Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking.

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Abstract
A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-{2-[(2-aminophenyl)methylcarbonylamino]ethylaminocarbonyl}-ethyl)phenyl]ethylamino-5''-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5''-N-ethylcarboxamidoadenosine (NECA) > (-)-N6-[(R)-1-methyl-2-phenylethyl]adenosine (R-PIA) > (+)-N6-[(S)-1-methyl-2-phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 .mu.M) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5''-N-ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4''-azido-2''-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors (see above) and is decreased in the presence of 10-4 M guanosine 5''-[.beta.,.gamma.-imido]triphosphate. Photoaffinity crosslinking of the A1 adenosine receptor binding subunit with 125I-labeled 8-{4-[2-(4-aminophenylacetylamino)ethyl]carbonylmethyloxphenyl}-1,3-dipropylxanthine (PAPAXAC) (an A1 selective photoaffinity probe) in the same tissue reveals a 38-kDa peptide that exhibits the appropriate A1 receptor pharmacology. 125I-labeled PAPA-APEC, therefore, has identified the A2 receptor binding subunit as a 45-kDa protein that is unique and distinct from the A1 binding subunit.