©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Domains of the Human A Adenosine Receptor That Are Important for Binding Receptor Subtype-selective Ligands Using Chimeric A/A Adenosine Receptors (*)

(Received for publication, June 2, 1995)

Scott A. Rivkees (§) Mark E. Lasbury Hemang Barbhaiya

From the Herman B Wells Center for Pediatric Research, Pediatric Endocrine Unit, James Whitcomb Riley Hospital and the Department of Biochemistry and Molecular Biology and the Program of Neurobiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To provide new insights into the regions of the human A(1) adenosine receptor (A(1)AR) involved in ligand binding, a series of chimeric human A(1) and rat A adenosine receptors (A(1)/A) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the AAR agonist [^3H]CGS-21680, the A(1)AR agonist [^3H]2-chloro-N^6-cyclopentyladenosine (CCPA), and the A(1)AR antagonist [^3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the AAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A(1)AR (A(1)TM1/A), [^3H]CGS-21680 and [^3H]CCPA binding was detectable. When an A(1)TM1-2/A construct was studied, [^3H]CGS-21680 binding was lost and [^3H] DPCPX binding appeared. Saturation studies using [^3H]CCPA revealed that the A(1)TM1/A construct had low affinity. However, with the subsequent addition of A(1)AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [^3H]DPCPX also revealed that the TMs 1-4 of the A(1)AR conferred wild-type receptor affinity. When the ligand binding properties of A(1)TM1-4/A, A(1)TM1-6/A, and wild type A(1)AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A(1)ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A(1)AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.


INTRODUCTION

The nucleoside adenosine exerts its potent biological effects via specific receptors that belong to the superfamily of G protein-coupled receptors(1) . To date, four adenosine receptor subtypes have been cloned(2, 3, 4, 5, 6, 7, 8, 9, 10) . These include the A(1) and A(3) receptors that inhibit adenylyl cyclase and the A and A receptors that stimulate adenylyl cyclase(1, 6) . Each adenosine receptor subtype has unique ligand binding properties and distinct patterns of tissue expression(11, 12) .

Recently, there has been considerable interest in A(1) adenosine receptors (A(1)ARs) (^1)for several reasons. In the brain, A(1)ARs are widely distributed (5, 7, 13) and are believed to tonically inhibit neuronal activity(13) . A(1)ARs may influence the pathogenesis of seizure disorders (14) and protect neurons against excitotoxic damage (13) . In the heart, A(1)AR activation influences the cardiac conduction system and may protect against ischemic damage(15, 16, 17) .

The A(1)AR was initially cloned from the thyroid gland of dogs (2, 4) and later isolated from several other species(5, 7, 18, 19, 20, 21) . In each species, the A(1)AR cDNA encodes a protein of 326 amino acids that contains seven transmembrane spanning regions. A(1)ARs have distinct ligand binding properties as compared to other adenosine receptor subtypes(12, 22, 23) . A(1)ARs have high affinity for N^6-substituted adenosine analogues and have high affinity for C^8-substituted xanthine antagonists (11, 12) . Several highly selective A(1)AR compounds are available including the agonist 2-chloro-N^6-cyclopentyladenosine (CCPA) and the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(24, 25) .

When compared to AARs, the amino terminus of the A(1)ARs is longer than that of AARs, while the carboxyl terminus of the A(1)ARs is shorter than that of the AAR(5, 7, 8, 18, 19, 20, 21) . At the amino acid level, A(1) and AARs are generally 50% identical over all and 60% identical within the transmembrane domains(5, 7, 8, 18, 19, 20, 21) . In contrast to A(1)ARs, AARs have high affinity for 5`-substituted adenosine agonists and low affinity for N^6-substituted compounds(11, 12) . Highly selective AAR agonists are available and include the compound CGS-21680(26) .

Presently our understanding of how ligands bind to A(1)ARs is at early stages. Similar to how other G protein-coupled receptors bind small molecule ligands(27) , it has been postulated that ligand-binding sites of A(1)ARs are within transmembrane regions(28, 29, 30) . Using computer modeling, Ijzerman and colleagues (28, 29, 30) have suggested that the adenosine ribose group interacts with transmembrane (TM) domain 5, whereas the N^6 position interacts with TM7.

