Cooperativity in agonist binding at the D2 dopamine receptor: evidence from agonist dissociation kinetics

Elodie Kara, Hong Lin, Philip G. Strange. University of Reading, School of Pharmacy, Reading, United Kingdom.

The D2 dopamine receptor is a G protein coupled receptor, and constitutes the main site of action of antipsychotic drugs. As for many other GPCRs, there is much evidence suggesting oligomerisation of the D2 receptor. In order to analyse that property further, we investigated non-competitive interactions between ligands at the D2S receptor in ligand dissociation studies. Membranes of CHO cells expressing the D2S dopamine receptor were incubated with the agonist ligand [3H]NPA (∼0.1 nM) for 3 hours in buffer A (20 mM Hepes, 1 mM EGTA, 1 mM EDTA, 6 mM MgCl2, 100 mM NMDG, 0.2 mM DTT pH 7.4) to reach equilibrium, washed to remove excess unbound radioligand and resuspended in ice cold buffer A at a concentration of 30 μg/100 μl. Aliquots of 100 μl of labelled membranes were placed in tubes and dissociation was induced by a 40-fold dilution with buffer A, in the absence or the presence of saturating concentrations of an antagonist/inverse agonist ((+)-butaclamol, or haloperidol), a partial agonist (aripiprazole), or an agonist (NPA, or dopamine) and the assay terminated by filtration at different times to follow the kinetics of dissociation. Data obtained for [3H]NPA dissociation in the absence or presence of (+)-butaclamol, or haloperidol, were best described by a one phase model with similar half-lives (470.8±37.2, 393.1±42.4 and 447.7±16.8 minutes for buffer alone or in the presence of (+)-butaclamol or haloperidol respectively, P>0.05), showing that (+)-butaclamol and haloperidol are acting competitively with respect to [3H]NPA dissociation. When dopamine, NPA and aripiprazole were included in the dilution buffer, data were best described by a model with two phases, where the fast phase corresponded to ∼30% of the total dissociation for dopamine and NPA, and ∼20% for aripiprazole. Moreover, the rate of the fast dissociation phase in the presence of dopamine (11.1±0.9 min) and aripiprazole (8.8±1.9 min) was faster than in the presence of NPA (13.8±0.5 min, P<0.05), showing that these ligands are inducing a different conformation of the receptor. The half-lives of the slow phase for the different ligands were not different and were similar to that seen for dilution only (P>0.05). These results cannot be explained by a ternary complex model and are consistent with an oligomeric receptor in which binding of [3H]NPA can be modulated cooperatively by ligands binding elsewhere in the oligomer.