chemical composition of the receptor has not changed. It should not depend on the efficacy of the competing agonist used or the downstream level at which the final response is measured. This premise has been the cornerstone of classic receptor pharmacology and has underpinned major advances in drug discovery and the definition of novel receptor subtypes (Arunlakshana and Schild, 1959; Black et al., 1965, 1972). However, recent observations with all three members of the -adrenoceptor family of G protein-coupled receptors (GPCRs) have begun to cast doubt on this basic pharmacological principle (Konkar et al., 2000; Lowe et al., 2002; Baker et al., 2003a,b; Baker, 2005a,b).
Changes in antagonist affinity measurements have been noted at all human -adrenoceptors. At both the human 1- and 3-adrenoceptor, responses to some agonists are readily inhibited by antagonists (giving high antagonist affinity values), whereas others are relatively resistant to antagonism (giving low antagonist affinity values for the same antagonist at the same receptor; Pak and Fishman, 1996; Konkar et al., 2000; Lowe et al., 2002; Baker et al., 2003a, Baker, 2005a,b). Furthermore, at both receptors, certain agonist ligands demonstrated two components in their concentration-response curves, one component of which is more readily antagonized than the other (Baker et al., 2003a; Baker, 2005a). These observations have led to the proposal that the 1- and 3-adrenoceptors can exist in at least two different states or conformations with different sensitivities to antagonists (Granneman 2001; Molenaar, 2003; Arch, 2004). At the human 2-adrenoceptor, antagonist affinities also differ 10-fold depending on the agonist used and the level response measured (Baker et al., 2003b). This seems to depend upon the length of agonist incubation, the efficacy of the competing agonist, and the response being measured, i.e., cAMP versus CRE-gene transcription measurements.
The observations with the -adrenoceptors suggest that the underlying principle on which original receptor classification was based is in need of re-examination. Antagonist affinity estimates at many GPCRs may indeed depend upon the nature of the agonist used. For example, it has been proposed that agonists can differentiate between different G protein-coupled conformations of a GPCR, thus leading to agonist trafficking (Kenakin et al., 1995; Berg et al., 1998). In this case, antagonist affinities may well vary for the different G protein-coupled conformations of the same receptor. Kenakin (2002) has also proposed that receptors can adopt a variety of different conformations and that different ligands may stabilize a specific set of conformations, leading to different responses. Thus again, antagonist affinity may vary in the presence of different agonists. Alternatively, the observations made with the 2-adrenoceptor may simply reflect receptor-specific alterations in the chemical structure of the receptor over time and with different agonists that may be a consequence of receptor phosphorylation or dephosphorylation (Iyer et al., 2006; Vaughan et al., 2006). In addition, the observations made concerning the 1- and 3-adrenoceptor may reflect physical differences in the conformation of the receptor recognized by different agonists as a consequence of the association with other signaling or scaffolding proteins (including receptor dimerization; Hall and Lefkowitz, 2002; Milligan, 2004; Bulenger et al., 2005). In short, it is not currently known how widespread the patterns of changing antagonist affinity measurements are, to what extent this applies to other families of GPCRs, or the effects of interactions of the same receptor with different G proteins.
The adenosine A1-receptor is a GPCR that primarily couples to Gi proteins and thus produces an inhibition in the production of intracellular cAMP (Libert et al., 1992; Olah and Stiles, 1995). However, the human A1-receptor has also been shown to couple to Gs-proteins in CHO-K1 cells (Cordeaux et al., 2000, 2004). Therefore, the A1-receptor provides a model system in which to evaluate whether antagonist affinity values vary for a Gi-coupled receptor and whether the values obtained vary with the response measured. Furthermore, because the receptor is known to exist in different G protein-coupled states, antagonist affinities can be examined in two different states of the receptor (Gi- and Gs-coupled) within the same assay. Therefore, we have studied the influence of different agonists on antagonist affinity measurements for Gi- and Gs-coupled pathways at the level of ligand binding, cAMP accumulation, and CRE-gene transcription.
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Materials and Methods
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Materials
Fetal calf serum was from PAA Laboratories (Teddington, Middlesex, UK). [3H]DPCPX, [3H]adenine, and [14C]cAMP were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). CGS 15943, DPCPX, DPPX, GR 79236, NECA, and CPA were from Tocris Cookson (Avonmounth, Bristol, UK). Adenosine amine congener (AAC), 2-chloroadenosine, 2-chloro-cyclopentyladenosine (2C-CPA), CHA, PIA, and XAC were from Sigma Chemical (Poole, Dorset, UK), which also supplied all other reagents.
