Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis

Abstract

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-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 36,570 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 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR 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 greater than 90%. In contrast, mutation of His-251 to Leu-251 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.

FIG. 1. Nucleotide and deduced amino acid sequence of the cloned bovine brain A₁AR

Translation of amino acids begins at the ATG providing the longest open reading frame. The stop codon is represented by @.

FIG. 2. Comparison of the deduced amino acid sequences of bovine, dog, and rat A₁AR

Dashes represent identical amino acids with differences noted in canine and rat sequences. Putative transmembrane domains (I–VII) are marked with a solid line. Italicized amino acids represent changes in the A₁ARs in these transmembrane regions. Possible sites for glycosylation in extracellular loop 2 are marked by asterisk. His-251 and His-278 are marked by ↑.

FIG. 3. Representative curves depicting [³H]XAC (A) and [¹²⁵I]APNEA (B) saturation binding assays with membranes prepared from COS7 cells transiently transfected with bovine A₁AR cDNA

For both curves, nonspecific binding was defined with 10 mM theophylline. Assays were performed as described under “Experimental Procedures.” Each curve was replicated at least five times.

FIG. 4. Agonist competition uersus [¹²⁶I]APNEA (~0.3 nM) in membranes prepared from COS7 cells transiently transfected with bovine brain A₁AR cDNA

Competing ligand was added at the concentration shown on the abscissa. The amount of [¹²⁵I]APNEA bound is on the ordinate. Assays are described under “Experimental Procedures.” This experiment is representative of four similar assays.

FIG. 5. Representative experiment demonstrating R-PIA-mediated inhibition of forskolin-stimulated adenylylcyclase activity in membranes prepared from CHO cells stably expressing bovine brain A₁AR cDNA

Assays were performed as described under “Experimental Procedures” using ~100 μg of protein per assay tube. Forskolin was present at 50 μM where indicated and the concentration of R-PIA was as shown. This experiment was performed four times with similar results.

FIG. 6. Northern blot analysis of tissue distribution of A₁AR mRNA

Poly(A)⁺ RNA (3–5 μg) was run on agarose/formaldehyde gels, transferred to nylon membranes, and probed with ³²P-labeled full-length bovine A₁AR cDNA. Washing conditions were described under “Experimental Procedures.” Tissues analyzed were bovine brain (B), heart (H), thyroid (T), spleen (S), kidney (K), lung (Lg), and liver (Lv). A is a photograph of film exposedt o theb lot for 12 h. B is a selected panel from the same blot exposed to the film for 48 h to allow visualization of transcripts present in heart, thyroid, and kidney. For comparison, an analysis is shown of transcript levels in DDT₁ MF-2 smooth muscle cells (D). Position of the 18 and 28 S ribosomal RNA is shown to the left of A.

FIG. 7. Representative [³H]XAC saturation assay in membranes prepared from COS7 cells transiently expressing wild-type and mutagenized (His-251 → Leu-251) A₁AR cDNA

Nonspecific binding was defined with 10 mM theophylline. The bestfit lines for nonspecific binding for both groups were superimposable. Approximately equivalent amounts (~3.5 μg) of protein were used per assay tube for both groups. Other experimental details are described under “Experimental Procedures.” Similar results were obtained in three other experiments.

FIG. 8. Representative [¹²⁵I]APNEA saturation assay in membranes prepared from COS7 cells transiently expressing wild-type and mutagenized (His-251 → Leu-251) A₁AR cDNA

Nonspecific binding was defined with 10 mM theophylline. The best-fit lines for nonspecific binding for both groups were superimposable. Approximately equivalent amounts (~3.5 μg) of protein were used per assay tube for both groups. Other experimental details are described under “Experimental Procedures.” This experiment was repeated three times.