Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor.

Abstract Ligand recognition has been extensively explored in G protein-coupled A1, A2A, and A2B adenosine receptors but not in the A3receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A3 adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His95, Trp243, Leu244, Ser247, Asn250, and Lys152. The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by ∼3–38-fold, suggesting that these residues were critical for the high affinity of A3adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A3 agonist 2-chloro-N 6-(3-iodobenzyl)-5′-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5′-γ-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp243 was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A3 receptor interactions.