Cell Culture
CHO cells stably expressing the human A1-adenosine receptor (Hill et al., 2003) were secondarily transfected with CRE-SPAP reporter gene using Lipofectamine and Opti-MEM (Invitrogen, Paisley, UK) as per manufacturer's instructions. Transfected cells were selected for 3 weeks using resistance to 1 mg/ml neomycin (for A1-receptor) and 200 µg/ml hygromycin (for CRE-SPAP reporter gene). A single clone was then isolated by dilution cloning. These cells, CHO-A1-CRE-SPAP cells (or CHO-A1 cells), were used throughout this study. A control CHO cell line stably expressing the CRE-SPAP reporter gene but not the A1-receptor was also used. Cells were grown in Dulbecco's modified Eagle's medium/nutrient mix F-12 (DMEM/F-12) containing 10% fetal calf serum and 2 mM L-glutamine in a humidified 5% CO2, 95% air atmosphere at 37°C.
[3H]DPCPX Whole-Cell Binding
CHO-A1 cells were grown to confluence in white-sided 96-well view plates. On the day of experimentation, the media were removed and replaced with 100 µl of serum-free media (i.e., DMEM/F-12 containing 2 mM L-glutamine only) containing the competing ligand and immediately followed by the addition of 100 µl of serum-free media containing [3H]DPCPX (to give a final concentration of [3H]DPCPX of 2.67–3.60 nM). Total and nonspecific binding (as defined by 10 µM XAC) were measured in every experiment. In saturation studies, 100 µl of serum-free media or serum-free media containing 20 µM XAC was added to each well, immediately followed by 100 µl of serum-free media containing [3H]DPCPX to give final well concentrations of 10 µM XAC (to define nonspecific binding) and [3H]DPCPX in the range from 0.005 to 43.89 nM. The cells were incubated for 90 min at 37°C in a humidified 5% CO2, 95% air atmosphere. After 90 min, the media and drugs were removed, and the cells were washed twice by the addition and removal of 2 x 200 µl of phosphate-buffered saline/well. A white base was then added to the plate, followed by 100 µl of MicroScint 20 per well, and a sealant film was placed over the wells. The plates were then counted the following day on a TopCount (Packard, Boston, MA) 2 min per well. The protein content was determined by the method of Lowry et al. (1951).
Binding studies were also carried out after pretreatment with pertussis toxin (PTX). Here, after the cells had grown to confluence, the media were removed and replaced with either 100 µl of serum-free media (to ensure that the serum-starving step did not cause any changes in the ligand affinities) or 100 µl of serum-free media containing 100 ng of PTX/ml. The cells were incubated for an additional 24 h before whole-cell binding was undertaken as described above.
[3H]cAMP Accumulation
Cells were grown to confluence in 24-well plates. Twenty-four hours before experimentation, the media were removed and replaced with serum-free media (with or without PTX at 100 ng/ml). On the day of experimentation, the media were removed, and the cells were prelabeled with [3H]adenine by incubation with 2 µCi/ml [3H]adenine in serum-free media (0.5 ml/well) for 3 h at 37°C (5% CO2). The [3H]adenine was removed, and each well was washed by the addition and removal of 1 ml of serum-free media. One milliliter of serum-free media containing 10 µM rolipram, with or without the final required concentration of DPCPX, was added to each well, and the cells were incubated for 30 min at 37°C (5% CO2). Agonist in 10 µl was added to each well, and the plates were incubated for 10 min at 37°C. Forskolin was then added to each well (except basal), and the plates were incubated for an additional 10 min at 37°C before the reaction was terminated by the addition of 50 µl of concentrated HCl per well. The plates were then frozen and thawed, and [3H]cAMP was separated from other 3H nucleotides by Dowex and alumina column chromatography, each column being corrected for efficiency by comparison with [14C]cAMP recovery as described previously (Donaldson et al., 1988).
In experiments where the response to DPCPX, XAC, CGS 15943, and DPPX was examined alone (e.g., Fig. 3), following the forskolin addition, the ligands were incubated for 1 h (37°C; 5% CO2) to maximize any observed responses. For the same reason, all [3H]cAMP experiments involving the control CHO-CRE-SPAP cells (i.e., those without the transfected A1-receptor) were also incubated for 1 h at 37°C.
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Fig. 3. [3H]cAMP accumulation in response to DPCPX (a and b) and XAC (c and d). a and c, CHO-A1-CRE-SPAP cells with and without a preincubation with 100 ng/ml PTX. b and d, CHO-CRE-SPAP cells (i.e., the parent cells without the transfected receptor) in the absence of PTX. Bars show basal [3H]cAMP accumulation, that in response to 10 µM forskolin both with and without preincubation with PTX. Data points are mean ± S.E.M. of triplicate values from a single experiment, and are they representative of four separate experiments in each case. CGS 15943 stimulated responses in both CHO-A1-SPAP cells (both with and without PTX) and CHO-CRE-SPAP cells in an identical manner to those seen with DPCPX. DPPX stimulated responses identical to those seen with XAC.
