Abnormalities of Dopamine D 3 Receptor Signaling in the Diseased Brain

Abstract

Dopamine D₃ receptors (D₃R) modulate neuronal activity in several brain regions including cortex, striatum, cerebellum, and hippocampus. A growing body of evidence suggests that aberrant D₃R signaling contributes to multiple brain diseases, such as Parkinson’s disease, essential tremor, schizophrenia, and addiction. In line with these findings, D₃R has emerged as a potential target in the treatment of neurological disorders. However, the mechanisms underlying neuronal D₃R signaling are poorly understood, either in healthy or diseased brain. Here, I review the molecular mechanisms involved in D₃R signaling via monomeric D₃R and heteromeric receptor complexes (e.g., D₃R-D₁R, D₃R-D₂R, D₃R-A_2aR, and D₃R-D₃nf). I focus on D₃R signaling pathways that, according to recent reports, contribute to pathological brain states. In particular, I describe evidence on both quantitative (e.g., increased number or affinity) and qualitative (e.g., switched signaling) changes in D₃R that has been associated with brain dysfunction. I conclude with a description of basic mechanisms that modulate D₃R signaling such as desensitization, as disruption of these mechanisms may underlie pathological changes in D₃R signaling. Because several lines of evidence support the idea that imbalances in D₃R signaling alter neural function, a better understanding of downstream D₃R pathways is likely to reveal novel therapeutic strategies toward dopamine-related brain disorders.

Introduction

In the 1950s, after the role of dopamine (DA) as a signaling molecule in the brain was demonstrated, ¹ it was reported that DA levels dramatically decrease in Parkinson’s disease (PD). ² Shortly after that, the DA precursor l-DOPA (l-3,4-dihydroxyphenylalanine) was started to be used to increase DA concentration in the brain of patients with PD. ² The temporary relief of PD symptoms by l-DOPA indicated that loss of DA signaling is the pathognomonic feature of PD. Evidence from animal models and humans has shown that, in addition to PD, DA signaling also plays a central role in several brain disorders including schizophrenia, essential tremor, attention deficit hyperactivity disorder, depression, and addiction. This plethora of DA-associated diseases closely matches with the function of the DA system in multiple brain functions such as motor coordination, emotions, memory, reward mechanisms, and neuroendocrine regulation.

The brain DA system is constituted by DA-containing nuclei (i.e., substance nigra pars compacta, ventral tegmental area, and arcuate nucleus) and by the target areas (i.e., cortex, basal ganglia, thalamus, limbic structures, and pituitary gland). ¹ On target areas, DA acts through five DA receptors, which belong to the superfamily of seven-transmembrane G protein–coupled receptors (GPCRs). Based on structural, biochemical, and pharmacologic criteria, DA receptors have been grouped into two main families: the D₁ (D₁ and D₅ subtypes) and D₂ (D₂, D₃, and D₄ subtypes) families. ³ Structurally, the major difference between D₃ receptor (D₃R) and D₁ family receptors is located in the third intracellular loop and carboxyl terminal tail: D₃R has a long third loop and short carboxyl terminus, whereas D₁ and D₅ receptors display opposite features showing short third intracellular loops and long carboxyl terminal tails. Compared with D₂ and D₄ receptors, D₃R has a distinctive third intracellular loop. ³

A growing body of evidence suggests that aberrant D₃R signaling contributes to several brain disorders. Consequently, D₃R has emerged as a potential therapeutic target in the treatment of major neurological disorders such as schizophrenia, ⁴ PD, ⁵ and addiction. However, the mechanisms underlying D₃R signaling are poorly understood, either in healthy or diseased brain. Here, I review recent reports on the molecular mechanisms involved in D₃R signaling via monomeric D₃R and heteromeric receptor complexes. Then, I illustrate examples of qualitative and quantitative changes in D₃R signaling that may contribute to several brain diseases. I conclude with a description of potential targets for the modulation of D₃R signaling. Unraveling the unknown downstream signaling pathways activated by D₃R in both the healthy and the diseased brain is likely to reveal new therapeutic strategies toward DA-associated disorders.

