Identification of A2a Adenosine Receptor Domains Involved in Selective Coupling to GS
ANALYSIS OF CHIMERIC A1/A2a ADENOSINE RECEPTORS*

(Received for publication, October 25, 1996)

Mark E. Olah

From the Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Responses to adenosine are governed by selective activation of distinct G proteins by adenosine receptor (AR) subtypes. The A2aAR couples via Gs to adenylyl cyclase stimulation while the A1AR couples to Gi to inhibit adenylyl cyclase. To determine regions of the A2aAR that selectively couple to Gs, chimeric A1/A2aARs were expressed in Chinese hamster ovary cells and ligand binding and adenylyl cyclase activity analyzed. Replacement of the third intracellular loop of the A2aAR with that of the A1AR reduced maximal adenylyl cyclase stimulation and decreased agonist potency. Restricted chimeras indicated that the NH2-terminal portion of intracellular loop 3 was predominantly responsible for this impairment. Reciprocal chimeras composed primarily of A1AR sequence with limited A2aAR sequence substitution stimulated adenylyl cyclase and thus supported these findings. A lysine and glutamic acid residue were identified as necessary for efficient A2aAR-Gs coupling. Analysis of chimeric receptors in which sequence of intracellular loop 2 was substituted indicated that the nature of amino acids in this domain may indirectly modulate A2aAR-Gs coupling. Replacement of the cytoplasmic tail of the A2aAR with the A1AR tail did not affect adenylyl cyclase stimulation. Thus, selective activation of Gs is predominantly dictated by the NH2-terminal segment of the third intracellular loop of the A2aAR.


INTRODUCTION

The endogenous release of adenosine or the administration of adenosine analogs produces a variety of physiological effects (1, 2, 3, 4). These responses result from the activation of cell surface adenosine receptors (ARs)1 that belong to the family of GPCRs. To date, four AR subtypes, A1, A2a, A2b, and A3, have been cloned from a variety of species (5, 6). Like other members of the GPCR family, the nature of the physiological responses to adenosine or its analogs is partly governed by the affinity of the individual AR subtypes for agonists, and perhaps more importantly, the selective coupling of the activated receptor subtype to distinct G proteins. The A1AR (7, 8) and A3AR (9, 10) are coupled to Gi proteins. The inhibition of adenylyl cyclase has been classically associated with A1AR activation (2) while a pertussis toxin-sensitive stimulation of phospholipase C has been described for cells endogenously expressing the A3AR (11). The A2aAR and A2bAR are coupled to Gs activation with the resulting stimulation of adenylyl cyclase, increase in cellular cAMP levels and protein kinase A activation (2). Activation of the A2aAR-Gs signal transduction pathway appears to be involved in adenosine-induced vasodilation (12, 13), inhibition of platelet aggregation (14), and modulation of neutrophil function (15, 16) although the complete signal transduction pathways have yet to be delineated.

In that most cell types possess a variety of G proteins, it is assumed that the fidelity of AR to G protein signaling is dictated by structural differences in specific regions of the receptors that physically contact specific G protein alpha  subunits. With the recent recognition of the importance of G protein beta gamma subunits in intracellular signaling, studies have also begun to focus on receptor regions which may interact with these subunits (17). The identification of regions of ARs involved in signaling to G proteins has not been reported. However, for several GPCRs such as the adrenergic receptors (18, 19, 20, 21, 22, 23, 24) and muscarinic receptors (25, 26, 27, 28, 29, 30), mutational analysis of receptor coupling to G proteins has been extensive. These studies have delineated the involvement of multiple and probably interdependent cytoplasmic domains of the GPCRs in transmitting signals to G proteins. In particular, genetic engineering of intracellular loop 2, intracellular loop 3, and the COOH-terminal tail has demonstrated the importance of these segments. Studies employing peptides whose design is based on these cytoplasmic domains of GPCRs (31, 32, 33, 34) have supported the results of mutagenesis studies. To a lesser extent, mutations in intracellular loop 1 (35, 36) and receptor transmembrane domains (37, 38) have also resulted in altered G protein signaling. This extensive analysis has indicated that the precise location of amino acids of the receptor involved in G protein activation is not the same among the individual GPCRs.

In order to define the regions of the A2aAR responsible for the selective activation of Gs, the present study profiles the functional responses of a series of chimeric ARs. From the analysis of chimeric ARs designed for this study, it is found that coupling of the A2aAR to Gs is predominantly dictated by amino acids constituting the NH2-terminal portion of intracellular loop 3. Individual amino acids in this region that have at least a partial role in Gs coupling are identified. The nature of the residues comprising the most COOH-terminal portion of intracellular loop 2 is also apparently critical. The relatively large cytoplasmic tail of the A2aAR appears to have little importance in the selective coupling of the receptor to Gs.


EXPERIMENTAL PROCEDURES

Materials

Vent DNA polymerase (New England Biolabs) and Taq DNA polymerase (Life Technologies) were used for the construction of all genetically engineered receptors. ZM241385 was prepared, radioiodinated, and subsequently purified by high performance liquid chromatography as recently described (39). All radiochemicals were from DuPont NEN. Cell culture supplies and pertussis toxin were from Life Technologies. Forskolin was from Calbiochem and R-PIA purchased from Boehringer Mannheim. NECA was a gift of Dr. R. Olsson (University of South Florida).

Mutagenesis

Sequences derived from the canine A2aAR (40) and human A1AR (41) were used to construct all chimeric receptors. Chimeric receptors employed in this study are identified as A2- (majority of structure derived from the A2aAR) or A1- (majority of structure derived from the A1AR) followed by the region in which the substitution of sequence from the donor receptor was made. For more restricted chimeras, an arrow (right-arrow) follows the specific amino acids of the A2aAR that were replaced with those of the A1AR. For point mutations, the A2aAR amino acid is given in single letter code followed by position number and then the residue used to make the substitution. The majority of chimeric receptors were constructed using a three-step polymerase chain reaction approach with oligonucleotides consisting of both A2aAR and A1AR sequences defining the splicing regions as described previously (42). For constructs containing limited amino acid sequence, a two-step polymerase chain reaction technique was employed with a single oligonucleotide specifying the base substitutions. Constructs were sequenced with Sequenase 2.0 (U. S. Biochemical Corp.) to confirm the presence of the desired mutations.

