Cloning, Expression, and Characterization of the Unique Bovine A1 Adenosine Receptor STUDIES ON THE LIGAND BINDING SITE BY SITE-DIRECTED MUTAGENESIS*

The bovine brain A1 adenosine receptor (AIAR) is distinct from other AIARs in that it displays the unique agonist potency series of NB-R-phenylisopropyladeno- sine (R-PIA) > A@-S-phenylisopropyladenosine (S-PIA) > 6’-N-ethylcarboxamidoadenosine and has a 6- 10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,670 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G pro-tein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 6.7 kb with a tissue distribution consistent with AIAR binding. Subcloning of the cDNA in a pCMVS expression vector with subsequent transfec- tion into both COS7 and Chinese hamster ovary cells revealed a fully functional AIAR which could inhibit adenylylcyclase and retained the unique pharmaco- logic properties of the bovine brain AIAR. The was in each of

The bovine brain A1 adenosine receptor (AIAR) is distinct from other AIARs in that it displays the unique agonist potency series of NB-R-phenylisopropyladenosine (R-PIA) > A@-S-phenylisopropyladenosine (S-PIA) > 6'-N-ethylcarboxamidoadenosine and has a 6-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,670 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 6.7 kb with a tissue distribution consistent with AIAR binding. Subcloning of the cDNA in a pCMVS expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional AIAR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain AIAR.
The AIAR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by >90%. In contrast, mutation of His-261 to Leu-26 1 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur. 11 To whom correspondence should be addressed Duke University A, adenosine receptors are integral membrane proteins which bind extracellular adenosine and then initiate a transmembrane signal via G proteins' to modulate the activity of a number of effector systems including adenylylcyclase (inhibition), K+ channels (opening), and Ca2+ channels (close) (1-3). These receptors are found in a diverse selection of cell types and tissues and are known to be dynamically regulated by a variety of pathophysiological conditions including agonist-induced desensitization (4, 5), antagonist-induced sensitization (6), and in altered thyroid states (7). Subpopulations of Al adenosine receptors have been postulated to exist based on distinct pharmacological profiles and on absolute agonist and antagonist affinities (8)(9)(10)(11).
One such subtype is the bovine AIAR which differs from the classical AIAR found in most tissues and species in that it binds antagonists such as XAC with subnanomolar affinities and has a unique agonist potency series of R-PIA > S-PIA > NECA (8)(9)(10)(11). This contrasts with the typical AIAR which has 5-10-fold lower affinities for antagonists and agonists and a potency series of R-PIA > NECA > S-PIA (1, 2).
We have previously documented that these unique properties of the bovine brain AIAR are displayed by the membranebound, solubilized, and purified receptor suggesting these characteristics are intrinsic to the receptor protein itself and not the environment in which it resides (10,11). AIARs of canine thyroid (12) and rat brain (13,14) have recently been cloned. For both clones, the expressed receptor displays the pharmacological properties described above for the classical Biochemical studies have documented that the AIAR is a glycoprotein which contains complex-type carbohydrate chains (10,15), likely contains disulfide bonds (16,17), and contains histidine residues in the ligand binding site (17). Klotz et al. (17) have suggested that there are at least 2 histidines in the receptor binding pocket and that agonists and antagonists interact with distinct transmembrane domain recognition sites.
In this paper, we report the cloning, expression, and characterization of the unique bovine brain AIAR, and through site-directed mutagenesis studies begin to probe the important amino acids in the receptor binding pocket.
Library Screening-A 60-mer antisense oligonucleotide based on a region (amino acids 201-221) of the putative third intracellular loop of the protein encoded by the canine RDC7 clone (19) was synthesized and labeled with [Y-~'P]ATP on the 5'-hydroxyl end by T4 polynucleotide kinase. This probe was used to screen approximately 1.1 X IO6 recombinants from a size-selected (2-4-kb) bovine brain cDNA library (XZAP) generously provided by Richard Dixon (Merck, Sharp & Dohme Research Laboratories) and Robert Lefkowitz (Duke University). Duplicate nylon filters (Biotrans, ICN) were used for phage lifts and were prehybridized in 6 X TEN (1 X TEN = 15 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8.3)/6 X Denhardt's solution, 0.1% SDS, 0.2 mg/ml salmon sperm DNA for 5 h at 45 "C. Hybridization with labeled probe (850,000 cpm/ml) was conducted in 6 X TEN, 6 X Denhardt's, 0.1% SDS, 0.5 mg/ml sodium pyrophosphate for 16 h at 45 "C. Final wash conditions for primary and final round screening were 2 X TEN, 0.1% SDS at 45 "C for 10 min and 0.5 X TEN, 0.1% SDS at 55 "C for 5 min, respectively. Third round screening allowed for the isolation of a single clone, which was determined by analysis on a 1% agarose gel to contain an insert of -2.0 kb. The insert was rescued from the phage vector using the R408 helper phage following the manufacturer's protocol (Stratagene).