Monomeric and Heteromeric D₃R Signaling

Brain D₃R messenger RNA (mRNA) is detected early in development (e.g., in the embryonic days 10 and 11 of rodents) ⁶ and continually expressed during the postnatal period.^7–10 In adult rats ¹¹ and mice, ¹² regions that affect emotional, cognitive, and endocrine functions express D₃R mRNA (e.g., nucleus accumbens, islands of Calleja, hippocampus, prefrontal cortex, hypothalamus, and striatum). Interestingly, in the mouse brain, the D₃R co-expressed with D₁R and D₂R with regional, sex, and age-dependent differences in the co-expression pattern. ¹² In the human brain, D₃R mRNA has been detected in nucleus accumbens and in the islands of Calleja. ¹³ Although D₃R is widely expressed in the brain, elucidating D₃R signal transduction mechanisms has been challenging because D₃R and D₂R have similar pharmacologic profiles due to the high sequence identity and homology shared by these receptors (e.g., homology of 52% overall and of 75% in the transmembrane domain of rat sequences). ¹¹ To specifically activate D₃R, many studies have been performed in heterologous expression systems. These experimental systems have revealed that D₃R can be linked to a wide array of intracellular signals via its coupling to multiple G protein α subunits, such as G_iα3, ¹⁴ G_o,^15,16 G_q,^17,18 and G_s. ¹⁹ Consistent with its ability to activate multiple G proteins, D₃R can modulate several downstream effectors including adenylyl cyclase, cyclin-dependent kinase 5 (CDK5), creatine kinase 1 (CK-1), protein kinase A (PKA), protein kinase B (PKB)/Akt, protein kinase C (PKC), phospholipase C (PLC), phospholipase D (PLD), protein phosphatase 2B (PP2B), and extracellular signal–regulated kinase (ERK).^{14–16,18,20–27} Moreover, under particular conditions, D₃R can mediate both opposite and synergistic interactions with signaling pathways related to the production of cyclic adenosine monophosphate (cAMP). ²¹ Despite having similar sequences and pharmacologic profiles, D₃R and D₂R exhibit significant biochemical differences. For instance, D₂R activates ERK via a G_iα-dependent pathway, whereas D₃R activates ERK by a mechanism that depends on G_βγ. ²⁶

In addition to the canonical signaling via monomeric GPCRs, several reports indicate that the interaction among GPCRs (forming dimers and higher-order entities) can influence GPCR signaling both quantitatively (i.e., heteromers exhibit increased affinity for the ligand) and qualitatively (i.e., heteromers use alternative signaling pathways).^28,29 In particular, D₃R functionally interacts with adenosine A_2a receptors (A_2aR), ³⁰ D₁R,^31,32 D₂R, ³³ and nicotinic acetylcholine receptors. ³⁴ The D₃R-D₁R interaction increases the affinity of D₁R for its ligand after D₃R activation, whereas no change is observed in the D₃R affinity to its ligand. ³¹ The activation of A_2aR in the D₃R-A_2aR macromolecular complex reduces both the affinity of D₃R for DA and the D₃R-mediated inhibition of adenylate cyclase, whereas A_2aR signaling is inhibited by D₃R activation. ³⁰ Moreover, D₃R interacts with its own alternatively spliced variant, named D₃nf, as demonstrated by co-immunoprecipitation and colocalization experiments.^35–37 The expression of D₃nf reduces the ligand-binding capacity of D₃R, possibly due to D₃R-D₃nf mislocalization from the plasma membrane to intracellular compartments. ³⁷

The D₃R gene contains 6 exons and 5 introns and produces at least 7 distinct alternatively spliced variants including the full-length D₃R, a shorter receptor isoform (D₃S) lacking 21 amino acids within the third intracellular loop (IL₃), and the truncated isoform D₃nf lacking transmembrane-spanning domains 6 and 7 by a premature stop codon. ³⁸ Interestingly, D₃R and D₃S bind DA with high affinity, whereas the 5 additional D₃R variants including D₃nf do not bind DA, but they may regulate receptor dimerization. ³⁹ Overall, this evidence supports the idea that receptor-receptor interactions at neuronal surface modulate D₃R signaling.

Multiple D₃R-associated pathways have been described in neurons.^22,27 Indeed, D₃R can modulate neural activity by acting on neurotransmitter receptors via PKA, as well as on ion channels via G_αi/o, as demonstrated by electrophysiological and imaging studies in isolated neurons and brain slices from rodents.^17,40–42 These studies, however, did not clarify whether D₃R effects are mediated by monomeric-canonical pathways or by heteromeric receptors. In medium spiny neurons (MSNs) of the striatum, D₃R modulates Ca²⁺ channels via PLC and PP2B ⁴³ and can also activate the Akt/mTOR/p70S6/4E-BP1 pathway, ⁴⁴ which play a key role in protein synthesis, synaptic plasticity, and memory. Also, D₃R suppresses synaptic transmission by reducing GABA_A receptor current via PKA-mediated endocytosis of GABA_A receptors in the nucleus accumbens ⁴⁰ and in CA1 pyramidal neurons from the hippocampus. ⁴¹ Actions of D₃R in the brain might be region specific ⁷ and may be quantitative and qualitatively modified under disease states, as discussed in the following sections.

