Agonist Versus Antagonist Action of ATP at the P2Y4 Receptor Is Determined by the Second Extracellular Loop

Abstract

UTP is a potent full agonist at both the human P2Y4 (hP2Y 4) and rat P2Y4 (rP2Y4) receptor. In contrast, ATP is a potent full agonist at the rP2Y4 receptor but is a similarly potent competitive antagonist at the hP2Y4 receptor. To delineate the structural determinants of agonism versus antagonism in these species homologues, we expressed a series of human/rat P2Y4 receptor chimeras in 1321N1 human astrocytoma cells and assessed the capacity of ATP and UTP to mobilize intracellular Ca2+. Replacement of the NH 2 terminus of the hP2Y4 receptor with the corresponding region of the rP2Y4 receptor resulted in a receptor that was activated weakly by ATP, whereas replacement of the second extracellular loop (EL2) of the hP2Y4 receptor with that of the rP2Y4 receptor yielded a chimeric receptor that was activated fully by UTP and near fully by ATP, albeit with lower potencies than those observed at the rP2Y 4 receptor. These potencies were increased, and ATP was converted to a full agonist by replacing both the NH2 terminus and EL2 in the hP2Y4 receptor with the corresponding regions from the rP2Y 4 receptor. Mutational analysis of the five divergent amino acids in EL2 between the two receptors revealed that three amino acids, Asn-177, Ile-183, and Leu-190, contribute to the capacity of EL2 to impart ATP agonism. Taken together, these results suggest that the second extracellular loop and the NH2 terminus form a functional motif that plays a key role in determining whether ATP functions as an agonist or antagonist at mammalian P2Y4 receptors.