DNA Sequencing-The rescued insert contained in the pBluescript vector was used in sequence analysis. Both strands of the cDNA insert were sequenced using [35S]dATP and T7 DNA polymerase  (20). COS7 cells were grown in 75-cm2 flasks in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and gentamicin (0.01 mg/ml), and cells were harvested 48-72 h posttransfection for radioligand binding assays. CHO cells, grown in Ham's F-12 media supplemented with 10% fetal bovine serum and penicillin (100 units/ ml)/streptomycin (100 pg/ml) were employed for stable expression of the cDNA. Approximately 300,000 cells in 25-cm2 flasks were transfected with 30 pg of the pCMV5 expression vector containing the AIAR cDNA insert and 3 pg of pSVneo by a calcium phosphate precipitation and 15% glycerol shock procedure (21) with minor modifications. Cells were then maintained in the presence of 250 pg/ ml G418 (Geneticin, GIBCO), and, after 14 days of treatment, resistant colonies were selected and replated. Clonal cell lines expressing physiologic levels of AIAR, as determined by radioligand binding assays, were used in adenylylcyclase studies.
Site-directed Mutagenesis of the AIAR-The method for the creation of mutagenized cDNA was based on that described by Nelson and Long (22). Two oligonucleotides complementary to the sense strand of the cloned A,AR were synthesized in order to produce two mutant AIARs. The first oligonucleotide (5' AGG ATG AGC AAG GGC AGC CAG CTG AGG GC 3') coded for the substitution of the histidine at position 251 (sixth transmembrane domain) with leucine. The second oligonucleotide (5' GCC GAG TTG CCG AGT GAG AGG AAG ATG GCG ATG GTA 3') was used to construct a mutated AIAR in which the histidine at position 278 (seventh transmembrane domain) was replaced with leucine. For both His-251 and His-278 mutants, an oligonucleotide complementary to the cDNA located 60 bp upstream from a unique BsmI site and the oligonucleotide coding for the His-Leu substitution were used as primers in a polymerase chain reaction using 100 ng of AlAR/pCMV5 plasmid as the template. The fragment created in the first-step polymerase chain reaction was gel-purified and then used as a primer in a second polymerase chain reaction (one cycle only) for the extension of the mutagenized cDNA with expression vector DNA used as template. This fragment was then amplified by continuing the polymerase chain reaction with the BsmI site oligonucleotide and an oligonucleotide complementary to a region of the cDNA located 60 bp downstream from a unique BstEII site used as primers. The final product of -750 bp was extracted with phenol and chloroform and ethanol-precipitated. It was then digested with BsmIIBstEII and ligated into the A1AR/pCMV5 expression vector which had been similarly digested and gel-purified. Sequence of the mutant cDNA was verified by double strand sequencing. COS7 cells were transfected with His-251 and His-278 expression constructs as described above.
Radioligand Binding Assays-Membranes from COS7 cells were prepared as follows. Media was aspirated from the flask, and the cells were washed with 10 ml of lysis buffer (10 mM Tris, 5 mM EDTA, pH 7.4 at 22 "C). A fresh 5 ml of ice-cold lysis buffer was added to the flask, and cells were scraped and transferred to a dounce homogenizer on ice. The cells were homogenized with 20 strokes, spun at 38,500 X g for 5 min, and resuspended in 6 ml of 50/10/1 buffer (50 mM Tris, 10 mM MgC12, 1 mM EDTA, pH 8.26 at 5 "C). The cells were further diluted 3-4.5-fold with 50/10/1 buffer.
