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The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.

Luciferase complementation assays have emerged as a simple means of monitoring receptor-effector interactions in living cells in a time-resolved manner. Here, we describe a nanoluciferase complementation assay capable of reporting on β-arrestin2 recruitment to the human dopamine D₃ receptor (D₃R) upon its activation in intact HEK293T cells. Using this assay in time-resolved experiments, we detect differences in arrestin response termination rates between the endogenous agonist dopamine and the synthetic D3R agonist FAUC-73. We also investigate the influence of exogenous GRK2 on β-arrestin2 recruitment to the D₃R. We find that, in contrast to the D₂R and D₄R, the potency of dopamine to induce arrestin recruitment to D₃R is not significantly influenced by GRK2 overexpression. In further agreement with a lack of GRK2 regulation of D₃R signalling and again contrary to the D₂R and D₄R, we do not observe dopamine-induced recruitment of GRK2 to D₃R. Conversely, dopamine concentration-dependently decreases the interaction between GRK2 and D₃R. Additionally, we examine both the Ser-9 and Gly-9 variants of the human D₃R, which, according to some earlier reports, differ in terms of dopamine affinity and functional potency. However, we find no difference in the concentration-response relationships between these two variants, neither when arrestin recruitment nor GRK2 interactions are studied. In summary, the present report demonstrates the utility of nanoluciferase complementation for studying D₃R pharmacology in living cells.

The dopamine D3 receptor (D3R) is expressed in the ventral striatum as well as the cerebral cortex of the brain and is an emerging target for the treatment of schizophrenia, Parkinson’s disease, cognitive deficits, restless legs, and drug abuse. However, in addition to therapeutic actions, D3R activation has been associated with adverse effects such as impulse control disorders and dyskinesia. The D3R signals via two distinct pathways, the classical G protein-dependent pathway and the more recently discovered arrestin pathway. The respective roles of either pathway in (patho)physiological functions as well as in desirable and undesirable drug effects remain unknown. Receptor mutants that signal selectively via either of the two pathways would be helpful tools for future exploration of this topic in vivo. Here, we used site-directed mutagenesis and a nanoluciferase complementation assay to find point mutations in the D3R that result in such characteristics. We identified one mutant, A131W, which is unable to signal via G proteins, while leaving arrestin recruitment intact. Confirmatory experiments indicated that this mutant is expressed on the cell surface at WT levels but is unable to elicit G protein-dependent downstream effects such as adenylate cyclase inhibition and potassium channel opening. If introduced into experimental animals, this D3R mutant may become valuable for future studies of behaviours known to be strongly modulated by D3R ligands, such L-DOPA-induced dyskinesia, reward-driven behaviour, and cognition.