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CRE-SPAP Gene Transcription
Cells were grown to confluence in 96-well plates in 100 µl of DMEM/F-12 containing 10% fetal calf serum and 2 mM L-glutamine. Once confluent, the media were removed and replaced with 100 µl of serum-free media (DMEM/F-12 containing 2 mM L-glutamine), and the cells were incubated for an additional 24 h. Where used, PTX was added to the serum-free media (100 ng/ml); thus, the cells received 24-h pretreatment with PTX. On the day of experimentation, the serum-free media were removed and replaced with 100 µl of serum-free media or 100 µl of serum-free media containing an antagonist at the final required concentration, and the cells were incubated for 1 h (37°C; 5% CO2). Agonist in 10 µl (diluted in serum-free media) was then added to each well, and the plate was incubated at 37°C for 10 min. Forskolin in 10 µl was then added to all but the basal wells, and the cells were incubated for 5 h (37°C; 5% CO2). After 5 h, the media and drugs were removed, 40 µl of serum-free media was added to each well, and the cells were incubated for an additional 1 h at 37°C. The plates were then incubated at 65°C for 30 min to destroy any endogenous phosphatase. The plates were then cooled to 37°C. One hundred microliters of 5 mM 4-nitrophenyl phosphate in diethanolamine buffer was added to each well, and the plates were incubated at 37°C until the yellow color developed. The plates were then read on a Dynatech MRX plate reader at 405 nm.
Data Analysis
[3H]DPCPX Whole-Cell Binding. Saturation curves for specific [3H]DPCPX binding were fitted to the following equation using Prism 2 (GraphPad Software Inc., San Diego, CA):
 | (1) |
where BMAX is the maximal specific binding, KD is the dissociation constant of [3H]DPCPX, and [L] is the concentration of [3H]DPCPX.
Curves for inhibition of specific binding of [3H]DPCPX by a range of A1-antagonists and agonists were fitted to the following equation:
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where [A] is the concentration of competing antagonist and IC50 is the concentration that inhibits specific binding by 50%. Antagonist dissociation constants (KB) were then determined from the following expression:
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where [L] and KD are the concentration and dissociation constants of [3H]DPCPX, respectively.
Functional Experiments. One-site concentration-response curves. Sigmoidal agonist concentration-response curves were fitted to the data using the following equation through computer-assisted nonlinear regression using the program Prism 2:
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where Emax is the maximal response, [A] is the agonist concentration, and EC50 is the concentration of agonist that produces 50% of the maximal response.
Antagonist KD values were then calculated from the shift of the agonist concentration responses in the presence of a fixed concentration of antagonist using the following equation:
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where DR (dose ratio) is the ratio of the agonist concentration required to stimulate an identical response in the presence and absence of a fixed concentration of antagonist [B].
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Fig. 1. a, [3H]DPCPX binding to whole CHO-A1-CRE-SPAP cells showing total binding and nonspecific binding determined in the presence of 10 µM XAC. Data points are mean ± S.E.M. of quadruplicate determinations. This single experiment is representative of six separate experiments. b and c, inhibition of [3H]DPCPX binding to whole cells by DPCPX, DPPX, CGS 15943, and XAC in CHO-A1-CRE-SPAP cells. In b, the cells have been serum starved for 24 h before experimentation. In c, the cells have been incubated with 100 ng/ml PTX in serum-free media for 24 h before experimentation. Bars represent total [3H]DPCPX binding and nonspecific binding as determined by the presence of 10 µM XAC, and data points are mean ± S.E.M. of triplicate determinations. The concentration of [3H]DPCPX was 3.60 nM, and these single experiments are representative of four (b) and four (c) separate experiments.
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In experiments where three different fixed concentrations of the same antagonist were used, Schild plots were constructed using the following equation:
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These points were then fitted to a straight line. A slope of 1 then indicates competitive antagonism (Arunlakshana and Schild, 1959).
Two-Site Agonist Curves. Because many concentration-response curves clearly contained two components, two-site analysis was required for most ligands. This was performed using the following equation:
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where basal is the response in the absence of agonist, FK is the response to a fixed concentration of forskolin, [A] is the concentration of A1-agonist, IC50 is the concentration of A1-agonist that inhibits 50% of the response to forskolin, SMAX is the maximal stimulation of the Gs-component of the response by the A1-agonist, and EC50 is the concentration of A1-agonist that stimulated a half-maximal Gs-response.