For saturation binding experiments, each assay tube contained 50 pl of H20 or 10 mM theophylline (to define nonspecific binding), 50 pl of radioligand, 50 pl of 50/10/1 buffer, and 100 pl (-5-10 pg of protein) of membrane suspension. In assays with mutagenized AIAR, 10 mM theophylline and 0.1 mM R-PIA gave similar results for nonspecific binding. For competition binding studies, each tube contained 50 p1 of competing ligand, 50 p1 of radioligand, 50 pl of 50/10/ 1 buffer, and 100 pl of the membrane suspension. All incubations were at 37 "C for 60 min and terminated by the addition of ice-cold 50/10/1 buffer containing 0.01% CHAPS and rapid filtration over 0.03% polyethylenimine-treated filters using a Brandel cell harvester. Data analysis was performed via a nonlinear least-squares fitting technique as previously described (23). Protein determinations were performed via the Bradford method (24). Adenylylcyclose Assay-Membranes from stably transfected CHO cells were prepared exactly as described above for COS7 cells. Adenylylcyclase assays were performed as previously described (25). cAMP accumulation experiments were performed using the cAMP radioimmunoassay kit as described by the manufacturer (Amersham). In both assays forskolin (50 pM) was used to activate the adenylylcyclase system.
Northern Analysis-RNA was prepared from bovine tissues that had been frozen in liquid nitrogen immediately upon dissection. Tissues were homogenized in ice-cold buffer consisting of 0.1 M Tris HC1, pH 7.5,4 M guanidinium thiocyanate, and 1% P-mercaptoetha-no1 with a Polytron set at full speed for a 30-5 burst. Sodium lauryl sarcosinate was added to the homogenate to give a final concentration of 0.5%, and the suspension was layered onto CsCl(5.7 M CsCl, 0.01 M EDTA, pH 7.5) in an ultracentrifuge tube. Samples were centrifuged at 175,000 X g for 18 h. The resulting RNA pellet was resuspended in 10 mM Tris, 1 mM EDTA, pH 7.5, and 0.1% SDS; diluted with an equal volume of 40 mM Tris-HC1, pH 7.6, 1.0 M NaCl, 2 mM EDTA and 0.2% SDS; and then applied to an oligo(dT)-cellulose column (Bethesda Research Laboratories) according to standard procedures (21). Poly(A)+ RNA was eluted with 0.5 ml of water and ethanol-precipitated and electrophoresed on a 1% agarose and 6.6% formaldehyde gel (3-5 pg of RNA per lane). RNA was transferred from the gel to a Zeta-probe membrane (Bio-Rad) with 10 X SSC (1 X SSC = 150 mM NaC1, 15 mM sodium citrate, pH 7.0) over 12 h. The RNA was fixed to the membrane by UV cross-linking and incubated in a prehybridization solution of 50% formamide, 6 X SSPE (1 X SSPE = 150 mM NaCl, 10 mM NaH2P0,, 1 mM EDTA, pH 7.4), 5 X Denhardt's solution, 0.5% SDS, and 0.25 mg/ml salmon sperm DNA at 42 "C overnight. The full-length bovine brain A,AR cDNA was labeled with [32P]ATP using the Amersham multiprime DNA labeling system and diluted in hybridization buffer (identical to prehybridization except for the omission of 5 X Denhardt's solution and increase of salmon sperm DNA to 0.5 mg/ml) to a final activity of 9.5 X 10' cpm/ml. Hybridization was conducted at 42 "C for 24 h after which the membrane was washed twice in 2 X SSC, 0.5% SDS at 22 "C for 15 min. This was followed by three sequential washes in the 0.1 X SSC, 0.1% SDS for 15 min at 55, 60, and 66 "C. and protein kinase A. A single consensus sequence for protein kinase A phosphorylation is also present in the second intracellular loop. These sites may represent important regions for AIAR regulation as a recent study has demonstrated the in vivo phosphorylation of the AIAR of DDTl MF-2 smooth muscle cells in association with agonist-induced desensitization (29). The cytoplasmic carboxyl terminus of the bovine brain AIAR is also relatively short which is a feature shared by several other receptors that are linked to the inhibition of adenylylcyclase via the Gi protein (28). This region of the receptor also contains a cysteine at position 309 which represents a possible site for palmitoylation (30).

Screening
To validate that the isolated cDNA did indeed code for the bovine brain AIAR, nearly the entire 2.0-kb insert was subcloned into the pCMV5 expression vector and the plasmid was transiently expressed in COS7 cells. As shown in Fig. 3A 0.16 nM and a B,,, of 1.00 f 0.28 pmol/mg (n = 5). A representative saturation curve is shown in Fig. 3B. Neither ligand bound to membranes prepared from nontransfected COS7 cells.
