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 bindin g studies on COS-7 cell membranes expressing either the wild-type or t he mutant receptor revealed that the affinity of the prototypic agonis t CGS21680 phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine) for the mutant receptor was 15-fold lower than for the wild-type receptor. Thi s was confirmed in functional studies with intact cells. The EC(50) va lues of CGS21680 for the stimulation of cAMP production differed in a similar way. Antagonists of various chemical structure were equally ef fective on both mutant and wild-type receptors, thus the mutation sele ctively diminishes agonist affinity, We propose an indirect perturbati on of the binding site, perhaps through a proton transfer mechanism as suggested by molecular modelling.