Regulation of agonist binding to A 2A adenosine receptors: Effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg 2+ ion

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Adenosine acts as a neuromodulator through at least two receptor subtypes, A 1 and A 2. A 2 receptors have been further divided into A 2A (high agonist affinity) and A 2B (low agonist affinity) receptors. Both A 1 and A 2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A G s protein couples the A 2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A 2A receptor and G s, we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A 2A receptor in rat striatal membranes, using [ 3H]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A 2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [ 3H]CGS 21680 binding to rat striatal membranes with about equal potency. The nonhydrolyzable analogs, GDP[S] and GTP[S], were equipotent inhibitors and approx. 100-times more potent than GDP and GTP. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demonstrated the presence of two binding sites in rat striatal membranes with mean values for K D of 5.6 and 343 nM and B max of 200 and 942 fmol/mg protein. The high-affinity binding site has the characteristics of the A 2A receptor. In the presence both of (0.1 mM) GDP[S] and GTP[S], the K D values for the high-affinity site were increased severalfold, whereas the low-affinity site was no longer detected in filtration assays. Dissociation studies revealed monophasic dissociation curves both in the absence and presence of 0.1 mM GDP[S]. However the K −1 value increased in the presence of guanine nucleotide. We also showed that in bovine striatal membranes agonist binding to A 2A receptors was modestly modulated by guanine nucleotides, suggesting differences of receptor G s-protein-coupling mechanism in different species. Divalent cations often increase agonist binding to different receptors, whereas Mg 2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [ 3H]CGS 21680 binding to the A 2A receptor and determined the requirement of these cations to obtain the modulation of binding by guanine nucleotides. We found that millimolar concentrations of divalent cations were required to obtain an effective interaction between the A 2A receptor and G s. The high-affinity binding of [ 3H]CGS 21680 to the A 2A recetor in rat striatal membranes was dependent on the presence of Mg 2+ ions. Guanine nucleotides modulated [ 3H]CGS 21680 binding to rat striatal membranes only in the presence of either MgCl 2 or CaCl 2. These findings demonstrate that in rat striatum the affinity state of the A 2A receptor for agonists is regulated by guanine nucleotides and divalent cations.