To document that the expressed receptor has the unique pharmacological profile expected, the ability of AIAR agonists to compete for [1251]APNEA binding sites was determined (Fig. 4) Fig. 5 demonstrates a dose-dependent inhibition of adenylylcyclase by R-PIA. The -30% attenuation is similar to that observed in previous studies of AIAR-mediated adenylylcyclase inhibition (4, 25). Similar results were obtained using cAMP accumulation as a determinant of AIAR functional activity (data not shown). The distribution of the AIAR mRNA in bovine tissues by  5. Representative experiment demonstrating R-PIAmediated inhibition of forskolin-stimulated adenylylcyclase activity in membranes prepared from CHO cells stably expressing bovine brain AIAR cDNA. Assays were performed as described under "Experimental Procedures" using -100 pg of protein per assay tube. Forskolin was present at 50 p~ where indicated and the concentration of R-PIA was as shown. This experiment was performed four times with similar results. Northern analysis is presented in Fig. 6 and is in agreement with the findings from previous ligand binding and functional studies (31). Two transcripts of 3.4 and 5.7 kb are abundant in brain with a significant amount also in heart. In the kidney, a 3.4-kb transcript was detected. The thyroid contained only a 5.5-kb message. No transcript was detected in bovine spleen, lung, or liver. DDTl MF-2 cells, a cell line known to contain AIAR, also contained two transcripts of 3.4 and 5.6 kb.
As mentioned in the Introduction, biochemical studies have suggested that histidine residues may be important for adenosine binding (17). Indicated in Fig. 2 are 2 histidines within the putative transmembrane domains (one each in transmembrane domains 6 and 7). In order to assess if these amino acids are important for agonist or antagonist binding, sitedirected mutagenesis was utilized to individually change the histidines to leucines as described under "Experimental Procedures." The mutagenized receptors were then expressed in COS7 cells, radioligand binding was performed, and the results were compared to the wild-type receptor. Antagonist ([3H]XAC) and agonist ([lZ5I]APNEA) binding to the His-278 + Leu-278 (seventh domain) mutant were both dramatically decreased compared to wild-type receptor (specific binding < 10%). This mutant was, therefore, not evaluated further.
Much different results were observed for the His-251 + Leu-251 mutant. In [3H]XAC saturation assays (Fig. 7), a 74% decrease in antagonist binding was observed and the Kd changed increased from 0.17 f 0.02 to 0.65 2 0.15 nM in mutant as compared to wild-type AIAR (n = 4). Agonist binding as assessed by [lZ5I]APNEA saturation assays (Fig.  8) decreased by 74%, while the K d was not affected (1.68 f 0.4 and 1.23 f 0.14 nM in wild-type and His-251 + Leu-251, respectively, n = 3). Full R-PIA competition curves uersus [3H]XAC were also constructed to completely examine the effect of the histidine substitution on agonist binding in that [lZ5I]APNEA saturation assays may only provide information regarding high affinity binding. In these experiments, the ICs0  The best-fit lines for nonspecific binding for both groups were superimposable. Approximately equivalent amounts (-3.5 pg) of protein were used per assay tube for both groups. Other experimental details are described under "Experimental Procedures." This experiment was repeated three times.
competing with [3H]XAC rather than ['261]APNEA is always lower even in native bovine membranes. Therefore, the point mutation had no apparent effects on agonist affinity. The His-251 Leu-251 also bound agonist ligands in the potency order noted above for wild-type AIAR, again indicating agonist recognition remained intact.

DISCUSSION
The bovine brain AIAR has long been recognized as a distinct form of the A,AR in that it binds agonists with a different potency series and has an -10-fold higher affinity for both agonists and antagonists compared to rat or other AIARs (8)(9)(10)(11). In this paper we describe the cloning and characterization of the cDNA for the bovine brain AIAR and through site-directed mutagenesis studies have begun to probe the binding pocket of the receptor.
Radioligand binding studies demonstrate that in COS7 cells, the cDNA codes for an adenosine receptor that displays a pharmacologic potency series which is identical to that ' the AlAR reported for the bovine brain A,AR in both its membrane (11) and purified (10) forms. This potency series of R-PIA > S-PIA > NECA is unique for bovine cortex as it differs from that reported for A 1 A b obtained from tissues of several other species in which NECA possesses a significantly greater affinity than S-PIA. The expressed receptor has all the pharmacological characteristics expected including high affinity agonist and antagonist binding and selective and stereospecific binding. In addition, the expressed receptor functionally couples to its G protein and can inhibit adenylylcyclase activity.