Site-directed mutagenesis also has been applied in efforts to identify sites involved in ligand binding. Olah and co-workers (31) have suggested that His and His are important for ligand binding. Townsend-Nicholson and Schofeld (32) and Tucker et al.(33) have reported that Thr may interact with the 5`-ribose position. Studies of chimeric A(1)/A(3) adenosine receptors by Olah and co-workers (34) have suggested the presence of a region in TM5 that influences the affinity for 5`-substituted compounds. Using chimeric A(1)/A(3) receptors it has also been suggested that the second extracellular loop may influence ligand binding(35) . Most recently, Tucker and colleagues (33) have suggested that the A(1)AR 270 amino acid interacts with the N^6 position of adenosine or the C^8 position of xanthines.

Although the above reports have provided important insights into adenosine-receptor interactions, we still do not have a comprehensive understanding of how adenosine receptors bind receptor subtype-selective ligands. For other G protein-coupled receptors, chimeric receptor studies have provided important insights about the transmembrane domains that are involved in ligand binding(36, 37, 38, 39) . By taking advantage of the distinct ligand binding properties of A(1) and AARs and the availability of highly selective A(1) and AAR radioligands, we have used a broad series of A(1)/AAR chimeric receptors to identify receptor regions involved in A(1)AR agonist and antagonist binding. We now provide evidence that the first four transmembrane domains of the human A(1)AR bind to and confer specificity for A(1)AR-selective ligands.


EXPERIMENTAL PROCEDURES

cDNAs

The cDNA encoding the full-length rat AAR was provided by Dr. J. S. Fink (Boston, MA)(8) . The cDNA encoding the full-length human A(1)AR was provided by Dr. S. M. Reppert (Boston, MA)(40) .

Generation of Chimeric Receptors

Chimeric receptors were made by the polymerase chain reaction (PCR) overlap-extension method of Ho et al.(41) using the primer pairs shown in Table 1. Common amino acids within extra- or intracellular loops were identified as sites of overlaps. Primers were designed such that there was no change in the reading frame. Oligonucleotides were synthesized using an Applied Biosystems Oligonucleotide Synthesizer (Foster City, CA).



To generate A(1)/A chimeric receptors, oligonucleotide primer pairs (primers A and B) were designed to generate a 5` fragment of the A(1)AR. Another set of oligonucleotide primer pairs (primers C and D) were made to generate a 3` fragment of the AAR receptor. To generate A/A(1) chimeric constructs, oligonucleotide primer pairs (A and B) were constructed to generate a 5` fragment of the AAR, and oligonucleotide primer pairs were designed to generate a 3` fragment of the A(1)AR receptor (C and D). Receptor fragments were generated using >1 µg of DNA as the substrate for PCR reactions, and PCR reactions were performed using the Gene Amp Kit (Perkin-Elmer) reagents. PCR was generally performed using 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. PCR products were then separated on a 1% agarose gel and eluted using NA45 paper (Schleicher and Schuell). Receptor fragments (A-B and C-D) were then combined in third PCR reaction to generate a full-length, chimeric receptor in a fusion reaction using flanking primers (A and D). To generate truncated A(1)ARs, a 5` fragment of the A(1)AR was generated using the A(1)AR A primer and a D primer that included a stop codon.

Flanking PCR primers contained HindIII (A primers) or XbaI (D primers) restriction endonuclease sites at the ends. After fusion reactions, PCR products were digested with HindIII and XbaI and were subcloned into the mammalian expression vector pcDNA1, (In Vitrogen, San Diego, CA). Chimeric receptors were then sequenced using Sequenase Version 2 (Amersham Corp.).

Acute Transfections

Chimeric cDNA expression was characterized using COS 6 M or COS 7 cells. COS cells were grown as monolayers in Dulbecco's minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml), in 5% CO(2) at 37 °C. Cells were acutely transfected using the DEAE-dextran method(7, 42) . 10-cm plates were individually transfected with 5-10 µg of DNA or were sham transfected. At 48 h after transfection, cells were tested by radioreceptor assay. Sham transfected COS 6 M and COS 7 cells did not bind any of the radioligands used (see below). Under these binding conditions, there was little evidence of receptor-G protein coupling, (^2)similar to as previously reported by others(33) .

Radioreceptor Assays

Radioligand binding studies were performed using intact cells similar to as described previously(7) . Medium was removed, and the plates were washed with phosphate-buffered saline. Phosphate-buffered saline was added to each dish, and the cells were mechanically harvested. Cells were pelleted (4000 revolutions/min, 10 min, 4 °C) and resuspended in binding buffer consisting of phosphate-buffered saline with 10 mM MgCl(2) and 5 units/ml of adenosine deaminase (Boehringer Mannheim) at 37 °C for 45 min with shaking. Cells were then pelleted and resuspended to a final concentration of 0.5 mg of protein/ml with fresh binding buffer.

