Differences in the GTP-regulation of membrane-bound and solubilized A1-adenosine receptors.

Using [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a 3H-labeled A1-selective adenosine antagonist with high affinity and extremely low non-specific binding, it was possible to quantitatively evaluate the effect of GTP on agonist binding. Competition experiments on [3H]DPCPX binding to guinea-pig cerebral cortical membranes in the absence of GTP showed a high- and a low-affinity state for adenosine receptor agonists (82/18% for N6-cyclopentyl-adenosine). Addition of 1 mmol/l GTP only partially converted the high-affinity state of the A1-adenosine receptor into a low-affinity state. This failure of complete conversion from high- to low-affinity state was also seen in membranes from rat testes under the same experimental conditions and, moreover, in guinea-pig brain membranes under different experimental conditions, such as in the presence of Na+ or when free Mg2+ has been reduced by EDTA. The only difference was that in the absence of Mg2+ the high-affinity state of the A1-receptor was markedly smaller than in the presence of Mg2+ (36% vs. 82%). By contrast, in the solubilized state of the receptor total conversion of all receptors into the low-affinity state was obtained upon addition of 1 mmol/l GTP. Reduction of binding of the agonist radioligand [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine with increasing concentrations of GTP and Gpp(NH)p demonstrated that the guanine nucleotide affinity to the solubilized A1-receptor was more than 100-fold higher than to the membrane-bound receptor. Hence, the incomplete transition of the high-affinity into the low-affinity state of the membrane-bound A1-receptor upon addition of GTP may be attributable to the low affinity of the membrane-bound receptor-G-protein complex for GTP.