The bovine brain AIAR has also been considered unique based on the binding of the radiolabeled antagonist, [3H] XAC. The affinity of this compound for bovine brain AIAR is several-fold greater than that reported for several other AIARs including those present in rat fat (5) and brain (9) and DDTl MF-2 smooth muscle cells (25). COS7 cells transfected with the bovine brain AIAR cDNA bind [3H]XAC with a Kd of -0.4 nM in agreement with that displayed for the native receptor. This would indicate that the greater affinity of this receptor for the antagonist is due to distinct intrinsic properties of the bovine AIAR protein and not merely differences provided by the milieu of the membrane in its native tissue.
Recently, the cloning of AIARs from canine thyroid (12) and rat brain (13,14) cDNA libraries has been reported. Mahan et al. (13) observed the typical potency order of R-PIA > S-PIA > NECA for displacement of [3H]DPCPX from binding sites in A9-L cells transiently expressing the rat brain AIAR cDNA. Reppert and coworkers (14) reported this same potency order following the expression of a rat brain AIAR cDNA in COS-6 M cells. This agonist series was not used in the characterization of the canine AIAR clone, RDC7, expressed in COS7 cells (12). However, this study reported a Ki value of 4.55 nM for NECA in competition assays with [3H] CHA. This value is similar to that reported for the high affinity agonist state of NECA (Ki = 4.3 nM) for the rat brain AIAR (13) and several fold lower than that found in the present study for bovine brain AIAR.
This tissue distribution of AIAR mRNA in bovine tissues as determined by Northern analysis corresponds with the results of radioligand binding and functional studies. The AIAR is abundant in brain while heart, thyroid and kidney also contain appreciable amounts of message. Northern analysis in rat (13,14) and canine (12) tissues also demonstrates AIAR mRNA in brain and heart. However, unlike the bovine tissue, neither canine nor rat kidney contained detectable AIAR mRNA. Also, rat spleen appeared to possess AIAR transcripts while no message is present in bovine spleen.
Based on the unique ligand binding properties of the bovine AIAR and apparent differences in tissue distribution of the mRNA for this receptor, we propose that the bovine AIAR is a distinct subclass of this receptor. The existence of distinct subtypes of AIARs is in keeping with what has been found in other G protein-coupled receptors. The presence of multiplereceptor subtypes has recently been appreciated in almost all receptor families. Cloning techniques have allowed for the recognition of multiple a1 (32,33) and a2 (34) adrenergic receptors as well as Dl and DP dopamine (35), muscarinic (36)(37)(38)(39)(40), and serotonergic (41)(42)(43) receptors. Such receptor subtypes are often demonstrated to be distinct either pharmacologically, functionally, or on the basis of tissue distribution. The differences in pharmacology described above exist despite the remarkable amino acid homology between the bovine brain AIAR and those recently cloned. The encoded proteins are nearly identical in molecular mass with 326 amino acids constituting all of the cloned AIARs. The three AIARs now cloned are 92% identical overall and in the putative trans-membrane regions the bovine AIAR differs from the rat and canine receptors by 7 and 8 amino acid substitutions, respectively. In comparison, only 3 amino acid differences exist in the transmembrane regions between the rat and canine AIARs. It is generally believed that the composition of the transmembrane domains of the G protein-coupled receptors determine their ligand binding characteristics and, therefore, confer the specificity of receptor subtype activation (26,27). It is possible that very few or perhaps only a single amino acid difference in these regions is (are) responsible for the differences in bovine brain AIAR pharmacology relative to other AIARs. Suryanarayana et al. (44) recently demonstrated that a single point mutation (Phe to Asn) in the seventh transmembrane domain of the a2 adrenergic receptor dramatically reduced the receptor's affinity for selective agonists while increasing its affinity for a P-adrenergic receptor antagonist 3000-fold.
Site-directed mutagenesis provides a powerful technique for assessing the importance and role of individual or groups of amino acids in the functioning of proteins. Klotz et al. (17) have previously documented that diethylpyrocarbonate, a histidine-specific agent, was capable of specifically decreasing both agonist and antagonist binding to the AIAR of rat brain membranes. Diethylpyrocarbonate treatment decreased the number but did not alter the affinity of the receptors for antagonists. This suggests a modification of the ligand binding site itself. The fact that there was a selective protective effect of antagonist binding by antagonists but not agonists and similarly a protective effect of agonist binding by agonists but not antagonists suggested that there were at least 2 histidine residues near the binding pocket.