For binding reactions, the radioligands used were [^3H]CCPA (DuPont NEN; specific activity 33 Ci/mmol), [^3H]DPCPX (DuPont NEN; specific activity 100 Ci/mmol), and [^3H]CGS-21680 (DuPont NEN; specific activity 30 Ci/mmol). Generally, 50 µl cells (75-150 µg of protein) were added to the radioligand in a final volume of 150 µl in Multiscreen plates (GF/B 1.2-µm glass fiber filters; Millipore; Bedford, MA). Reactions were incubated at 21 °C for 1 h with shaking. Bound radioactivity was separated from free by vacuum filtration and determined by liquid scintillation counting. All determinations were performed in quadruplicate. Protein concentration was measured using bicinconinic acid (Pierce) with bovine serum albumin standards.

Statistical Analysis

Saturation and competition binding data were analyzed by computer using an iterative nonlinear regression program(43) . Comparisons among multiple groups were performed by one-way ANOVA, with post test comparison among specific treatment groups performed by the Bonferroni method. The InStat statistics program (GraphPad; San Diego, CA) was used for statistical computations.


RESULTS

To define the regions of the A(1)AR involved in agonist and antagonist binding, a series of chimeric A(1) and A adenosine receptors were generated. These receptor subtypes were chosen because each receptor subtype has distinct ligand binding characteristics (11, 12, 22) and highly selective radioligands for each receptor subtype are available(24, 25, 26) .

We first generated a series of chimeric A(1)/A receptors in which 5` regions of the AAR were replaced by progressively larger 5` regions of the A(1)AR (Fig. 1). Receptor splicing took place at extra- or intracellular loops, and transmembrane domains were transferred intact in the proper reading frame. To identify receptor regions involved in ligand binding, chimeric constructs were initially screened using fixed concentrations of the AAR agonist [^3H]CGS-21680 (20 nM), the A(1)AR agonist [^3H]CCPA (5 nM), or the A(1)AR antagonist [^3H]DPCPX (5 nM) in four to six separate studies. COS 6M and COS 7 cells were used for these studies. All subsequent studies involved COS 7 cells. At the above concentrations of radioligands, there was no specific binding to non-transfected or sham transfected COS 6M or COS 7 cells.


Figure 1: Schematic representation of chimeric A(1)/A, A/A(1) constructs and truncated A(1)ARs. Beside each receptor is a representation of binding studies. cDNAs were acutely expressed in COS cells. Binding was assessed 48 h later by radioreceptor assay using the A(1) agonist [^3H]CCPA (5 nM), the A(1) antagonist [^3H]DPCPX (DPX, 5 nM]), and the A agonist [^3H]CGS-21680 (20 nM]). Specific binding is shown; -, no binding; + specific binding present at >50 fmol/mg of protein. Data shown are representative of four to six separate studies/construct. All samples were tested in quadruplicate in each separate study.



Using [^3H]CGS-21680, we found that when the region extending from the amino terminus to after the end of TM1 of the AAR receptor was replaced by that of the A(1)AR (CR1), low level binding (25-50 fmol/mg of protein) was present (Fig. 1). When both TMs 1 and 2 of the AAR were replaced by the corresponding A(1)AR regions (CR2), [^3H]CGS-21680 binding was no longer detectable showing that TMs 1 and 2 of the AAR are important for [^3H]CGS-21680 binding.

Using [^3H]CCPA, no binding was detected on cells that expressed the wild-type AAR. However, with the substitution of AAR TM1 by that of the A(1)AR (CR1), low level [^3H]CCPA specific binding was apparent (25-55 fmol/mg of protein). With the successive addition of TMs 2 and 3 (CR 2 and CR3), low level binding continued to be detected (30-75 fmol/mg of protein). With the subsequent additions of TMs 4, 5, 6, and 7 (CRs 4-7), [^3H]CCPA binding was present at considerably higher levels (75-250 fmol/mg of protein) than that observed with the CR1, CR2, or CR3.

Next, [^3H]DPCPX was used to examine antagonist binding to the chimeric receptor constructs (Fig. 1). When CR1 was examined, no specific binding was detectable. With the successive addition of A(1)AR TMs 2 and 3 (CRs 2 and 3), specific low level binding was detectable (50-100 fmol/mg of protein). With the subsequent additions of TMs 4, 5, 6, and 7 (CRs 4-7), the amount of [^3H]DPCPX binding increased markedly (300-1500 fmol/mg of protein).

