Identification of an adenosine receptor domain specifically involved in binding of 5'-substituted adenosine agonists

The bovine A1 adenosine receptor (A1AR) and rat A3 adenosine receptor (A3AR) display distinct agonist and antagonist binding properties. To identify regions involved in ligand recognition, A1AR/A3AR chimeric receptors were created, expressed in COS-7 cells, and analyzed by radioligand binding. A chimeric receptor in which the third intracellular loop of the A1AR was replaced with that of the A3AR bound agonists and the antagonist, [3H]xanthine amine congener, with affinities identical to wild-type A1AR. A chimeric receptor with the fifth transmembrane domain (TM5) and third intracellular loop of the A1AR replaced with that of the A3AR displayed antagonist affinity similar to wild-type A1AR. However, relative to the A1AR, this chimeric demonstrated much greater affinity for 5'-substituted adenosine analogs, whereas affinity for N6-substituted compounds was unaffected. Substitution of a 6-amino acid cassette of the exofacial half of TM5 of the A3AR into the A1AR produced enhanced binding of exclusively a 5'-substituted analog, indicating involvement of this specific region in ligand recognition. These findings suggest that the 5'- and N6-substituents of adenosine agonists bind to distinct regions of ARs and that TM5 of the A3AR interacts more favorably with 5'-substituted compounds than does that of the A1AR.