Cell Culture and DNA Transfection

CHO cells maintained in Ham's F-12 media supplemented with 10% fetal bovine serum and penicillin (100 units/ml)/streptomycin (100 µg/ml) were used for all studies. All cDNAs were subcloned into the pCMV5 expression vector (Dr. D. Russell, University of Texas Southwestern). For transient receptor expression, nearly confluent monolayers of CHO cells were transfected via a modified DEAE-dextran procedure (43) employing varying amounts (5-30 µg/75-cm2 flask depending on the construct) of receptor cDNA to obtain approximately the equivalent amount of receptor expression for functional studies. Membranes were prepared from cells approximately 72 h after transfection and employed immediately for radioligand binding and adenylyl cyclase assays.

Radioligand Binding and Adenylyl Cyclase Assays

A 75-cm2 flask of transfected CHO cells was washed twice with 10 ml of ice-cold 10 mM Tris, 5 mM EDTA, pH 7.4, at 5 °C and cells were then scraped into 6 ml of the same buffer. Cells were disrupted on ice by 20 strokes by hand in a glass homogenizer and aliquoted equally in two tubes for radioligand binding and adenylyl cyclase assays and the homogenates were centrifuged at 43,000 × g for 10 min. Membrane pellets were resuspended in 50 mM HEPES, 10 mM MgCl2, pH 6.8, or 50 mM Tris, 10 mM MgCl2, 1 mM EDTA, pH 8.26, at 4 °C for 125I-ZM241385 or [3H]DPCPX binding assays, respectively. All membrane preparations were then treated with 2 units/ml adenosine deaminase. Saturation and competition binding assays were performed exactly as described previously (39, 42). In general, competition binding assays performed in the presence and absence of 10 µM Gpp(NH)p were conducted for only those constructs displaying impaired adenylyl cyclase activity.

Assay of membrane adenylyl cyclase activity was performed via the method of Salomon (44) as described previously (45). Briefly, membrane pellets were resuspended in TNM buffer (75 mM Tris, 200 mM NaCl, 1.25 mM MgCl2, pH 8.12, at 5 °C) and treated with 2 units/ml adenosine deaminase for 5 min at 30 °C. Adenylyl cyclase assays consisted of 40 µl of membrane suspension, 40 µl of cyclase mixture (TNM buffer supplemented with 140 µM dATP, 5 µM GTP, 30 units/ml creatine kinase, 5 mM creatine phosphate, 2.2 mM dithiothreitol, 100 µM papaverine, and 1.5 µCi of [alpha -32P]ATP) and 20 µl of increasing concentrations of agonist or forskolin at a final concentration of 1 µM. Assays were conducted at 30 °C for 15 min and terminated by addition of a stop solution containing 20,000 cpm/ml [3H]cAMP. Labeled cAMP was isolated by sequential chromatography over Dowex and alumina columns and quantities determined by liquid scintillation counting. Protein concentrations were determined via the Bradford assay (46).

Pertussis Toxin Treatment

To determine the effects of pertussis toxin on adenylyl cyclase activity, two 75-cm2 flasks of CHO cells were transiently transfected with receptor constructs as described above. 24 h post-transfection, cells were detached with trypsin, pooled, and aliquoted into three 75-cm2 flasks. The following day, a single flask was treated with 200 ng/ml pertussis toxin. Following a 24-h incubation, adenylyl cyclase activity was determined in membranes obtained from control and pertussis toxin-treated cells. The remaining flask of cells was used for receptor quantification via antagonist radioligand saturation binding.

Data Analysis

125I-ZM241385 and [3H]DPCPX saturation binding curves and NECA versus 125I-ZM241385 competition binding data were analyzed via a computer modeling program as described previously (47). For adenylyl cyclase assays, maximum responses to agonist are reported as a percentage of the adenylyl cyclase activity induced by 1 µM forskolin and agonist dose-response curves were analyzed via the computer modeling system described above to determine EC50 values. An ANOVA was used to compare ligand binding and adenylyl cyclase parameters of all mutant receptors to those obtained for the WT A2aAR.


RESULTS

To begin to identify regions of the A2aAR which upon agonist binding are responsible for activation of adenylyl cyclase via Gs coupling, chimeric adenosine receptors in which cytoplasmic regions of the canine A2aAR were replaced with analogous segments of the human A1AR were constructed, transiently expressed in CHO cells, and pharmacologically characterized. Expression of receptor constructs was quantitated via saturation binding analysis with the antagonist radioligand, 125I-ZM241385 (39). As certain studies have demonstrated a correlation between the level of GPCR membrane expression and agonist activity in functional assays (48, 49, 50), approximately equivalent levels (~3.0-5.0 pmol/mg) of receptor expression were obtained for the majority of constructs by varying the amount of cDNA employed in transfections. As the expression of a limited number of constructs ranged from a low level of ~1.5 pmol/mg, WT A2aAR activity was also examined at this relatively lower receptor density.

Initial chimeric receptors contained the relatively large sequence substitution of the entire carboxyl terminus tail (A2-Tail) and entire third intracellular loop (A2-IC3) of the A2aAR with the corresponding segments of the A1AR (Fig. 1). The pharmacological profiles of WT A2aAR expressed at 2 levels, A2-Tail and A2-IC3, are shown in Table I. In membranes from untransfected CHO cells, NECA produced a maximal stimulation of adenylyl cyclase that was <5% of that induced by forskolin (data not shown). The WT A2aAR, expressed at a level of ~4.3 pmol/mg, responded to NECA with a maximal stimulation of adenylyl cyclase activity which was 87.6% ± 11.0% of that induced by 1 µM forskolin. This represented an approximate 7-fold increase in adenylyl cyclase activity above basal levels. The EC50 value for NECA was 256.3 ± 51.8 nM. Decreasing WT A2aAR expression by ~66% did not significantly affect these parameters of adenylyl cyclase stimulation. At either level, 125I-ZM241385 displayed high affinity binding as described previously (39). Replacement of the entire carboxyl terminus tail of the A2aAR with that of the A1AR (A2-Tail) did not diminish the maximal stimulation of adenylyl cyclase induced by NECA nor significantly affect the EC50 of the agonist. Conversely, replacement of the entire third intracellular loop of the A2aAR (A2-IC3) resulted in an ~75% reduction in maximal adenylyl cyclase stimulation relative to wild-type receptor. Additionally, at A2-IC3 the EC50 of NECA increased ~5-fold.