For analysis of the cyclic AMP responses to DPCPX and CGS 15943 in which a secondary inhibitory effect was observed at high concentrations, the equation fitted was as follows:
 | (8) |
where basal is the response in the absence of ligand, [A] is the concentration of DPCPX or CGS 15943, SMAX is the maximal stimulation produced by the inverse agonist, EC50 is the concentration of inverse agonist that produced 50% of its maximal response, IMAX is the maximal extent of inhibition observed at high concentrations, and IC50 is the concentration of DPCPX or CGS 15943 required to produce 50% of this maximal inhibitory effect.
A3 µM (maximal) forskolin concentration was included in each CRE-gene transcription plate and a 10 µM (maximal) forskolin included in each cAMP plate for each separate experiment. All data are presented as mean ± S.E.M. of triplicate or quadruplicate determinations, and n in the text refers to the number of separate experiments.
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Results
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[3H]DPCPX Whole-Cell Binding. Because an important aim of this study was to compare the antagonist affinity measurements obtained in different functional assays, the affinity of each antagonist was first determined directly by competition binding studies. The KD value of [3H]DPCPX determined from saturation binding experiments was 3.61 ± 0.28 nM (n = 6), and the expression of the human A1-adenosine receptor in this stable cell line was 3767 ± 310 fmol/mg protein (n = 6; Fig. 1a). The affinity of the A1-antagonists was then determined in the presence and absence of PTX from competition binding studies; the log KD values are shown in Table 1 (Fig. 1, b and c). PTX ADP ribosylates a cysteine residue in the C-terminal region of the G
i/o subunit that is responsible for receptor selectivity and coupling (Jajoo et al., 2006). Therefore, pretreatment with PTX prevents the A1-receptor coupling to Gi/o proteins and, as shown in the functional responses below, can be used to isolate the A1-Gs-coupled responses. The affinity of the agonists used was also determined to give an indication of relative efficacy of the agonists when comparing the EC50 values of functional studies later (Table 1).
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TABLE 1 Log KD values of A1-antagonists and agonists obtained from inhibition of specific [3H]DPCPX binding to the human A1-adenosine receptor in CHO A1-CRE-SPAP cells
The first column is from binding obtained 24 h after plating cells into 96-well plates, the second column is after serum starving for 24 h before experimentation, and the third column is following treatment with PTX in serum-free media for 24 h before experimentation. Values represent mean ± S.E.M. of triplicate determinations of n different experiments in each case.
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[3H]cAMP Accumulation. 2C-CPA inhibited forskolin-stimulated [3H]cAMP accumulation in CHO-A1 cells to yield a log IC50 value of –9.06 ± 0.05 (n = 10) in keeping with its agonist activation of the Gi-coupled A1-receptor (Fig. 2a). This response was inhibited by DPCPX to yield a log KD value for DPCPX of –8.62 ± 0.10 (n = 9; Fig. 2a). However, close examination revealed a small increase in cAMP at the higher concentrations of 2C-CPA. When the cells were preincubated with 100 ng/ml PTX for 24 h, the inhibitory response to 2C-CPA was abolished, confirming that this is due to Gi-coupling (Fig. 2b). However, the stimulatory response remained. Therefore, it is likely that this PTX-insensitive stimulatory response seen at higher concentrations of 2C-CPA is due to the A1-receptor coupling to Gs proteins. This stimulatory response was also inhibited by DPCPX to yield a log KD value of –8.54 ± 0.11 (n = 6), very similar to that seen for antagonism of the inhibitory response.
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Fig. 2. [3H]cAMP accumulation in response to 2C-CPA (a and b) and AAC (c and d) in the absence and presence of DPCPX in CHO-A1-CRE-SPAP cells. b and d, following 24-h pretreatment with 100 ng/ml PTX. Bars show basal [3H]cAMP accumulation, that in response to 10 µM forskolin, and that in response to DPCPX in the presence of 10 µM forskolin. Data points are mean ± S.E.M. of triplicate values from a single experiment, and they are representative of nine (a), six (b), four (c), and four (d) separate experiments.
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Similar responses were seen with a range of adenosine A1-receptor agonists (Fig. 2; Table 2), and all of these responses were inhibited by DPCPX to yield comparable log KD values (Fig. 2; Table 3). However, although the PTX-insensitive stimulatory response seen with AAC (Fig. 2d) was antagonized by DPCPX, a maximal response was not reached in the presence of DPCPX, thus making a log KD value for DPCPX unobtainable.