Because the above studies suggested that TMs 1 and 2 were important for ligand binding, we next tested another series of chimeric receptors in which TMs 1 and 2 of the A(1)AR were replaced by corresponding domains of the AAR. When A(1)AR TM1 alone was replaced (CR8), [^3H]CGS-21680 (50-75 fmol/mg of protein) and [^3H]DPCPX (75-125 fmol/mg of protein) binding was apparent, but [^3H]CCPA binding was not observed. When both TMs 1 and 2 of the A(1)AR were replaced by TMs 1 and 2 of the AAR (CR9), binding was not detected by any of the radioligands used.

Saturation studies were next performed to determine if the differences in binding levels observed using fixed doses of radioligands reflected differences in chimeric receptor affinity (K) or differences in receptor expression (B(max)). Although the CR1 construct bound [^3H]CCPA, ligand affinity was low (Table 2; p < 0.01, ANOVA). With the addition of A(1)AR TMs 2 and 3 (CRs 2 and 3), receptor affinity increased, but remained less than that observed for the wild-type A(1)AR (p < 0.05, ANOVA). With A(1)AR TM4 (CR4), the affinity for [^3H]CCPA increased further and approximated that of the wild-type A(1)AR (p > 0.05, ANOVA).



Next, saturation studies were performed using [^3H]DPCPX (Table 3). When TMs 1-2 of the A(1)AR were present (CR2), K values reflected very low affinity (p < 0.01 versus wild type A(1)AR, ANOVA). However, when A(1)AR TM3 was added (CR3), the affinity for [^3H]DPCPX increased, yet remained less than that observed for the wild-type A(1)AR (p < 0.05, ANOVA). With the addition of A(1)AR TM4 (CR4), receptor affinity increased 7-fold and was actually greater than that observed for the wild-type A(1)AR. Receptor expression also increased with the addition of A(1)AR TM4. With the progressive substitution of A(1)AR TMs 5, 6, and 7 (CRs 5-7), chimeric constructs had high affinity and high levels of expression.



Because the CR4 construct was expressed at high levels, and A(1)AR TMs 1-4 contain regions that confer high affinity for [^3H]CCPA and [^3H]DPCPX, we next tested if A(1)AR TMs 1-4 alone were sufficient for ligand binding. Two different truncated A(1)AR receptors (T1 and T2) that contained TMs 1-4 and portions of the second extracellular loop were generated and examined in parallel with the wild-type A(1)AR. Although we could detect [^3H]DPCPX binding to the wild-type A(1)AR after acute expression, we could not detect specific binding to either of the truncated receptors. Thus, although TMs 1-4 contain determinants for A(1)AR agonist and antagonist binding, TMs 5-7 may also be required for ligand binding.

After showing that TMs 1-4 of the A(1)AR alone are not sufficient for ligand binding, we next tested if A(1)AR TMs 1-4 were sufficient for conferring specificity for A(1)-selective ligands. Competition studies were therefore performed on chimeric constructs using 10 compounds selected for their ability to distinguish different adenosine receptor subtypes(11, 12) . These compounds included A(1)- and A-selective agonists and antagonists as well as non-selective adenosine agonists and antagonists (see Fig. 2and Table 4). Because CR4, CR6, and the wild-type A(1)AR had comparable affinity for [^3H]DPCPX and were expressed at high levels, competition studies were performed using these three constructs. Each construct was tested two or more times in side-by-side experiments using each drug.


Figure 2: Competition of adenosinergic compounds for [^3H]DPCPX [2 nM] binding to the wild-type A(1)AR and the A(1)TM1-4/A (CR4) and A(1)TM1-6/A (CR6) constructs. Binding is expressed as a percentage of total binding. The data shown are representative of three separate studies. bullet, DPCPX; , CPA; black square, NECA; , CGS-21680. The abbreviations used are as in Table 3.





Competition studies revealed the expected rank order of potency of an A(1)AR for each construct. We found that CR4, CR6, and the wild-type A(1)AR each had low affinity for A-selective compounds (CGS-21680, CSC, and DMPA) and had high affinity for A(1)-selective compounds (CPA, CGS-15943, and DPCPX). The K values for each construct also were similar to values previously reported for A(1)ARs(12) . For the compounds CPA, DPCPX, NECA, and CGS-21680, which were tested in three or four separate side-by-side studies, no significant differences among K values were found (p > 0.05; ANOVA). Thus, TMs 1-4 of the A(1)AR are sufficient to confer specificity for A(1)-selective compounds.

