Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A 2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A G(s) protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and G(s), we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [H-3]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [H-3]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 A2A 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 A2A 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 Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [H-3]CGS 21680 binding to the A2A 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 A2A receptor and G(s). The high-affinity binding of [H-3]CGS 21680 to the A2A recetor in rat striatal membranes was dependent on the presence of Mg2+ ions. Guanine nucleotides modulated [H-3]CGS 21680 binding to rat striatal membranes only in the presence of either MgCl2 or CaCl2. These findings demonstrate that in rat striatum the affinity state of the A2A receptor for agonists is regulated by guanine nucleotides and divalent cations.

Regulation of agonist binding to A2A adenosine receptors: effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion.

MAZZONI, MARIA ROSA;MARTINI, CLAUDIA;LUCACCHINI, ANTONIO
1993

Abstract

Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A 2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A G(s) protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and G(s), we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [H-3]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [H-3]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 A2A 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 A2A 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 Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [H-3]CGS 21680 binding to the A2A 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 A2A receptor and G(s). The high-affinity binding of [H-3]CGS 21680 to the A2A recetor in rat striatal membranes was dependent on the presence of Mg2+ ions. Guanine nucleotides modulated [H-3]CGS 21680 binding to rat striatal membranes only in the presence of either MgCl2 or CaCl2. These findings demonstrate that in rat striatum the affinity state of the A2A receptor for agonists is regulated by guanine nucleotides and divalent cations.
Mazzoni, MARIA ROSA; Martini, Claudia; Lucacchini, Antonio
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/205669
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