Fig. 1. A, alignment of amino acid sequences of the cytoplasmic tail of the canine A2aAR and human A1AR. TM7 indicates end of transmembrane domain 7. Dashes (-) represent conserved amino acids and asterisks (*) indicate the end of coding sequence. B, alignment of amino acid sequences of intracellular loop 3 of the canine A2aAR and human A1AR. TM5 and TM6 represent transmembrane domains 5 and 6, respectively. Dots (·) are a gap in A1AR sequence to permit alignment. C, sequence of receptors designed to study regions of intracellular loop 3 of the A2aAR. Underlined amino acids are those which were substituted into the A2aAR. A2aAR sequence was replaced with analogous A1AR sequence for all constructs except for A2ERright-arrowAA in which alanines replaced glutamic acid and arginine of the A2aAR. D, sequence of receptors designed to study NH2-terminal region of intracellular loop 3. A2aAR sequence was replaced with analogous A1AR sequence except for E212Q in which glutamic acid of the A2aAR was replaced with glutamine. E, sequence of the reciprocal chimera A1-IC3N.
[View Larger Version of this Image (46K GIF file)]


Table I.

Analysis of WT A2aAR, intracellular loop 3 and cytoplasmic tail A1/A2aAR chimeras in ligand binding and adenylyl cyclase assays

WT A2aAR was examined at two levels of expression. Description of receptor constructs is given in text. 125I-ZM241385 binding and adenylyl cyclase assays were performed as described under "Experimental Procedures." All values represent mean ± S.E. with the number of experiments given in parentheses following identification of receptor construct. Statistical analysis was applied to Kd values obtained for 125I-ZM241385 and the parameters of NECA-induced adenylyl cyclase activity.
Receptor 125I-ZM241385 binding
NECA stimulated adenylyl cyclase
Bmax Kd Maximal responsea EC50

pmol/mg nM % nM
WT A2aAR-high (10) 4.33  ± 0.26 1.74  ± 0.21 87.6  ± 11.0 256.3  ± 51.8
WT A2aAR-low (3) 1.54  ± 0.20 0.94  ± 0.30 72.0  ± 7.3 404.7  ± 118.6
A2-Tail (5) 3.66  ± 0.64 0.82  ± 0.18b 70.1  ± 8.0 255.2  ± 40.5
A2-IC3 (4) 4.07  ± 0.36 1.57  ± 0.14 22.3  ± 5.4c 1368  ± 346c

a  Represents response expressed relative to 1 µM forskolin.
b  p < 0.05, significantly different than WT A2aAR.
c  p < 0.01, significantly different than WT A2aAR.

To determine which regions of the third intracellular loop replacement constituting A2-IC3 may be responsible for this chimera's impaired stimulation of adenylyl cyclase, a series of more restricted chimeric receptors was created. Fig. 1 contains a sequence alignment of intracellular loop 3 of the canine A2aAR and human A1AR as well as a schematic representation of the mutants focusing on the third intracellular loop of the A2aAR. Of the four chimeric receptors studied (Table II), only A2-IC3N representing substitution of 20 amino acids at the NH2-terminal portion of the loop displayed diminished capacity to activate adenylyl cyclase. In response to NECA, A2-IC3N was able to mediate a maximal stimulation of adenylyl cyclase approximately 50% of that observed at the WT A2aAR. The EC50 for NECA (189.5 ± 34.0 nM), however, was similar to that displayed by the WT A2aAR. At receptors A2ERright-arrowAA and A2RSTLright-arrowQKYY that both consisted of sequence substitution in the mid-portion of the third intracellular loop, NECA induced a maximal stimulation of adenylyl cyclase similar to that at the WT A2aAR. The potency of NECA at A2ERright-arrowAA (EC50 = 492.7 ± 75.9 nM) was slightly lower than at WT A2aAR. Data obtained with A2-KVSAS (see below) also indicated that sequence in the mid-portion of the third intracellular loop had no role in the selectivity of G protein coupling by the A2aAR. A2-IC3C consisting of amino acid substitutions in the COOH-terminal segment of the third intracellular loop of the A2aAR stimulated adenylyl cyclase in response to NECA in a fashion nearly identical to that of the WT A2aAR.

Table II.

Analysis of A1/A2aAR chimeras focusing on regions of intracellular loop 3 in ligand binding and adenylyl cyclase assays

Schematic representation of receptor constructs is shown in Fig. 1. 125I-ZM241385 binding and adenylyl cyclase assays were performed as described under "Experimental Procedures." All values represent mean ± S.E. with the number of experiments given in parentheses following identification of receptor construct. Statistical analysis was applied to Kd values obtained for 125I-ZM241385 and the parameters of NECA-induced adenylyl cyclase activity. Values for WT A2aAR and A2-IC3 are shown for comparison.
125I-ZM241385 binding
NECA stimulated adenylyl cyclase
Bmax Kd Maximal responsea EC50

pmol/mg nM % nM
WT A2aAR (10) 4.33  ± 0.26 1.74  ± 0.21 87.6  ± 11.0 256.3  ± 51.8
A2-IC3 (4) 4.07  ± 0.36 1.57  ± 0.14 22.3  ± 5.4b 1368  ± 346b
A2-IC3N (4) 4.83  ± 0.26 2.67  ± 0.07c 41.1  ± 7.2c 189.3  ± 40.0
A2ER right-arrow AA (6) 2.98  ± 0.70 1.51  ± 0.13 70.6  ± 9.1 497.2  ± 75.9c
A2RSTL right-arrow QKYY (5) 3.42  ± 0.66 1.85  ± 0.44 65.1  ± 4.7 301.8  ± 66.6
A2-IC3C (3) 3.46  ± 0.15 1.68  ± 0.50 91.8  ± 12.6 177.0  ± 20.9

a  Represents response expressed relative to 1 µM forskolin.
b  p < 0.01, significantly different than WT A2aAR.
c  p < 0.05, significantly different than WT A2aAR.

