The human adenosine A1 receptor (A₁R) has served as a useful model of GPCR binding and activation. The aim of this study is to examine the dissociation kinetics of a [³H]agonist from A₁R-G protein complexes and to compare them with the dissociation of a [³H]antagonist from A₁ receptors in order to further the understanding of agonist binding to GPCRs.

Membranes from CHO cells expressing high levels (9 pmoles/mg protein) of the A₁ receptor were harvested as previously described (Browning et al, 2000; Cohen et al., 1996). Radioligand binding studies were carried out at 23^oC in a Na/Mg/Hepes buffer (Cohen et al., 1996). Membranes, prelabelled with the inverse agonist [³H]DPCPX ([³H]-8-cyclopentyl-1,3-dipropylxanthine, 9 nM) or the agonist [³H]CHA ([³H]-N⁶-cyclohexyladenosine, 5 nM) for 60 min, were diluted 20 fold into buffer containing non-radioactive CHA or DPCPX as a 'chase' ligand. In further experiments there was no addition of a chase ligand ('dilution only') or the addition of GTP (10^-4 M) to disrupt receptor-G protein interactions. Dissociation was allowed to progress for 5-180 min, at which times the membranes were harvested by rapid filtration.

The dissociation rate of [³H]DPCPX was mono-exponential (k_off 0.22 ± 0.01 min^-1, n = 5), unaffected by GTP or by the nature or presence of a chase ligand. In contrast, [³H]CHA dissociation, initiated by dilution, was incomplete, decaying mono-exponentially (k_obs 0.09 ± 0.01 min^-1, n = 2) to a plateau of 71 ± 1% of the initial binding, compared to the predicted level of 14%. This is the phenomenon of 'agonist locking', whose mechanism is unknown (Cohen et al., 1996).

[³H]CHA dissociation was enhanced relative to 'dilution only' in the presence of DPCPX or CHA. Importantly, CHA induced greater dissociation than DPCPX. Analysis of the data by a two-exponential model indicated that the greater enhancement of [³H]CHA dissociation by CHA was due to an increase in the amplitude of the faster component of dissociation (49 ± 3 % relative to 32 ± 2 % in the presence of DPCPX, n = 5). The enhancement of [³H]CHA dissociation by CHA and DPCPX occurred at low concentrations of the chase ligands. The log potency of CHA to enhance dissociation increased from 8.3 ± 0.2 at 10 min to 9.2 ± 0.1 at 180 min (n=8). Little enhancement of [³H]CHA dissociation by DPCPX occurred after 10 or 60 min: at 180 min its log potency was 9.3 ± 0.2 (n = 8). CHA- and DPCPX-induced dissociation of [³H]CHA binding was also observed in the presence of GTP ([³H]CHA binding decreasing by 68 ± 5% and 20 ± 7% respectively at 60 min, relative to dilution alone, n = 4).

The novel mechanism by which the chase ligands can enhance [³H]agonist dissociation from A1R-G protein complexes to differing extents implies the transfer of information between a high affinity chase binding site and the [³H]agonist binding site. This could occur through a direct association of receptors or via other proteins associated with the receptor.

Browning, C et al. (2000) Br. J. Pharmacol. 129, 42P.
Cohen, FR et al. (1996) Br. J. Pharmacol. 117, 1521-1529.