We next attempted to determine the relative importance of the individual TM domains in the amino half of the A(1)AR in conferring specificity for A(1)-selective ligands. Due to the low level of expression of the CR1, CR2, and CR3 constructs, rigorous competition studies were difficult to perform. Because saturation studies revealed that the addition of TMs 2 and 3 resulted in increased affinity for [^3H]CCPA or [^3H]DPCPX, we tested the relative importance of these TM domains. Chimeric receptors in which TM 2 or TM 3 of the A(1)AR was replaced by the corresponding TM domains of the AAR were therefore generated (CR10 and CR11). Although we could detect [^3H] DPCPX binding to the wild-type A(1)AR after acute expression, we could not detect specific binding to either of these TM-substituted A(1)ARs. These observations therefore suggest that both TMs 2 and 3 contain residues that are important for ligand binding.


DISCUSSION

Our observations provide new insights into the potential sites of ligand-receptor interactions for the human A(1)AR. Using a series of chimeric A(1)/A receptors, we have found that the region of the A(1)AR from the amino terminus to the end of the fourth transmembrane domain is important for ligand binding and conferring specificity for A(1)-selective agonists and antagonists.

Using the highly selective A(1)AR agonist [^3H]CCPA, we found that by replacing the region from the amino terminus to after the end of TM1 of the AAR with that of the A(1)AR (CR1), specific binding appeared. Supporting the notion that TM1 of the A(1)AR contains an agonist binding site, [^3H]CCPA binding was lost when TM1 of the A(1)AR was replaced by that of the AAR (CR8). Although the CR1 construct bound [^3H]CCPA, the affinity for this ligand was low (K = 26.4 nMversus 0.7 nM for wild-type A(1)AR). However, with the addition of A(1)AR TM2 (CR2), the affinity for [^3H]CCPA increased greatly (K = 4.5 nM). When A(1)AR TMs 3 and 4 (CR4) were added, the affinity for [^3H]CCPA increased again (K = 1.9 nM) and approximated that of the wild-type A(1)AR. CRs 6 and 7 also had K values close to that of the wild-type A(1)AR. CR5, in contrast, had lower affinity than the CRs 4, 6, and 7. Since this receptor was generated by a splice in the third cytoplasmic loop, it is possible that a conformational change in this construct resulted in reduced affinity for [^3H]CCPA.

Using [^3H]DPCPX, we also found that TMs 1-4 of the A(1)AR receptor were important for antagonist specificity. When the region from the amino terminus to the end of TM2 of the AAR was replaced by that of the A(1)AR, [^3H]DPCPX binding became detectable although this construct had low affinity for the antagonist (K = 11.1 nMversus 1.5 nM for the wild-type receptor). With the addition of A(1)AR TM3 (CR3), the affinity for [^3H]DPCPX increased 3-fold (K = 3.1). With the subsequent addition of A(1)AR TM4 (CR4; K = 0.5 nM), receptor affinity actually exceeded that of the wild-type A(1)AR. These observations are consistent with the findings of [^3H]CCPA binding studies and support a role for TMs 1-4 in binding A(1)-selective ligands.

Competition studies also showed that A(1)AR TMs 1-4 conferred ligand specificity. Using 10 drugs that distinguish among the different adenosine receptor subtypes, we failed to detect any major differences in the pharmacologic profiles of CR4, CR6, and the wild-type A(1)AR.

To better define the contributions of the domains within TMs 1-4, we also attempted competition studies using CR 1-3 constructs. However, the low level of expression of these constructs made rigorous competition studies difficult to perform. Studies using fixed doses of radioligands, however, revealed the presence of important binding sites for A(1)-selective ligands in TMs 1 and 2. When TMs 1-2 of the AAR were replaced by those of the A(1)AR (CR2), [^3H]CGS-21680 binding was lost suggesting that AAR agonist specificity and binding involves TMs 1-2. Furthermore, when TMs 1, 2, and 3 of the A(1)AR were replaced by those of the AAR, neither [^3H]CCPA nor [^3H] DPCPX binding was observed.