D₃R Signaling in Brain Diseases

Essential tremor

A critical insight into the role of D₃R signaling in brain dysfunction was provided by studying the D₃R Ser9Gly polymorphism in patients with essential tremor, the most commonly inherited movement disorder. In essential tremor, a subtle change in the D₃R sequence (Ser9Gly polymorphism) increases D₃R coupling to both the inhibition of cAMP formation and the activation of the MAPK pathway (Figure 1A). The enhanced D₃R function, associated with the Gly-9 variant, was linked with the risk and age at onset for essential tremor. ⁴⁵ Further supporting the primary role of the gain of function of D₃R in pathological DA signaling, the D₃R-Gly-9 variant has been associated with impulsive behavior in PD, the second most prevalent neurodegenerative disease. Thus, these data suggest that an enhanced D₃R signaling could impair reward-risk assessment in the mesolimbic system and contribute to the development of impulsive behavior, in carriers of this genotype. Overall, these data suggest that a gain of function in two D₃R signaling pathways significantly contributes to brain dysfunction. It is noteworthy that the association of the D₃R Ser9Gly polymorphism with essential tremor has been found to be significant in American, French, ⁴⁵ and Spanish ⁴⁶ but not in Italian ⁴⁷ or Asian ⁴⁸ populations.

Figure 1.

D₃R in brain diseases. Based on recent reports, all models show molecular D₃R changes/variants associated with specific pathological conditions. (A) The D₃R-Gly-9 variant has been linked with the risk and age at onset for essential tremor. Relative to the D₃R-Ser-9 variant, the D₃R-Gly-9 is more efficient in both the inhibition of cAMP formation and the activation of ERK. (B) Under normal DA levels (control striatum), D₃R interacts with D₃nf and modulates Ca_V1 (L-type) channels through a PLC/IP₃/Ca²⁺/PP2B signaling pathway, whereas after chronic DA depletion (as seen in PD), D₃R-D₃nf interaction is reduced as a result of the D₃nf downregulation. Membrane redistribution of the “D₃nf-free” D₃R may situate it near the Ca_V2.1 channels, thus allowing channels to sense phosphatidylinositol-4,5-biphosphate depletion and reduce their opening after PLC activation by D₃R. (C) Some postmortem studies suggest that brain D₃R levels may be elevated in schizophrenia. (D) Long-lasting neuroadaptations following chronic drug use include an enhancement in both D₃R expression and D₃R-dependent signaling (Akt and ERK activation). Also, the functionally enhanced D₃R-Gly-9 variant has been associated with drug-dependence. D₃R indicates D₃ receptor; DA, dopamine; ERK, extracellular signal–regulated kinase; PLC, phospholipase C.

Parkinson’s disease

Supporting the idea that a gain of function of D₃R can impair brain physiology, recent evidence suggests that D₃R activity is enhanced in PD,^43,49 a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Loss of dopaminergic neurons is accompanied by a dramatic reduction in DA levels in the striatum.^50–52 Several reports have shown that DA depletion induces compensatory mechanisms (e.g., an increase in the number and the affinity of receptors), similar to that observed after ligand depletion in other receptor-ligand systems.^53,54 For instance, positron emission tomography using [ ¹¹ C]raclopride has shown an increased density of D₂-class receptors in the putamen nucleus in PD. ⁵⁵ Also, biochemical, electrophysiological, and behavioral data have shown that DA depletion increases the sensitivity (supersensitivity) of D₂-class receptors in animals models of PD (e.g., DA depletion induced by reserpine, ⁵⁶ α-methyl-p-tyrosine, ⁵⁷ 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP], ⁵⁸ and 6-hydroxydopamine [6-OHDA]).^49,59,60 In the 6-OHDA model, electrophysiological recordings in striatal MSNs have shown that D₃R signaling contributes to the D₂-class supersensitivity,^43,49 thus suggesting that DA depletion may increase D₃R expression and activity.

