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J. Biol. Chem., Vol. 279, Issue 12, 11456-11464, March 19, 2004

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

Christopher L. Herold, Ai-Dong Qi, T. Kendall Harden, and Robert A. Nicholas

From the Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365

UTP is a potent full agonist at both the human P2Y₄ (hP2Y₄) 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 NH₂ 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 rP2Y₄ 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 rP2Y₄ 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.

Received for publication, February 19, 2003 , and in revised form, December 8, 2003.

^* This work was supported in part by National Institutes of Health Grants HL71131 (to R. A. N) and GM38213 (to T. K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of North Carolina, CB #7365, Chapel Hill, NC 27599-7365. Tel.: 919-966-6547; Fax: 919-966-5640; E-mail: nicholas{at}med.unc.edu.

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