Taken together, the above results suggest that the impairment of adenylyl cyclase observed with the chimera A2-IC3 occurred principally due to replacement of amino acids in the NH2-terminal portion of the third intracellular loop. In order to further identify residues in the 20-amino acid replacement of A2-IC3N which may confer coupling to Gs, more restricted chimeric receptors were created (Fig. 1, Table III). A2-KVSAS represents a substitution of solely the distal 5 amino acids constituting the A2-IC3N chimera. At A2-KVSAS, NECA induced a maximal stimulation of adenylyl cyclase and displayed a potency similar to that at the WT A2aAR. Based on this finding, subsequent mutagenesis focused on the first 15 amino acids of the third intracellular loop. Unfortunately, chimeric receptors and a deletion mutant constructed to further study this region did not display appreciable 125I-ZM241385 binding nor stimulate adenylyl cyclase in response to NECA regardless of the amount of transfected cDNA. It is assumed that such proteins did not undergo the processing or folding required for proper membrane insertion or orientation. The presently employed sequence alignment of the A1AR and A2aAR indicated that 4 of the 15 amino acids in this region are conserved among the receptors, thus several of the remaining nonconserved residues were targeted for point mutations (Fig. 1, Table III). For the three mutant receptors examined, amino acids of the WT A2aAR were substituted with the analogous residues of the A1AR with the exception of E212Q in which the sequence alignment contained a gap in this region. Thus, glutamine was selected to replace glutamic acid at position 212 of the WT A2aAR. At the double point mutation A2RLright-arrowEY, NECA demonstrated a stimulation of adenylyl cyclase comparable to that at WT A2aAR in regards to both potency and efficacy. At both K209N and E212Q, NECA displayed unimpaired maximal stimulation of adenylyl cyclase, however, EC50 values were increased. Relative to WT A2aAR, NECA was approximately 3- and 5-fold less potent at K209N and E212Q, respectively. It is noted that E212Q was the only construct examined in this study which demonstrated a substantial (~3.5-fold) decrease in affinity for the antagonist radioligand, 125I-ZM241385.

Table III.

Analysis of A1/A2aAR chimeras and A2aAR point mutations focusing on the NH2-terminal region of intracellular loop 3 in ligand binding and adenylyl cyclase assays

Schematic representation of receptor constructs is shown in Fig. 1. 125I-ZM241385 binding and adenylyl cyclase assays were performed as described under "Experimental Procedures." All values represent mean ± S.E. with the number of experiments given in parentheses following identification of receptor construct. Statistical analysis was applied to Kd values obtained for 125I-ZM241385 and the parameters of NECA-induced adenylyl cyclase activity. Values for WT A2aAR and A2-IC3N are shown for comparison.
Receptor 125I-ZM241385 binding
NECA stimulated adenylyl cyclase
Bmax Kd Maximal responsea EC50

pmol/mg nM % nM
WT A2aAR (10) 4.33  ± 0.26 1.74  ± 0.21 87.6  ± 11.0 256.3  ± 51.8
A2-IC3N (4) 4.83  ± 0.26 2.67  ± 0.07 41.1  ± 7.2b 189.3  ± 34.0
A2-KVSAS (4) 3.37  ± 0.61 1.40  ± 0.22 85.3  ± 11.7 460.0  ± 90.6
A2RL right-arrow EY (3) 4.95  ± 0.58 2.00  ± 0.15 70.0  ± 6.4 251.3  ± 92.0
K209N (3) 3.30  ± 0.85 1.49  ± 0.44 108.2  ± 7.0 786.7  ± 122c
E212Q (4) 3.61  ± 0.45 6.33  ± 1.00c 102.2  ± 16.4 1358  ± 333c

a  Represents response expressed relative to 1 µM forskolin.
b  p < 0.05, significantly dfferent than WT A2aAR.
c  p < 0.01, significantly different than WT A2aAR.

To determine if the impaired stimulation of adenylyl cyclase by those mutant receptors described above resulted from reduced affinity of the receptors for agonist, the ability of NECA to compete for 125I-ZM241385 binding was analyzed. An additional parameter of receptor-G protein coupling, the sensitivity of agonist high affinity binding to the nonhydrolyzable guanine nucleotide Gpp(NH)p, was also examined. Table IV contains the results of the competition binding studies. Using membranes from transiently transfected CHO cells, the binding of NECA to the A2aAR was best fit to a two-site model (KH = 11.8 ± 3.5 nM; KL = 193.0 ± 62.0 nM) with 52.6 ± 6.8% of the receptors in the high affinity state. Consistent with previous studies of the A2aAR (51, 52), agonist binding was not sensitive to guanine nucleotide treatment (KH = 22.1 ± 5.3 nM; KL = 266.8 ± 26.3 nM; %RH = 79.7 ± 9.6%). NECA competition studies with A2-IC3 were performed four times with the data of individual experiments twice best fit by a two-site model (KH = 18.5 ± 2.1 nM; KL = 385.3 ± 170.8 nM; %RH = 76.7 ± 0.8%) and twice by a one-site model (Ki = 20.1 ± 1.8 nM). In the presence of Gpp(NH)p, NECA binding at A2-IC3 was best fit by a one-site model with relatively high affinity binding remaining (Ki = 31.4 ± 6.3 nM). At A2-IC3N, NECA binding was consistently best fit to a two-site model with parameters very similar to those of the WT A2aAR (KH = 13.2 ± 3.4 nM; KL = 250.9 ± 99.1 nM; %RH = 82.6 ± 6.9%). Binding was not significantly affected by Gpp(NH)p. Agonist binding to both K209N and E212Q was best fit by a single-site model with Ki values of 35.2 ± 3.4 and 61.9 ± 18.3 nM, respectively. As shown in Table IV, the addition of Gpp(NH)p did not affect NECA binding at either of these two point mutants.

Table IV.

Agonist affinity at receptor constructs demonstrating impaired adenylyl cyclase activation

NECA versus 125I-ZM241385 competition binding assays were performed for all constructs displaying impaired adenylyl cyclase activation as assessed by blunted maximal response or a decrease in NECA potency relative to wild-type A2aAR. Binding assays were performed in the absence (Control) or presence of 10 µM Gpp(NH)p. Data were analyzed as described under "Experimental Procedures" and high affinity (KH), low affinity (KL) binding constants and percentage of receptors in the high affinity state (RH) were determined. All values represent mean ± S.E. with the number of experiments noted in parentheses following identification of the receptor construct.
Control
+Gpp(NH)p
KH KL RH KH KL RH

nM % nM %
WT A2aAR (4) 11.8  ± 3.5 193.0  ± 62.0 52.6  ± 6.8 22.1  ± 5.3 266.8  ± 26.3 79.7  ± 9.6
A2-IC3 (4)a 18.5  ± 2.1 385.3  ± 170.8 76.7  ± 0.8b 31.4  ± 6.3c
A2-IC3N (4) 13.2  ± 3.4 250.9  ± 99.1 82.6  ± 6.9b 14.6  ± 3.6 693.5  ± 352.0 86.3  ± 3.7
K209N (3) 35.2  ± 3.4b,c 40.8  ± 1.1c
E212Q (3) 61.9  ± 18.3b,c 83.8  ± 30.2c
A2GT right-arrow PR (3) 62.1  ± 20.6b 1271  ± 307b 14.8  ± 7.5b 1088  ± 285c
A2GT right-arrow AA (3) 30.5  ± 4.6b,c 32.0  ± 6.3c

a  Of four experiments with A2-IC3, data was twice best fit by a two-site model. See text for details.
b  p < 0.05 compared to value for WT A2aAR; for KI values comparison made to KH for WT A2aAR.
c  Data best fit by a one-site model; KI is shown.