The enhancement of D₃R signaling by DA depletion may be reflecting changes in pharmacologic parameters (potency, affinity, and receptor number). At present, however, there is no consensus on which pharmacologic parameter is mainly affected.^{10,11,61–64} Although unilateral lesion of the substantia nigra with 6-OHDA increases the potency of the selective D₃R agonist 7-OH-DPAT in striatum ⁶⁵ and cerebellum, ⁶⁶ pharmacologic depletion of catecholamines increases the affinity of putative D₃ sites with no increase in the total number of sites in striatum. ⁶⁷ An alternative mechanism underlying the enhancement of D₃R signaling following DA depletion may rely on a reduced interaction of D₃R with its regulatory splice variant D₃nf. Indeed, D₃nf protein levels were found to be reduced in striatal homogenates from 6-OHDA lesioned rats. ⁴³ In line with a reduction in D₃R-D₃nf interaction after DA depletion, the D₃R/D₃nf mRNA ratio increases when D₃R is pharmacologically blocked (i.e., mimicking “hypodopaminergic” states). ⁶⁸

According to recent reports, qualitative changes in D₃R signaling may also contribute to the pathophysiology of PD. In particular, a switch in D₃R signaling has been described in striatal MSNs and striatonigral terminals of hemiparkinsonian rats,^43,69 a PD model based on the 6-OHDA–mediated lesion of substantia nigra pars compacta in one hemisphere. In striatal MSNs, while physiological D₃R signaling inhibits Ca_V1 (L-type) Ca²⁺ channels by PP2B activation, D₃R additionally modulates Ca_V2.1 (P/Q-type) channels via the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP₂) after DA depletion (Figure 1B). This Ca_V2.1 modulation by the D₃R-PIP₂ pathway is not detected in neurons from control animals, thus suggesting that this pathway is induced by DA depletion. ⁴³ Because Ca_V2.1 channels mediate γ-aminobutyric acid (GABA) release from the terminals of striatal neurons, the PIP₂-dependent D₃R signaling may reduce GABA release and affect synaptic transmission between striatal neurons after DA depletion.

In striatonigral terminals, the switch in D₃R signaling is related to the D₃R-D₁R interaction. ⁶⁹ Although D₃R activation potentiates both the stimulation of GABA release and the production of cAMP by D₁R in terminals from the control (nonlesioned) hemisphere, D₃R inhibited both D₁R actions in terminals from the denervated hemisphere. ⁶⁹ Overall, data from MSNs and striatonigral terminals support the notion that DA depletion switches D₃R signaling in basal ganglia.

l-DOPA, the gold standard treatment for PD, reduces parkinsonian symptoms but in later stages induces dyskinesia, a side effect characterized by hyperkinetic involuntary movements. ⁷⁰ Notably, l-DOPA increases D₃R expression in the caudate/putamen nuclei and striatonigral MSNs.^{62,63,71–73} Recent evidence indicates that D₃R plays a major role in l-DOPA–induced dyskinesia, which can be reduced by blocking D₃R^73–75 and D₁R ⁷⁵ in parkinsonian models. Interestingly, in MPTP parkinsonian primates, a partial block of D₃R (using a partial agonist for D₃R) reduces dyskinesia without affecting the therapeutic effects of l-DOPA, whereas no recovery of motor disturbances is observed if D₃R is entirely blocked. ⁷⁴ These data emphasize that D₃R mediates different actions, participating in both dyskinesia and motor recovery following l-DOPA treatment, thus highlighting the importance of fine-tuning D₃R signals in PD-diseased brain.

Schizophrenia

Dopamine signaling is the main target in schizophrenia, a mental disorder characterized by hallucinations, bizarre delusions, and negative symptoms such as lack of motivation, reduction in spontaneous speech, and social withdrawal. Indeed, first-generation antipsychotics (e.g., haloperidol and chlorpromazine) bind to D₂-class receptors.^76,77 Since its cloning in 1990, D₃R emerged as a potential target for antipsychotics.^11,78 Although it is controversial whether D₃R levels are affected in schizophrenia, ⁷⁹ postmortem studies suggest that D₃R levels may be elevated in people with schizophrenia who are off antipsychotics (Figure 1C). ⁴ In contrast, the parietal cortex of postmortem tissue from long-term hospitalized patients with chronic schizophrenia expresses low D₃R levels. ⁸⁰ These data suggest that long-term antipsychotic medication may modify brain D₃R expression in schizophrenia. Interestingly, low D₃R mRNA levels in patients with schizophrenia have been associated with an enhanced D₃nf-specific splicing of D₃R pre-mRNA. ⁸¹