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TABLE 2 Log IC50 values (without PTX) and log EC50 values (following preincubation with PTX) for A1-agonist-induced changes in forskolin-stimulated [3H]cAMP accumulation in CHO A1-CRE-SPAP cells
All agonists produced a maximal inhibition of forskolin-stimulated [3H]cAMP accumulation and were therefore full agonists for the Gi-coupled response. -Fold over basal values for the Gs stimulatory responses are also shown. Values are mean ± S.E.M. of n separate experiments.
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TABLE 3 Log KD values for DPCPX from forskolin-stimulated [3H]cAMP accumulation measured in the presence of the different agonists at the inhibitory Gi-coupled receptor conformation (without PTX preincubation) and at the stimulatory Gs-coupled receptor conformation
Values are mean ± S.E.M. of n separate experiments.
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DPCPX alone, at concentrations of 10 and 30 nM, seemed to stimulate a response (Fig. 2). The response to DPCPX and the other antagonists used in this study was therefore examined more closely. DPCPX, XAC, CGS 15943, and DPPX all caused stimulatory responses in CHO-A1 cells (log EC50 = –8.43 ± 0.07, 1.61 ± 0.07-fold over basal; –7.80 ± 0.15, 1.51 ± 0.07-fold over basal; –8.61 ± 0.07, 1.45 ± 0.06-fold over basal; and –7.79 ± 0.09, 1.37 ± 0.05-fold over basal, respectively, all n = 4; Fig. 3). Pretreatment with PTX, however, abolished the stimulatory effect of these compounds (Fig. 3). However, higher concentrations of DPCPX and CGS 15943 caused a decrease in [3H]cAMP accumulation, regardless of whether the cells had been preincubated with PTX (Fig. 3). This was also seen in CHO cells not expressing the A1-receptor (CHO-CRE-SPAP cells; Fig. 3) and therefore is likely to be a nonspecific effect of the compounds at these high concentrations.
A1-CRE-Gene Transcription. 2C-CPA inhibited forskolin-stimulated CRE-gene transcription (SPAP) in CHO-A1 cells to yield a log IC50 value of –8.42 ± 0.08 (n = 7) in keeping with its agonist activation of the Gi-coupled A1-adenosine receptor (Fig. 4a). However, at higher concentrations of 2C-CPA, a substantial stimulatory response was also seen with a log EC50 of –6.66 ± 0.03 (n = 7; Fig. 4a). DPCPX antagonized both the inhibitory and stimulatory responses to 2C-CPA with similar affinities (log KD =–8.76 ± 0.05, n = 15 and –8.81 ± 0.05, n = 11, respectively). Furthermore, in experiments where several different concentrations of DPCPX were used, a Schild plot of the inhibitory component was constructed, and the slope was 1.04 ± 0.07 (n = 3), consistent with competitive antagonism. A Schild plot was not possible for the stimulatory component, as inhibition of the response by only two concentrations of DPCPX was possible within the concentration window available for the solubility of the compounds (Fig. 4a). When a similar experiment was performed after 24-h preincubation with PTX, the inhibitory response to 2C-CPA was abolished, confirming that this is due to Gi coupling. However, once again the stimulatory response remained, suggesting coupling to Gs proteins. This stimulatory response was also inhibited by DPCPX to again yield a similar log KD value for DPCPX of –8.84 ± 0.09 (n = 8).
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Fig. 4. CRE-SPAP gene transcription in response to 2C-CPA (a and b) and NECA (c and d) in the absence and presence of 3, 30, and 300 nM DPCPX in CHO-A1-CRE-SPAP cells. b and d, following 24-h pretreatment with 100 ng/ml PTX. Bars show basal CRE-SPAP gene transcription, that in response to 10 µM forskolin, and that in response to 3 or 30 nM or 300 nM DPCPX in the presence of 10 µM forskolin. Data points are mean ± S.E.M. of triplicate values from a single experiment, and they are representative of three separate experiments in each case.
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Once again, similar responses were seen to the other adenosine agonists (Figs. 4 and 5; Table 4). All of the inhibitory and stimulatory response yielded similar log KD values for DPCPX, and all inhibitory responses were found to be competitive (Figs. 4 and 5; Table 5). All log KD values determined in the presence of PTX were again found to be similar, with the exception of AAC, where once more a log KD value was not obtainable due to the decreasing maximums seen in the presence of DPCPX (Fig. 5d). When this study was extended to different antagonists (XAC, CGS 15943, and DPPX), similar antagonist affinity measurements were obtained for each antagonist at the inhibitory response and the stimulatory responses in both the absence and presence of PTX (Table 6).
