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J. Biol. Chem., Vol. 277, Issue 21, 19056-19063, May 24, 2002

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Identification by Site-directed Mutagenesis of Residues Involved in Ligand Recognition and Activation of the Human A₃ Adenosine Receptor^*

Zhan-Guo Gao^§, Aishe Chen, Dov Barak^§¶, Soo-Kyung Kim, Christa E. Müller, and Kenneth A. Jacobson^**

From the  Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and  Pharmaceutical Institute, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany

Ligand recognition has been extensively explored in G protein-coupled A₁, A_2A, and A_2B adenosine receptors but not in the A₃ receptor, 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 A₃ adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A₃ agonist 2-chloro-N⁶-(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'--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.

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