Addiction

Chronic drug use induces long-lasting neuroadaptations which has been associated with dopaminergic abnormalities. Although studies of D₃R expression in human cocaine-dependent subjects have had conflicting results, ³⁹ the use of the D₃R-preferring radioligand [ ¹¹ C](+)PHNO has shown higher number of available D₃R in the substantia nigra, hypothalamus, and amygdala of cocaine addicts, compared with healthy controls ⁸² (Figure 1D). Notably, substantia nigra D₃R levels correlated with years of cocaine use. ⁸² Consistent with the idea that chronic cocaine exposure leads to adaptive increases in D₃R expression, a 6-fold increase in D₃R mRNA levels was found in the nucleus accumbens of cocaine overdose victims, as compared with age-matched and drug-free control subjects. ⁸³ Similarly, an increased [ ¹¹ C](+)PHNO binding, reflecting higher D₃R levels, has also been observed in the substantia nigra of methamphetamine users, ⁸⁴ as well as in hypothalamus of alcohol-dependent patients. ⁸⁵ According to the heightened D₃R signaling in drug users, the functionally enhanced D₃R-Gly-9 variant was associated with the development of early-onset heroin dependence in Chinese population (Figure 1D). ⁸⁶

Studies in vitro and in animal models support the idea that drug use induces D₃R signaling abnormalities. In vitro, cocaine increases dendritic arborization and soma area in cultured dopaminergic neurons from mouse via D₃R-dependent activation of ERK and Akt. ⁸⁷ In rats, it has been suggested that D₃R play an important role in mediating nicotine’s effects on the brain. ⁸⁸ In both adolescents and adult rats, nicotine upregulates D₃R but reduces D₃nf mRNA levels in the nucleus accumbens, thus increasing the D₃R/D₃nf ratio (Figure 1D). ⁸⁸ It has also been proposed that D₃R and its alternatively spliced isoform D₃nf may play a role in cocaine addiction (a risk factor for schizophrenia) and behavioral sensitization (the progressive and long-lasting augmentation of certain behaviors following repetitive stimulant drug administration). ³⁹ Supporting this idea, D₃R has been found to enhance the reinforcing effect of cocaine, ⁸⁹ and blockade of D₃Rs inhibits cocaine’s rewarding effects and relapse to drug-seeking behavior in rats. ⁹⁰ A cornerstone study demonstrated the crucial role of D₃R signaling in the motivation to take drug induced by drug-related cues. ⁹¹ In this study, rats were trained to self-administer cocaine by a lever pressing. Next, progressively, a light stimulus was associated with cocaine self-administration. Light stimulus, which then becomes the conditioned stimulus, gains reinforcing properties and finally maintains drug-seeking behavior even without drug delivery. Remarkably, the selective D₃R partial agonist BP897 dose-dependently reduced cue-induced cocaine-seeking behavior in rats trained under this schedule of reinforcement. ⁹¹

Overall, multiple lines of evidence support the idea that D₃R plays a central role in addiction, thus driving a need to develop novel pharmacologic agents targeting this receptor. Since 2005, 110 patents or patent applications have been published on original compounds with D₃R selectivity. ⁷⁹ In contrast to the initial lack of D₃R-selective drugs, a list of compounds with relative high selectivity for D₃R currently includes the following: BP 897, SB-277011A, S33084, ABT-925, GSK598809, and F17141. ⁷⁹ In particular, GSK598809 has proven to transiently alleviate craving in smokers after overnight abstinence, thus providing the first clinical evidence for a usefulness of D₃R antagonist for the treatment of addiction. ⁹²

Emerging perspectives for improving the treatment of brain disorders focus on the understanding of pathophysiology mechanisms to identify disease pathways, which will finally facilitate the selection of therapeutic targets. ⁹³ In DA-related brain diseases, quantitative and qualitative changes in D₃R signaling may be reflecting direct effects on basic mechanisms that control D₃R activity at the cell surface, such as desensitization and internalization. A better understanding of these basic mechanisms in both the healthy and the diseased brain is likely to reveal new molecular targets for therapeutic.