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Fig. 5. CRE-SPAP gene transcription in response to GR 79236 (a and b) and AAC (c and d) in the absence and presence of 3, 30, and 300 nM DPCPX in CHO-A1-CRE-SPAP cells. b and d, following 24-h pretreatment with 100 ng/ml PTX. Bars show basal CRE-SPAP gene transcription, that in response to 10 µM forskolin, and that in response to 3 or 30 nM or 300 nM DPCPX in the presence of 10 µM forskolin. Data points are mean ± S.E.M. of triplicate values from a single experiment, and they are representative of four (a), three (b), seven (c), and five (d) separate experiments.
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TABLE 4 Log IC50 values and log EC50 values (determined from two-site concentration-response analysis, without PTX), and log EC50 values following preincubation with PTX (which abolished the inhibitory response) of A1-agonist-induced changes in forskolin-stimulated CRE-SPAP production in CHO A1-CRE-SPAP cells
All agonists stimulated a maximal inhibition of forskolin-stimulated CRE-SPAP production and were therefore full agonists for the Gi-coupled response. -Fold over basal values for the Gs stimulatory responses are also shown. Values are mean ± S.E.M. of n separate experiments.
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TABLE 5 Log KD DPCPX log KD values for DPCPX obtained in CHO-A1-CRE-SPAP cells from antagonism of the CRE-SPAP responses to a range of different A1-agonists in the presence of 3 mM forskolin
Measurements were made for both Gi-coupled and Gs-coupled (in the presence and absence of PTX preincubation) conformations of the human A1-receptor. Values are mean ± S.E.M. of n separate experiments. Schild plots were performed for the Gi-inhibitory component of each agonist in the presence of 3, 30, and 300 nM DPCPX, and the slopes for these plots shown.
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TABLE 6 Log KD values for XAC, CGS 15943, and DPPX obtained from antagonism of the CRE-SPAP responses to a range of different A1-agonists in the presence of 3 µ M forskolin
Measurements were made for both Gi-coupled and Gs-coupled (in the presence and absence of PTX preincubation) conformations of the human A1-receptor. Values are mean ± S.E.M. of n separate experiments.
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Lack of Responses in CHO-CRE-SPAP Cells. Full 6- or 7-point concentration-response curves were performed in CHO-CRE-SPAP cells (i.e., CHO cells stably expressing the CRE-SPAP reporter but not the human A1-adenosine receptor) both in [3H]cAMP accumulation and CRE-SPAP gene transcription assays in a manner identical to that performed in the CHO-A1 cells for every ligand used in this study (n = 3 for each ligand in each assay). There was no response to any compound except for the decrease in response to DPCPX and CGS 15943 mentioned above (Fig. 3) and a small decrease in response at the highest concentration (100 µM) of PIA and 2-chloroadenosine. This suggests that all of the stimulatory Gs and inhibitory Gi responses discussed above are occurring via the transfected human A1-adenosine receptor and not through a native receptor present in the parent cell line.
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Discussion
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Antagonist affinity measurements have traditionally been considered to be constant for a given receptor-ligand interaction, and this property has been used to define which receptors are present within a given tissue (Arunlakshana and Schild, 1959; Black et al., 1965, 1972; Kenakin et al., 1995; Hill, 2006). However, recent evidence obtained for the three Gs-coupled human -adrenoceptors indicates that this is not always the case (Konkar et al., 2000; Lowe at al, 2002; Baker et al., 2003a,b; Baker, 2005a,b). Changes in antagonist affinity at the human -adrenoceptors have been attributed to two agonist conformations or states of the receptor (1 and 3; Pak and Fishman, 1996; Konkar et al., 2000; Lowe et al., 2002; Baker et al., 2003a, Baker, 2005a,b), or they have been shown to vary depending upon the assay used, the time of incubation used, and the nature of the competing agonist (2; Baker et al., 2003b). A similar difference has been noted for the histamine H1-receptor where antagonist affinity estimates varied according to the response measured (inositol phosphate accumulation or nuclear factor-
B gene transcription; Bakker et al., 2001). Here, we have examined a Gi-coupled receptor (the human adenosine A1-receptor; Libert et al., 1992; Olah and Stiles, 1995; Cordeaux et al., 2000) and evaluated, using a range of different agonists, the extent to which antagonist affinity estimates depend on the level of the signaling cascade at which responses are measured, the length of incubation time, and the nature of the stimulating agonist.
