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, [ 3 H]8-cyclopentyl-1,3-dipropylxanthine ([ 3 H]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 μM, well within the therapeutic ranges of these compounds used to treat asthma. We have identified C B -(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. [ 3 H]Adenosine binds to purified H/F-A 1 AR-G protein complexes with a Ko 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/F-A 2A AR purified from mammalian cells incubated in [ 32 P]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.