Modulation of D₃R Signaling

Dopamine receptors are modulated by several mechanisms, such as homologous and heterologous desensitization, a reversible reduction in signal transduction after acute activation. Experimental and theoretical data suggest that D₃R desensitization (tolerance) can develop after its structural rearrangement following agonist binding, and that the magnitude of the second D₃R response can be reduced by 60% compared with the first response. ⁹⁴ Also, D₃R can be desensitized following phosphorylation by both PKC ⁹⁵ and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). ⁹⁶ Both PKC and CaMKII phosphorylate the 229-serine residue at the IL₃, a region that exhibits high divergence between D₂R and D₃R, and may play a crucial role in the unique signaling signatures of each receptor subtype. ⁹⁷ Downregulation of D₃R signaling by CaMKII depends on intracellular Ca²⁺ levels and, therefore, is associated with neuronal activity. ⁹⁶ Importantly, the CaMKII-mediated inhibition of D₃R is not observed after DA depletion. ⁶⁹

Although D₃R undergoes limited agonist-induced internalization, ⁹⁸ D₁R-D₃R heterodimers can be internalized in response to the paired stimulation of both D₁R and D₃R via a β-arrestin–dependent mechanism in human embryonic kidney 293 cells. ³² Also, a recent report showed that PKC-mediated phosphorylation of D₃R can induce clathrin-mediated D₃R endocytosis and lysosomal D₃R degradation. ⁹⁵ Similarly, via caveolin/clathrin-mediated D₃R internalization, dysbindin-1 reduces the magnitude and potency of DA-induced cAMP production and phosphorylation of ERK1/2 and Akt. ⁹⁹ Notably, dysbindin-1 is a candidate gene for schizophrenia. Palmitoylation is another posttranslational modification that can regulate D₃R activity. Compared with D₂R, D₃R undergoes a more extensive palmitoylation on its cysteine residues at the carboxyl terminus tail and, importantly, palmitoylation was found to be essential for cell surface expression, PKC-mediated endocytosis, agonist affinity, and agonist-induced tolerance of D₃R. ¹⁰⁰ Also, DA receptor–interacting proteins, such as AIP1 (ALG-2 interacting protein 1) could be substantial in D₃R stability and trafficking. ¹⁰¹

Concluding Remarks and Perspectives

In the past few years, great progress has been made in understanding D₃R signaling in heterologous systems. Although neural D₃R signaling is incompletely understood, a growing body of evidence indicates that changes in D₃R-induced pathways contribute to several neurological disorders. Future studies may delineate the relative contribution of monomeric-canonical versus heteomeric D₃R signaling to pathological brain states. Another intriguing aspect that remains to be clarified is whether sex-specific differences influence D₃R signaling in the diseased brain, as suggested by the association of the Gly allele of the D₃R Ser9Gly polymorphism with schizophrenia in female but not male patients.^102,103

According to several reports, changes in both D₃R signaling and DA levels are common factors in PD and schizophrenia. Similarly, quantitative and qualitative changes in D₃R signaling develop after DA depletion in the 6-OHDA hemiparkinsonian model.^43,69 However, it remains to be clarified whether the altered D₃R signaling is reflecting an adaptive mechanism to compensate changes in DA levels. Some properties of D₃R strongly suggest that D₃R is the main detector of changes in extracellular DA concentration and, consequently, D₃R signaling may be mainly affected by pathological changes in DA levels, as those observed in PD and schizophrenia. In particular, presynaptic and postsynaptic sites can contain D₃R,^104,105 which exhibits high affinity for DA (~25 nM).^11,97,106 Considering basal extracellular (5-10 nM) and synaptic (50 nM) concentrations of DA,^107–110 a fraction of D₃R may be constitutively activated, thus playing a crucial role in both tonic and phasic DA signaling. ⁶⁷ Interestingly, the occupancy of D₃R by DA is not completely abolished by catecholamine depletion using reserpine,^67,111 suggesting that even under severe reduction in DA level, D₃R is partially activated and able to perceive changes in DA concentration. Thus, a tempting but simplified model for PD may rely on an enhancement in striatal D₃R signaling as part of an adaptive mechanism to compensate the dramatic reduction in DA levels.

Although there is a clear role of D₃R signaling in drug-seeking behaviors and relapse in animal models, it is uncertain whether these findings translate to humans. Studies in humans could evaluate the significance of D₃R signaling in neurological diseases. However, direct evidence of molecular mechanisms in the human brain has been elusive, primarily due to methodological limitations. Notably, novel approaches allow the study of functional responses, including receptor signaling, in postmortem human tissue.^112,113 Using these novel approaches directly in human brain tissue, future studies will help to identify mechanisms that underlie pathological D₃R signaling, either induced by quantitative or qualitative changes.

In summary, many brain disorders are characterized by perturbations in D₃R signaling. A better understanding of how D₃R activity can be regulated may uncover causal factors and may even suggest novel treatments for DA-related diseases. In particular, elucidating the mechanisms that control expression, desensitization, and alternative splicing of D₃R may identify novel opportunities to modulate D₃R signaling.