Agonist occupied G protein-coupled receptors enhance the exchange of GDP by GTP at G proteins. The intermediate in this process is an agonist-receptor-G protein complex where no nucleotide is bound to the G protein. This complex, sensitive to the presence of divalent cations, exhibits a high affinity for agonists and can often be monitored in binding studies in membranes using a [ ³H]agonist. Here we report the results of initial studies of the effects of the incubation conditions on the potencies of GDP, GTP, and two 'nonhydrolysable' analogues, GDß S and GppNHp, for disrupting [ ³H]agonist-adenosine A₁ receptor(A₁R)-G protein complexes.

Membranes from CHO cells expressing high levels of the human A₁R (ca 8 pmoles/mg protein) were harvested as described previously (Browning et al, 2000). Radioligand binding studies were carried out at 23^o in a 100mM NaCl/20 mM Hepes buffer pH 7.4 containing 10mM MgCl₂ , no added Mg, or 10mM EDTA. Membranes were pretreated with adenosine deaminase, followed by incubation with the agonist [ ³H]PIA ([³H]-N⁶-(2-phenylisopropyl)adenosine, 3nM) in buffer containing 30 µ g/ml saponin in the presence or absence of guanine nucleotides for 15, 60 or 180 min. Results are reported as p(IC ₅₀) values ± sem(range/2) for n = 2-6 experiments in Table 1.

Under all incubation conditions high concentrations of guanine nucleotides inhibited >95% of the specific [ ³H]PIA binding. The inhibition curves had slope factors not different from 1. In 10mM EDTA, nucleotide potencies were independent of incubation time, suggesting that the nucleotides were stable and that there were no catalytic effects. The rank order of potency is GDP > GDPβS ³ GTP > GppNHp, with GDP unexpectedly being more potent than GTP. In the presence of added Mg²⁺ , the actions of the hydrolysable nucleotides decreased slightly with time but those of GDPβS and GppNHp increased. The potencies of the triphosphates were similar to those found in 10 mM EDTA but those of the diphosphates had been reduced 10-40 fold, such that GTP was more potent than GDP. The rank order of potency at 15 min (GTP > GppNHp > GDP GDPβS) has changed radically. The assays in the absence of added Mg ²⁺ indicated a 15-20 fold decrease in both GDP and GTP potencies between 15 min. and 60 min. This is incompatible with just GTP hydrolysis to GDP. At short incubation times the rank order of potency is GDPβS ≈ GTP ≈ GDP > GppNHp. In conclusion, the absolute and relative potencies of different guanine nucleotides to interact with, and disrupt, agonist-A₁R-G protein complexes are extremely sensitive to assay conditions.