SOLUBILIZATION AND CHARACTERIZATION OF THE A(2)-ADENOSINE RECEPTOR

Binding of [H-3] CGS 21680, an agonist radioligand selective for A2-ad enosine receptors (A2AR), to membranes and solubilized preparations fr om bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffecti ve in modulating agonist binding whereas cations, Na+ and Mg++, had di stinct effects. The addition of NaCl (200 mM) as well as the Mg++-free preparation of membranes led to a significant decrease, in binding af finity and the number of binding sites. Moreover, the presence of Nawas required for the demonstration of a guanine nucleotide effect, i.e . a decrease in maximal binding. Following solubilization, agonist-A2A R interactions were sensitive to guanine nucleotides even in the absen ce of Na+2; guanine nucleotides and Na+ had additive effects in reduci ng the number of binding sites. Moreover, the effect of GTP was revers ible, i.e. binding returned to control levels upon removal of the nucl eotide. This suggests the A2AR and its G protein (presumably G.) are s olubilized as a functional unit and may not dissociate even in the pre sence of GTP following solubilization. We, therefore, believe that a ' 'tight'' association exists between receptor and G protein (G.), and t hat guanine nucleotides and sodium act at different sites on the R-G c omplex. Drawing an analogy with similar observations on the avian beta -adrenergic receptor (Hertel et al, J.Biol.Chem. 265:17988-94, 1990; P arker & Ross, J.Biol.Chem. 266:9987-96, 1991) we postulate that the re gulatory features of the A2AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.