The analysis of the chimeric A1/A2aARs described above indicated that replacement of the third intracellular loop, and in particular its NH2-terminal domain, of the A2aAR with the analogous segment of the A1AR resulted in an impairment of NECA-stimulated adenylyl cyclase activity relative to the WT A2aAR. This diminished response of the chimera may have resulted from either the removal of amino acids in the A2aAR required for selective coupling to Gs or due to introducing amino acid sequence from the Gi-coupled A1AR or a combination of these factors. Upon activation by agonist, a chimeric AR containing sequence from the A1AR may at least partially produce a diminished adenylyl cyclase stimulation due to the activation of Gi proteins leading to inhibition of adenylyl cyclase. Additionally, it has been shown that chimeric GPCRs containing intracellular domains derived from parent receptors that individually couple to distinct G proteins may demonstrate a promiscuity in signal transduction (29). In order to identify the mechanism(s) responsible for the diminished functional responses observed with A2-IC3 and A2-IC3N, NECA-induced adenylyl cyclase stimulation at these chimeras was studied in membranes derived from pertussis toxin-treated cells. If these chimeras were coupled to Gi and thus upon activation by agonist produced partial inhibition of adenylyl cyclase, the stimulation of the enzyme may be increased in membranes in which pertussis toxin had abolished signaling via Gi proteins. Table V contains results from adenylyl cyclase assays performed with membranes prepared from cells maintained in the absence or presence of pertussis toxin (200 ng/ml × 24 h). These conditions for pertussis toxin treatment completely abolished the A1AR-mediated inhibition of forskolin-stimulated adenylyl cyclase observed in membranes obtained from CHO cells stably expressing the human A1AR (data not shown). As described under "Experimental Procedures," expression levels of WT A2aAR and A2a/A1AR chimeras were equivalent between control and treated cells. At the WT A2aAR, pertussis toxin treatment had no effect on the maximal stimulation of adenylyl cyclase by NECA. This finding indicates that even at high agonist concentrations, no dual coupling to Gs and Gi by the WT A2aAR was unmasked by pertussis toxin treatment. However, for both A2-IC3 and A2-IC3N, adenylyl cyclase stimulation was enhanced by pertussis toxin treatment relative to control. At A2-IC3, pertussis toxin increased adenylyl cyclase activity by ~78% relative to untreated cells although stimulation remained lower than that observed at the WT A2aAR. At A2-IC3N, pertussis toxin treatment likewise increased adenylyl cyclase stimulation by ~56% relative to control membranes. The maximal response induced by NECA at A2-IC3N in the presence of pertussis toxin (66.9 ± 9.6%) approached that obtained with the WT A2aAR. At WT A2aAR, A2-IC3, and A2-IC3N, pertussis toxin had no effect on the EC50 of NECA for the stimulation of adenylyl cyclase.

Table V.

Effect of pertussis toxin on adenylyl cyclase stimulation by WT A2aAR, A2-IC3, and A2-IC3N

Cells were incubated in the absence or presence of pertussis toxin (200 ng/ml × 24 hr) and 125I-ZM241385 saturation binding and adenylyl cyclase assays performed as described under "Experimental Procedures." All values represent the mean ± S.E. of experiments performed four (WT A2aAR, A2-IC3N) or three (A2-IC3) times.
Receptor Bmaxa NECA stimulated adenylyl cyclase
Control
+ Pertussis toxin
Increasec
Maximal responseb EC50 Maximal responseb EC50

pmol/mg % nM % nM %
WT A2aAR 4.71  ± 1.30 89.2  ± 2.3 269.4  ± 65.5 100.1  ± 12.2 385.8  ± 77.5 10.7  ± 13.0
A2-IC3 5.21  ± 1.00 16.5  ± 1.2 794.0  ± 125.0 29.4  ± 4.1 819.0  ± 147.4 77.8  ± 19.0
A2-IC3N 4.72  ± 0.81 44.5  ± 8.6 211.8  ± 30.2 66.9  ± 9.6 240.8  ± 44.6 56.0  ± 11.0

a  Receptor expression determined by 125I-ZM241385 saturation binding.
b  Represents response expressed relative to 1 µM forskolin.
c  Represents increase in maximal adenylyl cyclase stimulation obtained with pertussis toxin relative to control for each group.

Data derived following replacement of the entire third intracellular loop (A2-IC3) or solely the NH2-terminal portion of this loop (A2-IC3N) of the A2aAR with the analogous regions of the A1AR indicate the importance of these segments in adenylyl cyclase stimulation mediated by the A2aAR. However, it is possible that a loss of function by the A2aAR resulting from sequence substitution may have occurred due to nonspecific disruption of receptor structure. To determine if the identified regions when placed into the A1AR could mediate activation of adenylyl cyclase, the reciprocal chimeric receptors were created (Fig. 1), transiently expressed in CHO cells, and adenylyl cyclase activity in response to the agonist R-PIA was analyzed. Receptor expression levels were quantitated via radioligand binding with the A1AR selective antagonist, [3H]DPCPX. Adenylyl cyclase assays were performed with control and pertussis toxin-treated cells (Fig. 2). In the presence of pertussis toxin, any residual coupling of these chimeric receptors that are composed primarily of A1AR sequence to Gi proteins would be eliminated and thus make stimulation of adenylyl cyclase activity more readily apparent. Membranes prepared from pertussis toxin-treated cells expressing the WT A1AR displayed no stimulation of adenylyl cyclase activity in response to R-PIA (data not shown). Replacement of the entire third intracellular loop of the A1AR with that of the A2aAR produced a receptor, A1-IC3, that responded to R-PIA with a maximal increase in adenylyl cyclase activity that was 18.7 ± 4.4% of that induced by 1 µM forskolin. The EC50 of R-PIA at A1-IC3 was 9.75 ± 1.2 nM which is similar to that at the wild-type A1AR for the inhibition of adenylyl cyclase activity (53). In membranes prepared from pertussis toxin-treated A1-IC3 cells, maximal adenylyl cyclase stimulation by R-PIA was 27.9 ± 7.4% with an EC50 value of 21.3 ± 9.8 nM. In these experiments, A1-IC3 was expressed at a level of 1.31 ± 0.18 pmol/mg and displayed a Kd of 1.54 ± 0.28 nM for [3H]DPCPX. Replacement of the NH2-terminal region of the third intracellular loop of the A1AR with the analogous 15 amino acids (4 of which are conserved) of the A2aAR produced the chimeric receptor A1-IC3N that stimulated adenylyl cyclase to a level <5% of that observed with 1 µM forskolin. However, following pertussis toxin treatment of cells expressing A1-IC3N, R-PIA produced a maximal adenylyl cyclase stimulation of 19.6 ± 1.4% with an EC50 value of 25.8 ± 14.8 nM. A1-IC3N was expressed at 1.12 ± 0.16 pmol/mg with a Kd of 2.13 ± 0.52 nM for [3H]DPCPX.


