Purification and characterization of bovine cerebral cortex A1 adenosine receptor

Abstract

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.

FIG. 1

Radioligand binding characteristics of the purified A₁AR. (A) [³H]XAC saturation curves following hydroxylapatite chromatography. [³H]XAC was added at the concentrations shown on the abscissa and the amount bound is on the ordinate. Binding assays were performed as described under Materials and Methods. The assay contained 25 μl of hydroxylapatite eluate (~50 ng protein). Nonspecific binding was determined with 25 mM theophylline. This experiment is representative of five similar experiments. (B) Agonist competition curve analysis versus [³H]XAC in the hydroxylapatite-purified A₁AR. The assay contained ~0.5 nM [³H]XAC and 25 μl of hydroxylapatite eluate (~50 ng protein). Competing ligand was added to give the concentration shown on the abscissa and the amount of [³H]XAC bound is on the ordinate. This experiment was performed twice.

FIG. 2

SDS–PAGE analysis of receptor fractions following chromatography. (A) Specific fractions following affinity chromatography and hydroxylapatite chromatography were subjected to radioiodination with chloramine T and Na¹²⁵I and then subjected to SDS–PAGE/autoradiography on a 12% acrylamide gel as described under Materials and Methods. The A₁AR migrates as a M_r 36,000 protein. Molecular weight markers are shown to the left. This experiment is representative of five similar experiments. (B) Silver staining of gel containing purified receptor following hydroxylapatite chromatography. This sample was run on a 16% acrylamide gel and the mobility of molecular weight markers is shown to the left. The A₁AR migrates as a M_r 36,000 protein. The interface between the stacking and the separating gels is visible at the top of the lane.

FIG. 3

Photoaffinity labeling of the purified A₁AR. Following hydroxylapatite chromatography, an aliquot of eluted material was photoaffinity labeled with [¹²⁵I]PAPAXAC-SANPAH in the presence and absence (Control) of 5 mM theophylline as described under Materials and Methods. The sample was then subjected to SDS–PAGE/autoradiography. A single M_r 37,000 protein is specifically labeled. For comparison, a lane containing a Na¹²⁵I radioiodinated receptor is included. Receptors labeled by the two separate techniques comigrate on SDS–PAGE. Positions of molecular weight markers are shown to the left. This experiment was repeated three times.

FIG. 4

Glycoprotein nature of the purified A₁AR. The purified A₁AR was photoaffinity labeled with [¹²⁵I]PAPAXAC-SANPAH in the presence of 0.1 mM PMSF and then subjected to either endoglycosidase F 0.4 U for 4 h, α-mannosidase 2.5 U for 16 h or neuraminidase 1.25 U for 16 h. Control indicates no treatment. The samples were then prepared and subjected to SDS–PAGE on a 12% acrylamide gel followed by autoradiography. Positions of molecular weight markers are shown to the left. This experiment was performed three times.