Inverse agonism at dopamine D 2 receptors: a receptor recalcitrant to high levels of constitutive activation

Abstract

Neuroleptic drugs have been suggested to act as inverse agonists at the dopamine D 2 receptor. Nevertheless, the capacity with which inverse agonism at this receptor subtype can be resolved is limited. Modulation of the constitutive activation of the D 2 receptor was investigated in different cellular systems by monitoring either [ 35S]GTPγS binding responses at mutant Thr 343Ser D 2short receptor or inositol phosphates formation mediated by a chimeric D 2/α 1B 3ICL receptor. A weak (about −20% vs. basal [ 35S]GTPγS binding response) inverse agonist activity of putative dopamine antagonists (i.e., nemonapride, haloperidol or (+)-butaclamol) was observed with digitonin-permeabilized Chinese hamster ovary (CHO)-K1 cells stably expressing a mutant Thr 343Ser D 2short receptor only if a high (150 mM) KCl concentration was present in the binding buffer. No ligand-mediated decrease in basal [ 35S]GTPγS binding was observed on membrane preparations of the same cells. Markedly increased inverse agonist responses were obtained with a series of dopamine antagonists by exchange of the D 2short receptor's 3ICL by that of the α 1B-adrenoceptor and incorporation of an activating mutation (Ala 279Glu) in the distal BBXXB motif of its 3ICL and by co-expression with a G α11 protein. This chimeric D 2/α 1B receptor construct displayed a ligand binding profile comparable to that of the wt D 2short receptor and an effector activation profile close to that of the wt α 1B-adrenoceptor. Most of the putative dopamine antagonists attenuated by −54% to −59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved virtually as a silent antagonist (+5% vs. basal IP level) and antagonised both dopamine (p K B: 7.61)- and tropapride (p K B: 8.52)-mediated IP responses. Clozapine, olanzapine and raclopride displayed partial inverse agonist properties (−31%, −67% and −71% vs. tropapride 1 μM, respectively), whereas bromerguride (+63%) and (+)-UH 232 (+88%) demonstrated positive agonism.