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

Summary 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-cyclopentyladenosine). 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.Abbreviations CHAPS 3-3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate - CPA N⁶-cyclopentyl-adenosine - dATP deoxy-ATP - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - DPX 1,3-diethyl-8-phenylxanthine - Gpp(NH)p guanylylimidodiphosphate - HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid - ¹²⁵I]HPIA (–)-N⁶-3-(¹²⁵I]-iodo-4-hydroxyphenylisoprop-yl)-adenosine - NECA 5-(N-ethylcarboxamido)adenosine - PEI polyethylenimine - R-PIA (–)-N⁶-(R-phenylisopropyl)-adenosine - ³H]XAC ³H]xanthine amine congener Send offprint requests to Dr. Wolfgang Schütz at the above address