Role of histidine residues in the adenosine A2a receptor ligand binding site

The pH dependency of the binding of ligands to adenosine A2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'-N-ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N6-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.