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The four human adenosine receptors (ARs), A1, A2A, A2B, and A3, have been stably expressed in mammalian cells. Radioligand binding properties were examined for all four subtypes, including, for the first time the A2BAR. The A1AR-selective radioligand, 3H8-cyclopentyl-1,3-dipropylxanthine (3HCPX; A1; KD = 2 nM) also can be used to characterize recombinant A2BAR (KD = 40 nM). Theophylline and enprofylline both bind to A2BAR with KI of 7 μM, well within the therapeutic ranges of these compounds used to treat asthma. We have identified C8-(N-methylisopropyl)-amino-N6-(5′-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571) as the most selective antagonist of A1AR. Recombinant ARs have been extended with hexahistidine (H) and the FLAG (F) epitope to make H/F-A1 and H/F-A2A receptors that have been purified to near homogeneity under conditions that preserve radioligand binding. 3HAdenosine binds to purified H/F-A1AR-G protein complexes with a KD of 0.95 ± 0.3 nM; binding is not affected by the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Phosphoaminoacid analysis of H/F-A1AR and H/F-A2AAR purified from mammalian cells incubated in 32Pphosphate indicates that both receptors are phosphorylated on serine residues. Monoclonal antibodies were raised by using the purified H/F-A2AAR as an antigen. Antibodies with higher affinity than antipeptide antisera were generated and used for Western blotting and rat brain immunocytochemistry. Drug Dev. Res. 39:243–252, 1996. © 1997 Wiley-Liss, Inc.
Robeva et al. (Fri,) studied this question.