Fig. 2. Adenylyl cyclase stimulation in response to R-PIA in membranes prepared from cells expressing (A) A1-IC3 and (B) A1-IC3N. Sequences of constructs are shown in Fig. 1. A1-IC3 and A1-IC3N were expressed at levels of 1.31 ± 0.18 and 1.12 ± 0.16 pmol/mg, respectively. Where indicated, cells were exposed to 200 ng/ml pertussis toxin for 24 h prior to membrane preparation. Points represent the means of data obtained in duplicate for experiments performed three (A1-IC3) and four (A1-IC3N) times. Bars represent standard error.
[View Larger Version of this Image (16K GIF file)]


A series of chimeric ARs was also created in order to examine the role of the second intracellular loop of the A2aAR in coupling to the stimulation of adenylyl cyclase. As shown in Fig. 3, amino acids constituting the mid-portion of intracellular loop 2 are conserved among the A2aAR and A1AR, suggesting these residues are not involved in the fidelity of G protein coupling. Thus, focus of this mutational analysis to study selective Gs coupling was on the NH2- and COOH-terminal portions of this region (Fig. 3). Results obtained with these chimeras and subsequently analyzed point mutations are shown in Table VI. The 4-amino acid substitution from the A1AR constituting the A2-IC2N chimera designed to study the NH2-terminal residues had no effect on NECA-stimulated adenylyl cyclase activity relative to WT A2aAR. The importance of the COOH-terminal section of the second intracellular loop was initially examined in three mutant receptors. A2-IC2C, in which 5 amino acids over a 7-residue region of the A2aAR were replaced with A1AR amino acids, also responded to NECA with a stimulation of adenylyl cyclase characteristic of that of the WT A2aAR. Similarly, the more restricted chimera A2NGLright-arrowKMV demonstrated unimpaired functional response. However, substitution of Gly-118 and Thr-119 that are the amino acids at the junction of intracellular loop 2 and transmembrane domain 4 in the A2aAR with the analogous amino acids of the A1AR (proline and arginine, respectively), substantially altered adenylyl cyclase activation. At A2GTright-arrowPR, NECA induced a relatively unimpaired maximal stimulation of adenylyl cyclase (84.5 ± 12.9%) but was approximately 50-fold less potent (EC50 = 12,600 ± 5190 nM) than at the WT A2aAR. This rightward shift of the NECA dose-response curve was approximately 10-fold greater than that observed at any other construct examined in this study. Competition binding assays with A2GTright-arrowPR also reflected altered receptor-G protein coupling (Table IV). Relative to WT A2aAR, both the KH and KL values obtained for NECA at A2GTright-arrowPR increased approximately 5-fold. Additionally, only 14.8 ± 7.5% of the A2GTright-arrowPR population was in the agonist high affinity state which is an ~75% decline relative to WT A2aAR. In an attempt to identify an individual substitution in A2GTright-arrowPR responsible for its impaired signaling, two single point mutations were constructed. However, at both G118P and T119R the parameters for NECA-induced adenylyl cyclase stimulation including EC50 values were identical to those obtained at the WT A2aAR. The construct A2GTright-arrowAA in which Gly-118 and Thr-119 were both replaced with alanine rather than the analogous A1AR residues activated adenylyl cyclase in response to NECA in a fashion similar to that of the WT A2aAR. NECA competition binding at A2GTright-arrowAA was best fit by a one-site model and remained of relatively high affinity (Ki = 30.5 ± 4.6 nM).


Fig. 3. A, alignment of amino acid sequences of intracellular loop 2 of the canine A2aAR and human A1AR. Dashes (-) represent conserved amino acids. Position of threonine 119 is shown. TM3 and TM4 represent transmembrane domains 3 and 4, respectively. B, sequence of receptors designed to study regions of intracellular loop 2 of the A2aAR. Underlined amino acids represent sequence substituted into the A2aAR. A2aAR sequence was replaced with analogous A1AR sequence except for A2GTright-arrowAA in which alanines replaced glycine and threonine of the A2aAR.
[View Larger Version of this Image (23K GIF file)]


Table VI.

Analysis of A1/A2aAR chimeras and A2aAR point mutations focusing on intracellular loop 2 in ligand binding and adenylyl cyclase assays

Schematic representation of receptor constructs is shown in Fig. 3. 125I-ZM241385 binding and adenylyl cyclase assays were performed as described under "Experimental Procedures." All values represent mean ± S.E. with the number of experiments given in parentheses following identification of receptor construct. Statistical analysis was applied to Kd values obtained for 125I-ZM241385 and the parameters of NECA-induced adenylyl cyclase activity. Values for WT A2aAR are shown for comparison.
Receptor 125I-ZM241385 binding
NECA stimulated adenylyl cyclase
Bmax Kd Maximal responsea EC50

pmol/mg nM % nM
WT A2aAR (10) 4.33  ± 0.26 1.74  ± 0.21 87.6  ± 11.0 256.3  ± 51.8
A2-IC2N (3) 3.34  ± 1.00 2.41  ± 0.46 74.1  ± 5.8 213.0  ± 106
A2-IC2C (6) 4.46  ± 0.84 2.69  ± 0.45 71.5  ± 4.5 224.3  ± 29.3
A2NGL right-arrow KMV (3) 3.90  ± 1.30 1.43  ± 0.47 78.1  ± 7.4 105.3  ± 33.6
A2GT right-arrow PR (5) 4.24  ± 0.42 2.56  ± 0.31 84.5  ± 12.9 12,600  ± 5190b
G118P (3) 4.62  ± 0.51 2.20  ± 0.50 80.3  ± 7.3 156.1  ± 59.7
T119R (3) 4.58  ± 1.50 1.38  ± 0.27 73.5  ± 10.4 172.2  ± 64.2
A2GT right-arrow AA (5) 3.61  ± 0.49 1.46  ± 0.25 92.1  ± 11.8 399.4  ± 69.3

a  Represents response expressed relative to 1 µM forskolin.
b  p < 0.01, significantly different than WT A2aAR.


