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The adenosine A 1 and A 2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A 1 receptor at 3.3 Å resolution with PSB36, an A 1 -selective xanthine-based antagonist. This is compared with structures of the A 2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A 1 R and A 2A R were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T270 7.35 in A 1 R (M270 7.35 in A 2A R) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A 1 R or A 2A R antagonists., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The options for investigating solubilised G protein-coupled receptors (GPCRs) by biophysical techniques have long been hampered by their instability. A thermostabilised adenosine A2A receptor expressed in insect cells, purified in detergent and reconstituted into high-density lipoprotein (HDL) particles was immobilised onto a Surface Plasmon Resonance sensor chip. This allowed measurement of affinities and kinetics for A2A antagonists with affinities ranging from 50 pM to almost 2 μM. Compared with other formats, reproduction of affinities, and dissociation and association rate constants are good, reasonable and poor respectively, indicating stabilised receptors in HDL particles are useful for investigating specific aspects of GPCR-ligand interactions., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)