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. 2003 May;63(5):1021-31.
doi: 10.1124/mol.63.5.1021.

Identification of essential residues involved in the allosteric modulation of the human A(3) adenosine receptor

Zhan-Guo Gao  1 , Soo-Kyung Kim, Ariel S Gross, Aishe Chen, Joshua B Blaustein, Kenneth A Jacobson
Affiliations

Identification of essential residues involved in the allosteric modulation of the human A(3) adenosine receptor

Zhan-Guo Gao et al. Mol Pharmacol. 2003 May.

Abstract

We examined the effects on allosteric modulation and ligand binding of the mutation of amino acid residues of the human A(3) adenosine receptor (A(3)AR) 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, N(6)- (4-amino-3-[(125)I]iodobenzyl)-5'-N-methylcarboxamidoadenosine, were compared at wild-type (WT) and mutant A(3)ARs. 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 approximately 80% inhibition of agonist binding in WT A(3)ARs 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 [(3)H]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 A(3)ARs, but not the D58N(2.50) mutant receptor. The results were interpreted using a rhodopsin-based molecular model of the A(3)AR to suggest multiple binding modes of the allosteric modulators.

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Figures

Fig. 1
Fig. 1
Chemical structures of allosteric modulators.
Fig. 2
Fig. 2
The dissociation of the agonist radioligand [125I]I-AB-MECA from WT and mutant ARs in the absence and presence of various allosteric modulators. (■, control; ▲, DU124183; ▼, HMA; ◆, VUF5455). The dissociation rates of [125I]I-AB-MECA from N30A1.50 and H95A3.37 were determined at 4°C, whereas WT and other mutant receptors were measured at 25°C. The concentration of [125I]I-AB-MECA was 0.5 nM. The mean k−1 values calculated from three independent experiments are listed in Table 2.
Fig. 3
Fig. 3
The dissociation of the antagonist radioligand [3H]PSB-11 from WT (A) and D58N (B) mutant ARs in the presence or absence of sodium ions and HMA. The final concentration of [3H]PSB-11 used was 8 nM and the incubation temperature was 25°C. The mean k-1 values calculated from three independent experiments are listed in Table 3.
Fig. 4
Fig. 4
Effect of sodium ions on the equilibrium binding of the agonist radioligand, [125I]I-AB-MECA to WT and mutant ARs. [125I]I-AB-MECA (0.5 nM) was incubated with membranes (8 –20 µg of protein) from COS-7 cells expressing WT or mutant receptors in total assay volume of 200 µl of Tris-HCl buffer, pH 8.0, containing 10 mM MgCl2 and 1 mM EDTA at 25°C for 60 min. Data were from three independent experiments performed in triplicate.
Fig. 5
Fig. 5
Competition of allosteric modulators for the binding of [125I]I-AB-MECA to WT and H95A and W243A mutant ARs. A, HMA; B, DU124183; C, VUF5455. The final concentration of [125I]I-AB-MECA used was 0.5 nM. Data are from a representative experiment in duplicate. The Ki values listed in Table 1 are from three independent experiments in duplicate.
Fig. 6
Fig. 6
Rhodopsin-based molecular model of the A3AR, showing the previously defined docking mode of the A3-selective agonist Cl-IB-MECA (colored according to atom type) and a putative allosteric binding site for VUF5455 (orange) on TM 7. Shown are selected residues near the allosteric modulator and residues F182 and N274, which were found to be important for the allosteric action of VUF5455. The secondary structure of each TM is displayed by ribbon using different colors, which indicate TMs 1, 2, 3, 4, 5, 6, and 7 with red, orange, yellow, green, cyan, blue, and purple color, respectively. The coordinates of this A3AR model are available as from the Protein Data Bank ftp site under the name pdblo74.ent.Z. See http://www.rcsb.org/pdb/pdb_news2002.html#models_removal2 for more information.
Fig. 7
Fig. 7
Major interhelical H-bonding networks proposed for the hA3AR based on the rhodopsin structure, in the D582.50 region (A) and the DRY motif (B). The H-bonds are represented in yellow.
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