DISCUSSION

Through the analysis of a series of chimeric adenosine receptors composed of human A1AR and canine A2aAR sequence, the present study has identified regions of the A2aAR required for efficient coupling to Gs and thus the stimulation of adenylyl cyclase. In the A2aAR, intracellular loop 3 and in particular the NH2-terminal region of this domain appears to have the predominant role in conferring selective receptor coupling to Gs with the nature of amino acids in the most COOH-terminal portion of intracellular loop 2 also being important. This is the first description of a structural analysis of AR-G protein signal transduction. This study focused on determinants of the selective coupling of the A2aAR to Gs. Thus, data generated from chimeras that did not demonstrate impaired adenylyl cyclase stimulation do not indicate that the targeted regions have no role in G protein signaling but rather suggest they are not involved in maintaining the fidelity of specifically Gs activation. Such regions which may be conserved among certain receptor subtypes may be involved in a general activation of G protein alpha  subunits or perhaps involved in contact with G protein beta gamma subunits.

The structure of the A2aAR is interesting in that it possesses a cytoplasmic tail approximately 80 amino acid residues longer than any other cloned AR including the similarly Gs-coupled A2bAR. However, this tail does not appear to be involved in selective Gs signaling by the A2aAR as its complete replacement with that of the A1AR (A2-Tail) in this study did not impair NECA-induced adenylyl cyclase stimulation. In a study of chimeric beta 2/alpha 2-adrenergic receptors, Liggett and co-workers (20) found the proximal portion of the beta 2-adrenergic receptor cytoplasmic tail to have a partial role in mediating the stimulation of adenylyl cyclase in response to isoproterenol. Alternative splicing of the cytoplasmic tail of the EP3 prostaglandin E receptor was reported to produce receptor subtypes that differentially couple to G proteins including Gs (54). Conversely, studies of receptor chimeras generated from endothelin receptor subtypes (55) as well as vasopressin receptor subtypes (56) have shown that solely the replacement of the cytoplasmic tail of the Gs-coupled subtype with that of the Gi-coupled subtype resulted in no impairment of adenylyl cyclase stimulation relative to wild-type receptor. Even with the data presented in this study, the function of the relatively large cytoplasmic tail of the A2aAR has yet to be defined.

In contrast to the results obtained for the cytoplasmic tail, intracellular loop 3 of the A2aAR appears to be critical for full activation of Gs and the resulting adenylyl cyclase stimulation. Replacement of the entire loop of the A2aAR with that of the A1AR resulted in an ~75% decrease in the maximal adenylyl cyclase response elicited by NECA as well as a 5-fold shift to the right of the agonist dose-response curve. In a large part although not completely, the diminished maximal adenylyl cyclase response observed at A2-IC3 appears to arise as a result of replacement of amino acid sequence in the NH2-terminal portion of the loop. The effect of replacement of NH2-terminal residues in intracellular loop 3 (~50% decrease in maximal stimulation) did not fully mimic the response observed with the entire loop substitution. However, analysis of 4 chimeras targeting remaining portions of intracellular loop 3 suggested little or no role for additional sequence in this cytoplasmic segment in selective coupling of the A2aAR to Gs. It is possible that the lack of detection of an impaired adenylyl cyclase response may have arisen due to the design of the chimeras A2-KVSAS, A2ERright-arrowAA, A2RSTLright-arrowQKYY, and A2-IC3C in that creation of chimeras with slightly altered boundaries of sequence substitution may have signaled differently. Conversely, the NH2-terminal portion of intracellular loop 3 may indeed be the primary point of contact with Gs and this region may possibly be presented to the G protein in different conformations in chimeras A2-IC3 and A2-IC3N as a result of the differences in the adjacent sequence constituting the remainder of the loop.

The impaired adenylyl cyclase response observed with A2-IC3 and A2-IC3N relative to WT A2aAR appeared to result from both the removal of sequence responsible for Gs coupling as well as the introduction of A1AR sequence that promotes Gi activation. Following pertussis toxin treatment, both A2-IC3 and A2-IC3N demonstrated enhanced adenylyl cyclase stimulation suggesting a dual coupling of these chimeras to Gs and Gi. This response was not observed with the WT A2aAR. No mutation studies of A1AR-Gi coupling have been reported, thus it is not known which regions of the A1AR are involved in Gi coupling. Although generalizations may not be made with certainty, the third intracellular loop of other Gi-coupled receptors has been implicated in Gi activation (27, 32, 34, 55).

The "gain of function" displayed by the two reciprocal chimeric receptors, A1-IC3 and A1-IC3N, strongly support the notion that the third intracellular loop and in particular the NH2-terminal portion of this region of the A2aAR interacts with Gs resulting in adenylyl cyclase stimulation. Both A1-IC3 and A1-IC3N were shown to stimulate adenylyl cyclase to a level approximately 25% that of the wild-type A2aAR. This level of activity of A1-IC3N was observed only upon pertussis toxin treatment of the cells. This requirement likely arises from the presence in A1-IC3N of substantial amounts of A1AR sequence, particularly in the unmodified regions of the third intracellular loop, that retain the ability to productively couple to Gi. Despite the degree of A1AR sequence present in both A1-IC3 and A1-IC3N, the potency of R-PIA at these chimeras is identical to that at the wild-type A1AR (53) indicating a highly efficient coupling to Gs.

In agreement with the present results with the A2aAR, several studies with other GPCRs have detailed the importance of the NH2-terminal portion of intracellular loop 3 in selectivity of G protein coupling. This region appears to have a predominant role in signaling by beta -adrenergic receptor subtypes (21, 25, 29), the alpha 1-adrenergic receptor (22) and several muscarinic receptor subtypes (26, 27, 30, 31, 57), although the latter two receptor families are not coupled to Gs. Additionally, an 11-amino acid cassette in the NH2-terminal region of intracellular loop 3 of the alpha 2A-adrenergic receptor has been identified as apparently being responsible for the Gs-coupled component of this receptor's dual signaling activity (24). Mutagenesis studies of other Gs-coupled receptors have also identified the third intracellular loop as being crucial in signal transduction although more restricted structural analysis of this region was not performed (55, 56).

