Reciprocal Regulation of Dopamine D1 and D3 Receptor Function and Trafficking by Heterodimerization

Abstract

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET 2 ), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET 2 signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [ 3 H]R-(ϩ)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/ D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves ␤-arrestin binding because it was blocked by mutant ␤-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R-and D3R-related dysfunctions. Dopamine (DA) controls various physiological functions, including locomotor activity, learning and memory, and motivation and reward; dopaminergic dysfunctions have been implicated in the development of Parkinson's disease, schizophrenia, and drug abuse. DA acts through five receptors, belonging to the G protein-coupled receptor (GPCR) family, that are divided into D1-like (D1 and D5) and D2-like (D2, D3, and D4) subtypes. Each receptor displays unique properties, including affinity for DA and specificity for G protein coupling and signaling and shows a peculiar neuronal distribution (Missale et al., 1998). The D1 receptor (D1R) is the most abundant and widespread DA receptor in the brain, where it is found at high density in both motor and limbic areas (Missale et al., 1998). The D3 receptor (D3R) is less abundant and exhibits a more restricted pattern of distribution with high concentrations in the ventral striatum, particularly in the shell of the nucleus accumbens and islands of Calleja (Sokoloff et al., 1990;Lévesque et al., 1992) and lower expression in other brain regions (Sokoloff et al., 1990;Lévesque et al., 1992;Schwartz et al., 1998). Both D1R and D3R have been implicated in the regulation of rewarding mechanisms and motivated behavior and in the modulation of emotional and cognitive p...

Identification of Dopamine D1–D3 Receptor Heteromers

“…Evidence was also obtained for a D 3 receptor-mediated potentiation of D 1 receptor signaling (cAMP accumulation) and modifications of agonist-mediated internalization with D 1 -D 3 receptor heteromerization (43).…”

“…During the resubmission of this manuscript another research group also demonstrated D 1 -D 3 receptor heteromerization in co-transfected cells with BRET techniques (43). Evidence was also obtained for a D 3 receptor-mediated potentiation of D 1 receptor signaling (cAMP accumulation) and modifications of agonist-mediated internalization with D 1 -D 3 receptor heteromerization (43).…”

G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

“…It is conceivable that the ability of GRK4 to target both receptors might help explain the observed functional synergism between activated D 1 R and D 3 R in both in vitro and in vivo studies. Co-expression of these receptors in HEK-293 cells increases the affinity of dopamine to D 1 R compared with cells expressing the D 1 R alone and promotes greater adenylyl cyclase stimulation (48). Unlike the other D 2 -like receptors, activated D 3 R weakly inhibits cAMP production, although it robustly inhibits adenylyl cyclase 5 (49), an isoform that is not expressed in the renal proximal tubule (50).…”

Heteromeric Dopamine Receptor Signaling Complexes: Emerging Neurobiology and Disease Relevance

“…The D1R and D3 receptors (D3R) show prominent colocalization in certain neurons of the direct striatonigral pathway (Ridray et al, 1998) and have been shown to form D1-D3 heteromers both in cells by BRET and FRET and to coimmunopecipitate from rat striatum (Fiorentini et al, 2008;Marcellino et al, 2008b). The physiological effect of this interaction was a D3R-stimulated increase in D1R-mediated responses in neurons that coexpressed both receptors (Fiorentini et al, 2008;Marcellino et al, 2008b).…”

“…The physiological effect of this interaction was a D3R-stimulated increase in D1R-mediated responses in neurons that coexpressed both receptors (Fiorentini et al, 2008;Marcellino et al, 2008b). Although this heteromer was suggested to have potential therapeutic value as a drug target in Parkinson's disease (Ferré et al, 2010), these findings may also have significant future implications for disorders involving striatal D1R transmission, the most notable being drug addiction, where D1R signaling has such a critical role in drug reward.…”