PT - JOURNAL ARTICLE AU - Zhan-Guo Gao AU - Soo-Kyung Kim AU - Ariel S. Gross AU - Aishe Chen AU - Joshua B. Blaustein AU - Kenneth A. Jacobson TI - Identification of Essential Residues Involved in the Allosteric Modulation of the Human A<sub>3</sub> Adenosine Receptor AID - 10.1124/mol.63.5.1021 DP - 2003 May 01 TA - Molecular Pharmacology PG - 1021--1031 VI - 63 IP - 5 4099 - http://molpharm.aspetjournals.org/content/63/5/1021.short 4100 - http://molpharm.aspetjournals.org/content/63/5/1021.full SO - Mol Pharmacol2003 May 01; 63 AB - We examined the effects on allosteric modulation and ligand binding of the mutation of amino acid residues of the human A3adenosine 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 F182A5.43 and N274A7.45 mutations eliminated the allosteric effects of all three modulators but had little effect on agonist binding. The N30A1.50 and D58N2.50mutations abolished the allosteric effects of DU124183 and VUF5455, but not HMA, whereas the D107N3.49 mutation abolished the effects of DU124183, but not HMA or VUF5455. The T94A3.36, H95A3.37, K152AEL2, W243A6.48, L244A6.49, and S247A6.52 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 D58N2.50, D107N3.49, and F182A5.43 mutant receptors. In contrast, NaCl induced a modest increase of agonist binding in N30A1.50 and N274A7.45 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 D58N2.50mutant receptor. The results were interpreted using a rhodopsin-based molecular model of the A3AR to suggest multiple binding modes of the allosteric modulators. U.S. Government