To date, structure function studies of A(1)ARs have largely focused on sites within TMs 5-7. Studies of chimeric A(1)/A(3) adenosine receptor studies have suggested that 5` substituted compounds interact with TM5(34) . Site-directed mutagenesis studies have suggested that His in TM6 may play a role in binding antagonists(31) . Within TM7, differences in the amino acid at position 270 among cows (Ile) and dogs (Met) are believed to account for differences in affinity for A(1)-selective drugs among these species(33) . It has been suggested that this position interacts with the N^6 position of adenosine and the C^8 position of xanthines(33) . Two separate studies (32, 33) have also suggested that the amino acid at position 277 interacts with the 5`-ribose position. When His of bovine A(1)AR is converted to Ala, poor affinity for agonists and antagonists is demonstrated, although expression of this construct is not known(31) .

Although the aforementioned reports suggest the importance of sites in TMs 5-7 in ligand binding, it is unlikely that they will distinguish among A(1)- and A-selective ligands. Comparison of the primary amino acids sequence among A(1) and AARs reveals that the corresponding amino acids of the A(1)AR at positions 256, 270, 277, and 278 are identical in AARs(8) . Furthermore, our competition studies show that TMs 1-4 are sufficient to confer the ligand binding characteristics of an A(1)AR.

We do, however, agree with the concept that the carboxyl half of the receptor is important for ligand binding. When we tested two A(1)ARs truncated after TM4, we did not detect either [^3H]DPCPX or [^3H]CCPA binding. While it is possible that these constructs did not express, the removal of binding sites in TMs 5-7 may have resulted in the loss of binding.

Based on studies of other small molecule receptors(27) , we anticipate that adenosine- and xanthine-binding sites will reside within the transmembrane domains of A(1)ARs. However, because our constructs contained parts of extra- and intracellular loops in addition to TM domains, it is possible that non-transmembrane regions may influence ligand binding. Furthermore, chimeric A(1)/A(3) receptors have suggested that the second extracellular loop between TMs 4 and 5 may influence receptor binding (35) . Thus, future studies involving smaller receptor substitutions will be needed to examine the relative contributions of transmembrane and non-transmembrane domains on ligand binding specificity.

Currently, the orientation of adenosine with different receptor regions is not known. Site-directed mutagenesis studies of amino acid 270 have suggested that this site interacts with the N^6 position of agonists and the C^8 position of xanthine antagonists(33) . It also has been suggested that the Thr position in TM 7 interacts with the 5`-ribose of NECA(33) . Considering the expected close proximity of these sites in TM7 (28) and the size and confirmation of adenosine analogs(28, 44) , it is unlikely that both the N^6 and the 5`-ribose positions will interact with this region. Pharmacology studies show that the N^6-adenosine and C^8-xanthine positions are important for conferring receptor subtype ligand binding specificity. However, because the 270 and 277 amino acids are the same in A(1) and AARs, it is unlikely that these sites are the primary determinants of the N^6 or C^8 binding.

In contrast to previously proposed models of adenosine-receptor interactions(28, 29, 30, 33) , based on our findings we hypothesize that the N^6 and C^8 positions interact with sites within the first four transmembrane domains of the A(1)AR and that the 5`-ribose position interacts with TM7. If the 5`-ribose position is oriented toward TM7, as previously suggested (33) , this would also allow the ribose group to interact with TMs 1 and 2, if TMs 1 and 7 are juxtaposed. The observation that [^3H]CCPA binds to the CR1 construct, whereas [^3H]DPCPX does not, supports this hypothesis since CCPA contains a ribose group whereas DPCPX does not. If the ribose group binds to a TM7/TM1-2 region, the N^6 group could then interact with TMs 2-4. Because the C^8 position xanthines are believed to interact at sites to the N^6 adenosine position(12) , we would also expect xanthines to interact with this region. The N^3 or N^9 positions of adenosine and the N^1 or N^2 positions of xanthines, which are important for ligand binding but are not important for receptor subtype selectivity(12) , could then interact with sites in TMs 5-6.

Overall, our findings suggest that specificity for A(1)AR subtype-selective ligands is conferred by first four TMs of the A(1)AR. With the background information provided by these studies, we anticipate that individual transmembrane and single residue substitutions in the amino half of the A(1)AR will lead to the identification of the sites that confer specificity for receptor subtype-specific ligands.


FOOTNOTES

*
This work was supported by a Grant-in-aid from the American Heart Association, the James Whitcomb Riley Memorial Association, and National Institutes of Health Grant RO1NS326224. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and X68485[GenBank].

§
To whom correspondence should be addressed: Riley Hospital, Rm. 5984, 702 Barnhill Dr., Indianapolis, IN 46202. Fax: 317-274-5378; srivkee{at}Indyvax.IUPUI.edu.