As expected, stimulation of the A1-receptor by agonists caused a decrease in forskolin-stimulated [3H]cAMP accumulation in keeping with a Gi-coupled GPCR. In addition, a small augmentation of forskolin-stimulated [3H]cAMP accumulation was observed at higher agonist concentrations. Previous studies of [35S]guanosine 5'-O-(3-thio)triphosphate binding to Gs proteins have shown that the A1-receptor expressed in CHO-K1 cells can also couple directly to Gs proteins (Cordeaux et al., 2000, 2004). Experiments performed in the presence of PTX removed the inhibitory coupling to adenylyl cyclase via Gi proteins and enhanced the observed stimulation of [3H]cAMP accumulation. This confirmed that the stimulatory component was due to an A1-receptor-mediated stimulation of adenylyl cyclase via the Gs protein. However, because much higher concentrations of agonist were required to achieve this Gs coupling, the agonist efficacy (in terms of the ratio of agonist dissociation constant KD determined from binding compared with its IC50 and EC50 values seen in the functional assays; Clark et al., 1999), was, by definition, lower at the A1-Gs-coupled conformation of the receptor than at the equivalent A1-Gi-coupled conformation. For example, in the case of 2C-CPA, the KD/IC50 ratio for the Gi response was 437, whereas that for the Gs response (KD/EC50) was 2. The other agonists gave very similar estimates of efficacy for the two responses (Table 7).
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TABLE 7 Agonist efficacy estimates for Gi- and Gs-mediated responses based on the extent of signal amplification achieved by each agonist for each response relative to the KD value determined for each agonist from inhibition of [3H]DPCPX ligand binding
For the Gi efficacy estimates, the KD values were obtained from inhibition of [3H]DPCPX binding (see Table 1, column 2), and for Gs efficacy estimates, the KD values were obtained from [3H]DPCPX binding in the presence of PTX (see Table 1, column 3). Efficacy estimates are provided as log KD – log IC50 (or log EC50). For example, the value of 2.64 for 2C-CPA for inhibition of cyclic AMP accumulation indicates an amplification of 2.64 orders of magnitude (437-fold) over the ligand binding KD value, whereas the value of 0.4 for the Gs effect indicates an amplification of 2-fold.
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Close examination of the effect of DPCPX alone showed an increase in forskolin-stimulated [3H]cAMP accumulation. This could have been secondary to DPCPX acting as either an A1-Gi-inverse agonist or a weak A1-Gs-agonist. An alternative explanation could be antagonism of secreted endogenous adenosine from the cells themselves. Concentration-response curves were therefore constructed to the antagonists alone. An augmentation of forskolin-stimulated [3H]cAMP accumulation was observed in response to all four antagonists. Furthermore, the log EC50 values of the antagonists for this response were very similar to the log KD values obtained from whole-cell binding. Closer examination of the responses to DPCPX and CGS 15943 indicated that the response curves were biphasic. When the responses were examined in the parent CHO-CRE-SPAP cells (i.e., those with the CRE-SPAP reporter but without the A1-receptor), a similar decrease in [3H]cAMP was seen at matching higher concentrations. This suggests that the decrease in [3H]cAMP is a non-A1 receptor-mediated event and that DPCPX and CGS 15943 may well have cytotoxic properties at these higher concentrations, properties not shared by XAC and DPPX. When the responses in CHO-A1 cells were examined in the presence of PTX, the stimulatory component to all antagonists was abolished. Therefore, unlike the Gi-coupled conformation, there was no suggestion of agonist effects on Gs constitutive activity for the A1-receptor. This suggests that all four ligands were acting as Gi-coupled inverse agonists, although blockade of endogenous adenosine cannot be ruled out. Inverse agonist effects of DPCPX, XAC, and CGS 15943 have also been observed on A1-receptor-mediated guanosine 5'-O-(3-thio)triphosphate binding in CHO cells (Shryock et al., 1998).
When the ability of DPCPX to inhibit the 10-min [3H]cAMP agonist responses was examined, very similar log KD values were obtained for DPCPX, regardless of the competing agonist used to stimulate the A1-receptor. These were similar to the log KD value determined for DPCPX from whole-cell binding with [3H]DPCPX. However, the agonist-induced Gs-mediated [3H]cAMP response were too small to obtain a reliable measurement of DPCPX affinity. To isolate this Gs response and obtain antagonist affinity measurements at this conformation of the A1-receptor, the cells were preincubated with PTX for 24 h before experimentation. With the Gi"break" removed, the [3H]cAMP response to A1-agonists was far greater. However, it was striking that the antagonist affinity measurements for DPCPX obtained from antagonism of the Gs-coupled A1-response were very similar to those obtained from the Gi-mediated inhibition of cAMP accumulation.
It has previously been noted that tiny (3–5% maximum) -adrenoceptor Gs-coupled partial agonist cAMP responses can be markedly amplified (to 50% maximum) when longer term (5-h) Gs-coupled CRE-gene transcription is studied (Baker et al., 2003a, 2004; Baker, 2005a). Therefore, A1-CRE-gene transcription responses were examined, and in keeping with this, both the Gi and Gs-mediated responses to A1-agonists were clearly seen without the requirement for PTX preincubation to isolate the Gs component. It is also interesting to note, however, that there was no obvious amplification (in terms of EC50 values) in either the Gi or Gs responses to any of the A1-agonists when comparing cyclic AMP accumulation with CRE-mediated gene transcription.
