Vol. 63, Issue 5, 1021-1031, May 2003

Identification of Essential Residues Involved in the Allosteric Modulation of the Human A₃ Adenosine Receptor

Zhan-Guo Gao, Soo-Kyung Kim, Ariel S. Gross, Aishe Chen, Joshua B. Blaustein, and Kenneth A. Jacobson

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland

We examined the effects on allosteric modulation and ligand binding of the mutation of amino acid residues of the human A₃ adenosine receptor (A₃AR) that are hypothesized to be near one of three loci: the putative sodium binding site, the putative ligand binding site, and the DRY motif in transmembrane helical domain 3. The effects of three heterocyclic allosteric modulators [the imidazoquinoline 2-cyclopentyl-4-phenylamino-1H-imidazo[4,5-c]quinoline (DU124183), the pyridinylisoquinoline 4-methoxy-N-[7-methyl-3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF5455), and the amiloride analog 5-(N,N-hexamethylene)-amiloride] on the dissociation of the agonist radioligand, N⁶- (4-amino-3-[¹²⁵I]iodobenzyl)-5'-N-methylcarboxamidoadenosine, were compared at wild-type (WT) and mutant A₃ARs. The F182A^5.43 and N274A^7.45 mutations eliminated the allosteric effects of all three modulators but had little effect on agonist binding. The N30A^1.50 and D58N^2.50 mutations abolished the allosteric effects of DU124183 and VUF5455, but not HMA, whereas the D107N^3.49 mutation abolished the effects of DU124183, but not HMA or VUF5455. The T94A^3.36, H95A^3.37, K152A^EL2, W243A^6.48, L244A^6.49, and S247A^6.52 mutations did not influence allosteric effects of the modulators. Sodium ions (100 mM), which modulate agonist binding at a variety of receptors, caused an ~80% inhibition of agonist binding in WT A₃ARs but did not show any effect on D58N^2.50, D107N^3.49, and F182A^5.43 mutant receptors. In contrast, NaCl induced a modest increase of agonist binding in N30A^1.50 and N274A^7.45 mutant receptors. NaCl decreased the dissociation rate of the antagonist radioligand [³H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) at the WT A₃ARs, but not the D58N^2.50 mutant receptor. The results were interpreted using a rhodopsin-based molecular model of the A₃AR to suggest multiple binding modes of the allosteric modulators.