(^1)
The abbreviations used are: AR, adenosine receptor; TM, transmembrane; PCR, polymerase chain reaction; CCPA, 2-chloro-N^6-cyclopentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; 3`, 3`-deoxyadenosine; 5MT, 5`-deoxy-5`-methyl-thioadenosine; CSC, 8-(3-chlorostylryl)caffeine; DMPA, N^6-[2-(3,5-dimetoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine; NECA, 5`-N-ethylcarboxamidoadenosine: ANOVA, one way analysis of variance.

(^2)
S. A. Rivkees, unpublished results.


ACKNOWLEDGEMENTS

We are indebted to Dr. R. Frederick Bruns for valuable discussions and suggestions. We thank Rebecca McClain for expert technical assistance and Dr. Mark Kelley for assistance with PCR studies.


REFERENCES

  1. Londos, C., Cooper, D. M., and Wolff, J. (1980) Proc. Natl. Acad. Sci.U. S. A 77,2551-2554 [Abstract]
  2. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande Maenhaut, C., Dumont, J. E., and Vassart, G. (1989) Science 244,569-572 [Medline] [Order article via Infotrieve]
  3. Maenhaut, C., Van Sande, J., Libert, F., Abramowicz, M., Parmentier, M., Vanderhaegen, J. J., Dumont, J. E., Vassart, G., and Schiffmann, S. (1990) Biochem. Biophys. Res. Commun. 173,1169-1178 [Medline] [Order article via Infotrieve]
  4. Libert, F., Schiffmann, S. N., Lefort, A., Parmentier, M., Gerard, C., Dumont, J. E., Vanderhaeghen, J. J., and Vassart, G. (1991) EMBO J. 10,1677-1682 [Abstract]
  5. Mahan, L. C., McVittie, L. D., Smyk-Randall, E. M., Nakata, H., Monsma, F. J., Jr., Gerfen, C. R., and Sibley, D. R. (1991) Mol. Pharmacol. 40,1-7 [Abstract]
  6. Stiles, G. L. (1992) J. Biol. Chem. 267,6451-6454 [Abstract/Free Full Text]
  7. Reppert, S. M., Weaver, D. R., Stehle, J. H., and Rivkees, S. A. (1991) Mol. Endocrinol. 5,1037-1048 [Abstract]
  8. Fink, J. S., Weaver, D. R., Rivkees, S. A., Peterfreund, R. A., Pollack, A. E., Adler, E. M., and Reppert, S. M. (1992) Brain Res. Mol. Brain Res. 14,186-195 [Medline] [Order article via Infotrieve]
  9. Stehle, J. H., Rivkees, S. A., Lee, J. J., Weaver, D. R., Deeds, J. D., and Reppert, S. M. (1992) Mol. Endocrinol. 6,384-393 [Abstract]
  10. Zhou, Q. Y., Li, C., Olah, M. E., Johnson, R. A., Stiles, G. L., and Civelli, O. (1992) Proc. Natl. Acad. Sci.U. S. A. 89,7432-7436 [Abstract]
  11. Bruns, R. F. (1990) Ann. N. Y. Acad. Sci. 603,211-25 [Medline] [Order article via Infotrieve]
  12. Trivedi, B. K., Bridges A. J., Bruns, R. F. (1990) in Adenosine and Adenosine Receptors (Williams, M. C.) pp. 57-105 Humana Press, Clifton, NJ
  13. Jarvis, M. F., Williams, M. (1990) in Adenosine and Adenosine Receptors (Williams, M. C., ed) pp. 423-474, Humana, Clifton, NJ
  14. Young, D., and Dragunow, M. (1994) Neuroscience 58,245-261 [Medline] [Order article via Infotrieve]
  15. Tucker, A. L., and Linden, J. (1993) Cardiovasc. Res. 27,62-67 [Medline] [Order article via Infotrieve]
  16. Sparks, H. V., Jr., and Bardenheuer, H. (1986) Circulation Res. 58,193-201 [Abstract]
  17. Hori, M., and Kitakaze, M. (1991) Hypertension 18,565-574 [Abstract]
  18. Olah, M. E., Ren, H., Ostrowski, J., Jacobson, K. A., and Stiles, G. L. (1992) J. Biol. Chem. 267,10764-10770 [Abstract/Free Full Text]
  19. Tucker, A. L., Linden, J., Robeva, A. S., D'Angelo, D. D., and Lynch, K. R. (1992) FEBS Lett. 297,107-111 [CrossRef][Medline] [Order article via Infotrieve]