The affinity of DPCPX for both of these A1-receptor conformations was the same irrespective of the agonist used. The bigger window for Gi-coupled response allowed Schild analysis to be undertaken and confirmed that the interaction of DPCPX with the receptor was competitive. Although this was not possible for the less well coupled Gs-conformation, it also seemed competitive (Figs. 4 and 5). Preincubation with PTX also isolated the Gs component of the CRE-gene transcription response, and the agonist EC50 values obtained were very similar to those determined from the two-component fits of the responses obtained in the absence of PTX. Furthermore, the affinity of DPCPX was also the same. When this analysis was extended to the other A1-antagonists, a similar pattern was observed.
Interestingly, antagonist affinities were similar for both Gi and Gs-mediated responses, and these values did not change when longer term assays of CRE-mediated gene transcription were undertaken in the same cell background. In the 2-adrenoceptor, longer term gene transcription assays reveal differences in antagonist affinities that depend on agonist efficacy but that are not evident in shorter term assays of [3H]cAMP accumulation. It seems that certain agonists cause a chemical modification of the 2-adrenoceptor over time that alters antagonist affinity (Baker et al., 2003b). The fact that no similar effect is seen with the adenosine A1-receptor for both a well coupled (Gi) and a poorly coupled (Gs) response suggests that no chemical modification has been induced in the A1-receptor. Indeed, unlike the 2-adrenoceptor, the adenosine A1-receptor is resistant to agonist-induced receptor phosphorylation in CHO-K1 cells (Kahout and Lefkowitz, 2003; Palmer et al., 1996). Both the cAMP and gene transcription A1-receptor responses were measured in the presence of forskolin, which will have elevated cAMP levels within the cell. However, the affinity constants for A1-antagonists obtained from each functional studies were similar to those obtained from inhibition of [3H]DPCPX binding (in the absence of forskolin). These data suggest that this has no major influence on the outcome of this study.
The data obtained with the A1-receptor confirm one of the longest-held principles of pharmacology (i.e., that provided the chemical nature of the receptor is not changed the affinity of an antagonist for a particular receptor should be constant regardless of the response being measured and the agonist used to stimulate that response). The fact that this has been demonstrated for two different G protein-bound conformations of the A1-receptor and that the observation seems to be independent of the time of incubation with agonists and antagonists, the efficacy of the agonist, or the conformational state of the A1-receptor, provides strong evidence that the ligand binding site/conformation of the human A1-receptor is not chemically altered as a consequence of G protein coupling or prolonged incubation with agonists of different efficacy. The conformational of this basic pharmacological principle for the A1-receptor strengthens the data obtained for other GPCRs where there are variations in antagonist affinity. For example, the -adrenoceptors, studied in a similar CHO-CRE-SPAP cell background, have variations in antagonist affinity that must therefore be due to a change in the chemical or conformational nature of the ligand binding pocket (e.g., agonist-induced conformations or sites of action, second messenger-induced changes, agonist-induced auxiliary protein coupling, or heterodimerization).
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Acknowledgements
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We thank Mr Richard Proudman for technical assistance.
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Footnotes
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J.G.B. is a Wellcome Trust Clinician Scientist Fellow.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.113589.
ABBREVIATIONS: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; CRE, cAMP response element; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; CGS 15943, 5-amino-9-chloro-2-(2-furyl)1,2,4-trizolo[1,5-c]quinazoline; DPPX, 1,3-dipropyl-8-phenylxanthine; GR 79236, N-[(1S,2S)-2-hydroxycyclopentyl]adenosine; NECA, 5'(N-ethylcarboxamideo)adenosine; CPA, N6-cyclopentyladenosine; AAC, adenosine amine congener; 2C-CPA, 2-chloro-N6-cyclopentyladenosine; CHA, N6-cyclohexyladenosine; PIA, N6-2-phenylisopropyladenosine; XAC, xanthene amine congener; SPAP, secreted placental alkaline phosphatase; DMEM/F-12, Dulbecco's modified Eagle's medium/nutrient mix F-12; PTX, pertussis toxin; 2-CA, 2-chloroadenosine.
Address correspondence to: Dr. Jillian G. Baker, Institute of Cell Signaling, Queen's Medical Centre, Nottingham NG7 2UH, UK. E-mail: jillian.baker{at}nottingham.ac.uk
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References
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Abstract
Materials and Methods