Results with the chimeric receptors described above suggest that 15 amino acids constituting the proximal portion of intracellular loop 3 of the A2aAR are crucial in coupling of the receptor to Gs. Of these 15 residues, 4 are conserved between the A2aAR and A1AR and several others represent apparently conservative substitutions. Targeting several of the remaining nonconserved residues resulted in the identification of single amino acids which appear to have a role in Gs coupling. Replacement of lysine and glutamic acid at positions 209 and 212, respectively, of the A2aAR each resulted in significant decreases in the potency of NECA for the stimulation of adenylyl cyclase. These residues are probably not solely responsible for Gs coupling by the A2aAR as the effects observed with the larger chimeric substitutions were not reproduced by the individual point mutations. It is probable that multiple amino acids in a specific conformation are required for the most efficacious coupling to Gs. As with all mutagenesis studies of this nature, the precise role of amino acid(s) may not be unequivocally assigned as certain responses may occur due to indirect effects on overall protein architecture.

The nature of the impairment of adenylyl cyclase stimulation observed upon distinct mutations of intracellular loop 3 of the WT A2aAR varied. The most profound disruption of functional response was observed with substitution of the entire loop as maximal adenylyl cyclase response as well as potency of NECA were both substantially affected. However, the more restricted chimera A2-IC3N responded to NECA with a diminished maximal response although the EC50 of NECA was unaffected. Conversely, point mutations K209N and E212Q both responded to agonist with intact maximal adenylyl cyclase stimulation but with a decrease in NECA potency. Thus, it is possible that multiple amino acids of the WT A2aAR must be responsible for full activation of Gs (as judged by maximal adenylyl cyclase response) whereas individual amino acid mutations may disrupt the efficiency of the coupling. It has been shown that certain single point mutations in intracellular loop 3 of the M1 muscarinic receptor disrupted receptor function to a greater extent than larger amino acid replacements that contained the same residue substitution (58). Thus, the context of the mutation may influence the functional response.

Distinctions in the process of receptor coupling to G protein are also indicated by a comparison of the parameters of functional response to those of agonist binding. Despite the impaired stimulation of adenylyl cyclase at A2-IC3 and A2-IC3N, these chimeras did not display a marked loss of high affinity agonist binding relative to the WT A2aAR. In competition binding assays with A2-IC3 and A2-IC3N, both the agonist high affinity state and percentage of receptors in this population were not decreased relative to the WT A2aAR. Treatment with Gpp(NH)p did not modulate agonist binding to the WT A2aAR or A2-IC3N but did result in NECA binding to A2-IC3 being best fit to a one-site model. Point mutations in the third intracellular loop of the A2aAR that produced a loss of NECA potency in adenylyl cyclase assays (K209N and E212Q) also altered receptor-Gs coupling as determined in competition binding assays. NECA binding to both K209N and E212Q was best described by a single site model and was not sensitive to guanine nucleotide treatment. Interestingly, the differences between the Ki for NECA at K209N and E212Q relative to its KH at the WT A2aAR, ~3- and 5-fold respectively, are similar to the shifts in the EC50 of NECA observed in adenylyl cyclase assays with these mutant receptors.

Finally, this study examined the role of the second intracellular loop in signaling by the A2aAR. Focus was on the COOH-terminal portion of intracellular loop 2 as it is possible this segment may be in close proximity to the NH2-terminal of intracellular loop 3 in the membrane embedded receptor. Additionally, this region has been implicated in mutagenesis studies of receptor-G protein coupling (25, 29). It was found that tandem replacement of Gly-118 and Thr-119 at the junction of intracellular loop 2 and transmembrane domain 4 with the analogous residues of the A1AR greatly disrupted coupling of the A2aAR to Gs. The effect of this substitution must be profound as not only was NECA-induced adenylyl cyclase activation disrupted but affinity of the agonist for receptor decreased as did the percentage of receptors in the high affinity state. Gly-118 and Thr-119 of the A2aAR may not be directly involved in Gs activation and it is possible that their substitution with proline and arginine, respectively, may produce conformational changes in intracellular loop 2 and disrupt G protein coupling at other regions of the receptor. Several observations support this hypothesis. First, the individual replacement of either glycine or threonine with proline and arginine, respectively, did not affect receptor function relative to the WT A2aAR. Second, replacement of these residues with alanines also did not disrupt receptor stimulation of adenylyl cyclase or high affinity NECA binding. Substitution with proline, an amino acid that disrupts alpha -helical structure, in combination with the threonine replacement may alter overall protein conformation of the A2aAR although this amino acid combination is that present in the WT A1AR. Indeed, the proline and arginine substitution was also present in the A2-IC2C chimera that demonstrated unimpaired adenylyl cyclase stimulation. As discussed above, this finding again indicates the importance of the context in which an amino acid(s) substitution is made in affecting functional responses.

In summary, the coupling of the A2aAR to Gs is predominantly dictated by sequence in intracellular loop 3 of the receptor. In particular, the NH2-terminal region of the third intracellular loop of the A2aAR appears to be responsible for the fidelity of G protein coupling. Lysine and glutamic acid residues in this region have significant roles in the efficiency of A2aAR-Gs coupling. The analysis of multiple chimeric A1/A2aARs did not suggest a significant role for other cytoplasmic domains of the A2aAR in selective activation of Gs. However, the nature of amino acids constituting the COOH-terminal region of intracellular loop 2 is critical as it may affect conformation of this domain. Future mutagenesis studies directed at examining sequence substitutions and point mutations made in combination may more precisely define structural requirements of A2aAR-Gs coupling.


FOOTNOTES

*   This study was supported by National Heart, Lung, and Blood Institute SCOR Grant P50HL54314 in Ischemic Disease. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1    The abbreviations used are: AR, adenosine receptor; CHO, Chinese hamster ovary; G protein: guanine nucleotide-binding protein; GPCR, G protein-coupled receptor; Gpp(NH)p, guanylyl imidodiphosphate; WT, wild-type; NECA, 5'-N-ethylcarboxamidoadenosine; R-PIA, (-)-R-N6-(phenylisopropyl)adenosine; ZM241385, 4-(2-[7-amino-2-{2-furyl} {1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol.

Acknowledgments

Much appreciation is given to Dr. Gary L. Stiles for valuable discussions and providing use of laboratory equipment. Many helpful discussions with Dr. Tim Palmer are also appreciated.


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