  20. Libert, F., Van Sande, J., Lefort, A., Czernilofsky, A., Dumont, J. E., Vassart, G., Ensinger, H. A., and Mendla, K. D. (1992) Biochem. Biophys. Res. Commun. 187,919-926 [Medline] [Order article via Infotrieve]
  21. Townsend-Nicholson, A., and Shine, J. (1992) Brain Res. Mol. Brain Res. 16,365-370 [Medline] [Order article via Infotrieve]
  22. Linden, J. (1991) FASEB J. 5,2668-2676 [Abstract/Free Full Text]
  23. Koch, W. J., Inglese, J., Stone, W. C., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268,8256-8260 [Abstract/Free Full Text]
  24. Lohse, M. J., Klotz, K. N., Schwabe, U., Cristalli, G., Vittori, S., and Grifantini, M. (1988) Naunyn-Schmied. Arch. Pharmacol. 337,687-689 [Medline] [Order article via Infotrieve]
  25. Lohse, M. J., Klotz, K. N., Lindenborn-Fotinos, J., Reddington, M., Schwabe, U., and Olsson, R. A. (1987) Naunyn-Schmied. Arch. Pharmacol. 336,204-210 [Medline] [Order article via Infotrieve]
  26. Jarvis, M. F., Schulz, R., Hutchison, A. J., Do, U. H., Sills, M. A., and Williams, M. (1989) J. Pharmacol. Exp. Ther. 251,888-893 [Abstract]
  27. Hibert, M. F., Trumpp-Kallmeyer, S., Bruinvels, A., and Hoflack, J. (1991) Mol. Pharmacol. 40,8-15 [Abstract]
  28. Ijzerman, A. P., van Galen, P. J., and Jacobson, K. A. (1992) Drug Design Disc. 9,49-67 [Medline] [Order article via Infotrieve]
  29. van der Wenden, E. M., Ijzerman, A. P., and Soudijn, W. (1992) J. Med. Chem. 35,629-635 [Medline] [Order article via Infotrieve]
  30. van Galen, P. J., van Vlijmen, H. W., Ijzerman, A. P., and Soudijn, W. (1990) J. Med. Chem. 33,1708-1713 [Medline] [Order article via Infotrieve]
  31. Olah, M. E., Ren, H., Ostrowski, J., Jacobson, K. A., and Stiles, G. L. (1992) J. Biol. Chem. 267,10764-10770 [Abstract/Free Full Text]
  32. Townsend-Nicholson, A., and Schofield, P. R. (1994) J. Biol. Chem. 269,2373-2376 [Abstract/Free Full Text]
  33. Tucker, A. L., Robeva, A. S., Taylor, H. E., Holeton, D., Bockner, M., Lynch, K. R., and Linden, J. (1994) J. Biol. Chem. 269,27900-27906 [Abstract/Free Full Text]
  34. Olah, M. E., Jacobson, K. A., and Stiles, G. L. (1994) J. Biol. Chem. 269,18016-18020 [Abstract/Free Full Text]
  35. Olah, M. E., Jacobson, K. A., and Stiles, G. L. (1994) J. Biol. Chem. 269,24692-24698 [Abstract/Free Full Text]
  36. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., and Lefkowitz, R. J. (1988) Science 240,1310-1316 [Medline] [Order article via Infotrieve]
  37. Wong, S. K., Parker, E. M., and Ross, E. M. (1990) J. Biol. Chem. 265,6219-6224 [Abstract/Free Full Text]
  38. MacKenzie, R. G., Steffey, M. E., Manelli, A. M., Pollock, N. J., and Frail, D. E. (1993) FEBS Lett. 323,59-62 [CrossRef][Medline] [Order article via Infotrieve]
  39. Huang, R. R., Yu, H., Strader, C. D., and Fong, T. M. (1994) Mol. Pharmacol. 45,690-695 [Abstract]
  40. Rivkees, S. A., Lasbury, M. E., Stiles, G. S., Henergariu, O., and Vance, G. (1995) Endocrine , in press
  41. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., Pease, L. R. (1989) Gene (Amst.) 77,51-59 [CrossRef][Medline] [Order article via Infotrieve]
  42. Cullen, B. R. (1987) Methods Enzymol. 152,684-704 [Medline] [Order article via Infotrieve]
  43. McPherson, G. A. (1985) J. Pharmacol. Methods 14,213-228 [CrossRef][Medline] [Order article via Infotrieve]
  44. Bruns, R. F. (1980) Can. J. Physiol. Pharmacol. 58,673-691 [Medline] [Order article via Infotrieve]

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