Molecular characterization of recombinant human adenosine receptors

The four human adenosine receptors (ARs), A(1), A(2A), A(2B), and A(3), have been stably expressed in mammalian cells. Radioligand binding properties were examined for all four subtypes, including, for the first time the A(2B)AR The A(1)AR-selective radioligand, [H-3]8-cyclopentyl-1,3-dipropylxanthine ([H-3]CPX; A(1); K-D = 2 nM) also can be used to characterize recombinant A(2B)AR (K-D = 40 nM). Theophylline and enprofylline both bind to A(2B)AR with K-1 of 7 mu M, well within the therapeutic ranges of these compounds used to treat asthma. We have identified C-8-(N-methylisopropyl)-amino-N-6-(5'-endohydroxy) -endonorbornan-2-yl-9-methyladenine (WRC-0571) as the most selective antagonist of A(1)AR. Recombinant ARs have been extended with hexahistidine (H) and the FLAG (F) epitope to make H/F-A(1) and H/F-A(2A) receptors that have been purified to near homogeneity under conditions that preserve radioligand binding. [H-3]Adenosine binds to purified H/A(1)AR-G protein complexes with a K-D 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-A(1)AR and H/FA(2A)AR purified from mammalian cells incubated in [P-32]phosphate indicates that both receptors are phosphorylated on serine residues. Monoclonal antibodies were raised by using the purified H/F-A(2A)AR as an antigen. Antibodies with higher affinity than antipeptide antisera were generated and used for Western blotting and rat brain immunocytochemistry. (C) 1997 Wiley-Liss, Inc.