In this study we have now been able to directly study the role of the 2 histidine residues in transmembrane domains 6 and 7. Our data demonstrate that the His-278 (seventh domain) appears to be extremely important for the binding of both agonists and antagonists. Substituting leucine for this histidine leads to the almost but not quite complete loss of agonist and antagonist binding. In contrast, His-251 is important for both agonist and antagonist binding in that agonist and antagonist bindings are both decreased by substitution of Leu for His, but not to the same extent as that of His-278 -+ Leu-278. At the present time, no antibodies to the AIAR are available for immunodetection of the receptor protein. Therefore, agonist and antagonist radioligand binding studies are the most suitable methods available to quantitate membrane AIARs. It is possible, therefore, that the reduction in receptor number as assessed by [1251]APNEA and [3H]XAC binding may indicate a decline in the actual quantity of AIAR protein rather than the loss of a specific ligand-receptor interaction. Such a decline could result if the process by which the mutant AIAR cDNA is transcribed, processed, and translated in the COS7 cells is different from that for the wildtype cDNA. In addition, the mutated protein may not undergo the proper folding necessary for transport to and insertion in the membrane in the appropriate orientation. This latter possibility would be difficult to detect, for even immunologic techniques would not likely be able to distinguish between AIARs differing only in their three-dimensional structure. To minimize the occurrence of the possibilities described above, leucine, which is an aliphatic and relatively nonbulky amino acid that is prevalent in protein transmembrane domains, was selected to substitute for the histidine. However, even with all these caveats we believe that the mutant studies provide new insights into AIAR structure and function.
In addition to the effects on AIAR B,,,, the His-251 + Leu-251 substitution has differential effects on agonist and antagonist binding affinity. As judged from K d values for [3H] XAC, the mutated AIAR demonstrates approximately a 4fold decline in antagonist affinity. However, both [1251]AP-NEA and R-PIA uersus [3H]XAC competition curves indicate that the His-251 + Leu-251 substitution produces no effect on agonist affinity. The ability of His-251 + Leu-251 to bind agonists with high affinity demonstrates that the functional properties of the ligand binding pocket remain intact, and, furthermore, the mutated receptor does couple to a G protein, presumably Gi. The latter finding is reasonable based on previous analysis of structure and function of G proteincoupled receptors which indicates that transmembrane domain regions are involved in ligand binding, whereas intracellular regions are critical for G protein activation (25,26). The differential effects of the His-251 + Leu-251 substitution on agonist/antagonist binding are in agreement with previous studies on ligand-receptor interactions of the AIAR. These findings extend those of Klotz et al. (17) by specifically examining the histidine residues involved. Not only has sequencing allowed the location of the histidines to be determined but site-directed mutagenesis permits their individual contributions to be studied. The histidine at position 251 appears to be directly involved in antagonist binding since antagonist affinity is significantly altered. Mutation at this site does not alter agonist affinity, only receptor number. Changes in Emax are much more complicated in this system (AIAR) than changes in Kd since it is known that the quantity of receptor detected by both agonist and antagonist radioligands is dependent on receptor-G protein coupling, ion concentration, and the presence or absence of guanine nucleotides (45). Interestingly, Barrington et al. (46), using proteolytic digestions of agonist and antagonist occupied AIAR in native bovine brain membranes, demonstrated that although these ligands may share the same binding pocket they induce unique conformational changes of the AIAR. The conformational changes induced will of necessity determine not only the apparent affinity for a given ligand but also its coupling to the G protein and hence the number of receptors detected.
In summary, this paper provides evidence for the existence of a subtype of the A1 adenosine receptor which is in agreement with data obtained with the native bovine receptor. Apparently, minor variations in amino acid sequence may account for the differences in pharmacologic properties of the bovine AIAR as compared to the canine and rat AIARs previously described. Furthermore, this study significantly extends previous findings regarding AIAR-ligand binding interactions. His-278 in transmembrane domain 7 is apparently crucial for ligand recognition. His-251 in transmembrane domain 6, though involved in part in agonist binding, plays a much more important role in A1AR-antagonist interaction. Future studies probing other domains of the AIAR should provide a more detailed description of its ligand binding pocket.