Distinct Dopamine D2 Receptor Antagonists Differentially Impact D2 Receptor Oligomerization

Abstract

Dopamine D2 receptors (D2R) are known to form transient homodimer complexes, of which the increased formation has already been associated with development of schizophrenia. Pharmacological targeting and modulation of the equilibrium of these receptor homodimers might lead to a better understanding of the critical role played by these complexes in physiological and pathological conditions. Whereas agonist addition has shown to prolong the D2R dimer lifetime and increase the level of dimer formation, the possible influence of D2R antagonists on dimerization has remained rather unexplored. Here, using a live-cell reporter assay based on the functional complementation of a split Nanoluciferase, a panel of six D2R antagonists were screened for their ability to modulate the level of D2LR dimer formation. Incubation with the D2R antagonist spiperone decreased the level of D2LR dimer formation significantly by 40–60% in real-time and after long-term (≥16 h) incubations. The fact that dimer formation of the well-studied A2a–D2LR dimer was not altered following incubation with spiperone supports the specificity of this observation. Other D2R antagonists, such as clozapine, risperidone, and droperidol did not significantly evoke this dissociation event. Furthermore, molecular modeling reveals that spiperone presents specific Tyr1995.48 and Phe3906.52 conformations, compared to clozapine, which may determine D2R homodimerization.

Striatal Dopamine D2-Muscarinic Acetylcholine M1 Receptor–Receptor Interaction in a Model of Movement Disorders

“…This assay utilizes two inactive fragments of a split NL, which, when fused to two interacting proteins, come into close proximity and reassemble into a functional protein (Wouters et al, 2019b). As shown in Figure 1C, co-expression of M 1 R and D 2 R fused to the large and small subunits of a split NL (M 1 R-LgBiT and D 2 R-SmBiT, respectively) yielded a high luminescent signal (Figure 1C) when compared to HEK293T cells expressing either constructs for M 1 R and CB 1 R (M 1 R-LgBiT + CB 1 R-SmBiT) or D 2 R and CB 2 R (D 2 R-SmBiT + CB 2 R-LgBiT), as previously reported (Wouters et al, 2019a). In addition, very low signals were observed in cells expressing either M 1 R (19 ± 3.5) or D 2 R (9 ± 1.7), along with HaloTag-SmBiT or HaloTag-LgBiT, respectively.…”

“…The construct was verified by restriction digest and Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). This resulted in the fusion of the split NanoLuciferase (NL) fragment LargeBiT (LgBiT; 18 kDa) to the C-terminus of M 1 R. The constructs of cannabinoid types 1 and 2 receptors (CB 1 R and CB 2 R, respectively) and D 2 R fused with LgBiT or Small BiT (SmBiT; 1 kDa) were previously developed and described by our research group (Cannaert et al, 2016;Wouters et al, 2019a).…”

“…In all conditions, the construct encoding for the fluorescent protein Venus was co-transfected (5% of the total DNA transfected). The NanoBiT R assay was performed as described previously (Wouters et al, 2019a). Briefly, 48 h posttransfection, the cells were washed two times with PBS, detached, and centrifuged for 5 min at 1,000g at RT.…”

Dopamine Receptor Subtypes, Physiology and Pharmacology: New Ligands and Concepts in Schizophrenia

“…It has allowed to show the presence of an alternative mechanism of D3 receptor internalization independent of β-arrestin and used by group II GPCR ( Xu et al., 2019 ). Considering the excess D2 homodimers detected in schizophrenia ( Wang et al., 2010 ), the effects of DA antagonists on these entities has been specifically explored using bivalent ligands ( Pulido et al., 2018 ; Wouters et al., 2019 ). A molecular model of the homodimer has been also generated for D2 to provide docking information relative to bivalent ligands with different pharmacological properties (for example orthosteric and allosteric agents) ( Kaczor et al., 2016 ).…”

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor

“…The ability to form multimeric receptor assemblies affects physiological aspects [ 193 , 194 , 195 , 196 ] but is also associated with pathophysiological/pharmacological issues [ 197 , 198 , 199 , 200 , 201 ]. GPCR oligomerization can lead to modified ligand binding [ 187 , 202 , 203 , 204 ], G protein selectivity [ 205 ], signaling [ 206 , 207 , 208 ], or cell surface expression [ 209 , 210 ] and internalization [ 211 ]. Allosteric or cooperative effects between individual protomers in oligomeric receptor constellations have been observed [ 212 , 213 ].…”