Site-directed mutagenesis of the human adenosine A(2A) receptor. Critical involvement of Glu(13) in agonist recognition

A glutamic acid residue in the first transmembrane domain of the human adenosine A(2A) receptor was mutated to glutamine. Radioligand binding studies on COS-7 cell membranes expressing either the wild-type or the mutant receptor revealed that the affinity of the prototypic agonist CGS21680 (2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine) for the mutant receptor was 15-fold lower than for the wild-type receptor. This was confirmed in functional studies with intact cells. The EC(50) values of CGS21680 for the stimulation of cAMP production differed in a similar way. Antagonists of various chemical structure were equally effective on both mutant and wild-type receptors, thus the mutation selectively diminishes agonist affinity, We propose an indirect perturbation of the binding site, perhaps through a proton transfer mechanism as suggested by molecular modelling.