Immunological identification of A2 adenosine receptors by two antipeptide antibody preparations

Abstract

Two antipeptide antibody preparations were raised against deduced amino acid sequences within the presumed second extra-cellular loop (antibody TP/1) and the carboxyl-terminal domain (antibody TP/2) of the canine-derived A2 adenosine receptor (A2AR) cDNA species termed RDC8. Immunoblotting of canine liver plasma membranes with both TP/1 and TP/2 identified a single band of 52 kDa, which co-migrated with 125I-2-[4-[2-[2-[(4- azidophenyl)methylcarbonylamino]ethylaminocarbonyl]ethyl] phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine-labeled receptor. However, in membranes prepared from canine striatum, photoaffinity labeling and immunoblotting with TP/2, but not TP/1, revealed a single band of 34 kDa; the identity of the band observed on the immunoblot as an A2AR was confirmed by the ability of TP/2 to specifically immunoprecipitate photoaffinity-labeled receptor from crude canine striatal membranes. The size difference between liver and striatal A2ARs was not due to tissue-specific proteolysis, because membranes from striatum were prepared with a protease inhibitor cocktail previously shown to be effective in inhibiting endogenous A2AR proteolysis during membrane preparation. Also, the protease-sensitive carboxyl-terminal region of the receptor had remained intact, because the peptide used to raise TP/2 antibodies resides in this domain of the molecule. The difference in size was also not due to a greater carbohydrate content of the liver receptor, because treatment of liver and striatal membranes with endoglycosidase F produced small mobility shifts for both receptors. Removal of N-linked carbohydrate chains also did not alter the inability of TP/1 to recognize the striatal A2AR. Hence, we conclude that the A2AR present in liver, which displays the predicted immunoreactivity of RDC8, is immunologically distinct from the A2AR expressed in striatum and that the latter may represent an additional A2AR subtype.

Fig. 1

Presumed membrane topography of the canine A₂AR. The predicted membrane-spanning topography of the canine A₂AR is shown, with the amino acid sequences used for antibody generation indicated. Branched amino adds indicate predicted sites for N-linked glycosylation. Shaded amino acids represent residues that are conserved between the canine A₁AR and A₂AR; serine and threonine residues in bold type represent potential phosphorylation sites. The figure is adapted from ref. .

Fig. 2

Co-migration of A₂ARs identified by photoaffinity labeling and immunobiotting. Membranes from canine liver (A) or canine striatum (B) either were photoaffinity-labeled using ¹²⁵I-azido-PAPA-APEC, in the absence (lane 1) or in the presence (lane 2) of 5 mm theophylline, or were immunoblotted with 8 µg/ml affinity-purified TP/1 (lane 3) or 4 µg/ml affinity-purified TP/2 (lane 4). B, Crude membranes were used for the photoaffinity-labeling experiments and Ficoll gradient-purified synaptosomal membranes were used for the immunobiotting experiments. Each-panel is representative of at least three experiments performed on different membrane preparations, using at least three preparations of affinity-purified antibodies.

Fig. 3

Immunoprecipitation of canine striatal A₂ARs with TP/2. Crude membranes prepared from canine striatum were subjected to photoaffinity labeling using the A₂AR-selective agonist ¹²⁵I-azido-PAPA-APEC. A, Each 700-µg sample of labeled membranes was solubilized in 500 µl of HPEN containing 2% Triton X-100 and, after centrifugation to sediment insoluble material, 400 µl of the supernatant were subjected to immunoprecipitation with either 20 µg/ml TP/2 (lane 1) or 20 µg/ml nonimmune IgG (lane 2), as described in Experimental Procedures. B, Samples were immunoprecipitated with 20 µg/ml TP/2 in the absence of added peptide (lane 1) or in the presence of carboxyl-terminal domain peptide at 20 µg/ml (lane 2). Samples were also immunoprecipitated with 20 µg/ml TP/2 in the presence of second extracellular loop peptide at 20 µg/ml (lane 3). The sequences of the peptides are given in Experimental Procedures and Fig. 1.

Fig. 4

Sensitivity of endogenous canine A₂ARs to endoglycosidase F. A, Purified plasma membranes (500 µg) from canine liver were treated in the absence (lane 1) or presence (lanes 2 and 3) of 10 units/ml endoglycosidase F, at 37°, for 4 hr (lane 2) or 8 hr (lane 3) before samples were prepared with SDS for electrophoresis and immunoblotting with 4 µg/ml TP/2. B, Purified synaptosomal membranes (500 µg) from canine striatum were treated in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 10 units/ml endoglycosidase F for 10 hr at 37° before analysis by SDS-PAGE and immunoblotting with either 4 µg/ml TP/2 (lanes 1, 2, and 3) or 8 µg/ml TP/1 (lanes 4 and 5). B, lane 1, result of solubilizing 500 µg of canine striatal synaptosomal membranes in Triton X-100 on ice for 1 hr, followed by the addition of sample buffer to the supernatant and incubation at room temperature for 15 min before loading for electrophoresis.