The dopamine transporter gene SLC6A3: multidisease risks.

The human dopamine transporter gene SLC6A3 has been consistently implicated in several neuropsychiatric diseases but the disease mechanism remains elusive. In this risk synthesis, we have concluded that SLC6A3 represents an increasingly recognized risk with a growing number of familial mutants associated with neuropsychiatric and neurological disorders. At least five loci were related to common and severe diseases including alcohol use disorder (high activity variant), attention-deficit/hyperactivity disorder (low activity variant), autism (familial proteins with mutated networking) and movement disorders (both regulatory variants and familial mutations). Association signals depended on genetic markers used as well as ethnicity examined. Strong haplotype selection and gene-wide epistases support multimarker assessment of functional variations and phenotype associations. Inclusion of its promoter region's functional markers such as DNPi (rs67175440) and 5'VNTR (rs70957367) may help delineate condensate-based risk action, testing a locus-pathway-phenotype hypothesis for one gene-multidisease etiology.

Novel Potent Dopamine-Norepinephrine and Triple Reuptake Uptake Inhibitors Based on Asymmetric Pyran Template and Their Molecular Interactions with Monoamine Transporters.

We have carried out a structural exploration of (2S,4R,5R)-2-(bis(4-fluorophenyl)methyl)-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-473) to investigate the influence of various functional groups on its aromatic ring, the introduction of heterocyclic aromatic rings, and the alteration of the stereochemistry of functional group on the pyran ring. The novel compounds were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting monoamine neurotransmitter uptake. Our studies identified some of the most potent dopamine-norepinephrine reuptake inhibitors known to-date like D-528 and D-529. The studies also led to development of potent triple reuptake inhibitors such as compounds D-544 and D-595. A significant influence from the alteration of the stereochemistry of the hydroxyl group on the pyran ring of D-473 on transporters affinities was observed indicating stereospecific preference for interaction. The inhibitory profiles and structure-activity relationship of lead compounds were further corroborated by molecular docking studies at the primary binding sites of monoamine transporters. The nature of interactions found computationally correlated well with their affinities for the transporters.

SLC6 transporter oligomerization.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Molecular Mechanisms of Amphetamines.

There is a plethora of amphetamine derivatives exerting stimulant, euphoric, anti-fatigue, and hallucinogenic effects; all structural properties allowing these effects are contained within the amphetamine structure. In the first part of this review, the interaction of amphetamine with the dopamine transporter (DAT), crucially involved in its behavioral effects, is covered, as well as the role of dopamine synthesis, the vesicular monoamine transporter VMAT2, and organic cation 3 transporter (OCT3). The second part deals with requirements in amphetamine's effect on the kinases PKC, CaMKII, and ERK, whereas the third part focuses on where we are in developing anti-amphetamine therapeutics. Thus, treatments are discussed that target DAT, VMAT2, PKC, CaMKII, and OCT3. As is generally true for the development of therapeutics for substance use disorder, there are multiple preclinically promising specific compounds against (meth)amphetamine, for which further development and clinical trials are badly needed.

D-578, an orally active triple monoamine reuptake inhibitor, displays antidepressant and anti-PTSD like effects in rats.

Significant unmet needs exist for development of better pharmacotherapeutic agents for major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) as the current drugs are inadequate. Our goal in this study is to investigate behavioral pharmacological characterization of a novel triple reuptake inhibitor (TRI) D-578 which exhibits nanomolar potency at all three monoamine transporters (Ki; 16.2. 16.2, 3.23 nM, and 29.6, 20.6, 6.10 nM for the rat brain and cloned human dopamine, serotonin and norepinephrine transporters, respectively) and exhibited little to no affinity for other off-target CNS receptors. In a rat forced swim test, compound D-578 upon oral administration displayed high efficacy and not stimulating in locomotor behavior. The effects of D-578 and paroxetine were next evaluated in a rat model for traumatic stress exposure - the single prolonged stress (SPS) model - which has been shown to have construct, predictive, and behavioral validity in modeling aspects of PTSD. Our results show that SPS had no effect on the acquisition of conditioned fear, but impaired extinction learning and extinction retention of fear behavior compared to sham treatment. D-578, but not paroxetine, attenuated the extinction and extinction-retention deficit induced by SPS. These findings suggest that D-578 has greater efficacy in normalizing traumatic stress-induced extinction-retention learning in a model for PTSD compared to paroxetine. Overall these results suggest that D-578, in addition to producing a robust and efficacious antidepressant effect, may attenuate maladaptive retention of fearful memories and support further testing of this agent for the pharmacotherapy of depression and PTSD.

Biased signaling agonist of dopamine D3 receptor induces receptor internalization independent of ß-arrestin recruitment.

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration - which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while ß-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608-promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.

Selective activation of Dopamine D3 receptors and norepinephrine transporter blockade enhances sustained attention.

Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of ∼500 nM and behaves as a NET substrate. Systemic dosing of SK609 (4 mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4 mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05 mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25 mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8 mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.

Design, Synthesis, and Pharmacological Characterization of Carbazole Based Dopamine Agonists as Potential Symptomatic and Neuroprotective Therapeutic Agents for Parkinson's Disease.

We have developed a series of carbazole-derived compounds based on our hybrid D2/D3 agonist template to design multifunctional compounds for the symptomatic and disease-modifying treatment of Parkinson's disease (PD). The lead molecules (-)-11b (D-636), (-)-15a (D-653), and (-)-15c (D-656) exhibited high affinity for both D2 and D3 receptors and in GTPγS functional assay, the compounds showed potent agonist activity at both D2 and D3 receptors (EC50 (GTPγS); D2 = 48.7 nM, D3 = 0.96 nM for 11b, D2 = 0.87 nM, D3 = 0.23 nM for 15a and D2 = 2.29 nM, D3 = 0.22 nM for 15c). In an animal model of PD, the test compounds exhibited potent in vivo activity in reversing hypolocomotion in reserpinized rats with a long duration of action compared to the reference drug ropinirole. In a cellular antioxidant assay, compounds (-)-11b, (-)-15a, and (-)-15c exhibited potent activity in reducing oxidative stress induced by neurotoxin 6-hydroxydopamine (6-OHDA). Also, in a cell-based PD neuroprotection model, these lead compounds significantly increased cell survival from toxicity of 6-OHDA, thereby producing a neuroprotective effect. Additionally, compounds (-)-11b and (-)-15a inhibited aggregation and reduced toxicity of recombinant alpha synuclein protein in a cell based in vitro assay. These observations suggest that the lead carbazole-based dopamine agonists may be promising multifunctional molecules for a viable symptomatic and disease-modifying therapy of PD and should be further investigated.

Tamoxifen Directly Interacts with the Dopamine Transporter.

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 µM) and amphetamine-stimulated efflux (59% reduction at 10 µM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS.

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [3H]DA and inhibitor [3H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [3H]DA and [3H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.

Functional properties of dopamine transporter oligomers after copper linking.

Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA Km at ~ 0.5 µM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2ß-carbomethoxy-3ß-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than fivefold decreased (Kd of CFT = 109 nM after cross-linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.

Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding.

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.

A Novel Iron(II) Preferring Dopamine Agonist Chelator as Potential Symptomatic and Neuroprotective Therapeutic Agent for Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder, and development of disease-modifying treatment is still an unmet medical need. Considering the implication of free iron(II) in PD, we report here the design and characterization of a novel hybrid iron chelator, (-)-12 (D-607) as a multitarget-directed ligand against PD. Binding and functional assays at dopamine D2/D3 receptors indicate potent agonist activity of (-)-12. The molecule displayed an efficient preferential iron(II) chelation properties along with potent in vivo activity in a reserpinized PD animal model. The compound also rescued PC12 cells from toxicity induced by iron delivered intracellularly in a dose-dependent manner. However, Fe3+ selective dopamine agonist 1 and a well-known antiparkinsonian drug pramipexole produced little to no neuroprotection effect under the same experimental condition. These observations strongly suggest that (-)-12 should be a promising multifunctional lead molecule for a viable symptomatic and disease modifying therapy of PD.

Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists.

Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 µM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit ß-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure-activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.

Effects of diet and insulin on dopamine transporter activity and expression in rat caudate-putamen, nucleus accumbens, and midbrain.

Food restriction (FR) and obesogenic (OB) diets are known to alter brain dopamine transmission and exert opposite modulatory effects on behavioral responsiveness to psychostimulant drugs of abuse. Mechanisms underlying these diet effects are not fully understood. In this study, we examined diet effects on expression and function of the dopamine transporter (DAT) in caudate-putamen (CPu), nucleus accumbens (NAc), and midbrain regions. Dopamine (DA) uptake by CPu, NAc or midbrain synapto(neuro)somes was measured in vitro with rotating disk electrode voltammetry or with [3 H]DA uptake and was found to correlate with DAT surface expression, assessed by maximal [3 H](-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane binding and surface biotinylation assays. FR and OB diets were both found to decrease DAT activity in CPu with a corresponding decrease in surface expression but had no effects in the NAc and midbrain. Diet treatments also affected sensitivity to insulin-induced enhancement of DA uptake, with FR producing an increase in CPu and NAc, likely mediated by an observed increase in insulin receptor expression, and OB producing a decrease in NAc. The increased expression of insulin receptor in NAc of FR rats was accompanied by increased DA D2 receptor expression, and the decreased DAT expression and function in CPu of OB rats was accompanied by decreased DA D2 receptor expression. These results are discussed as partial mechanistic underpinnings of diet-induced adaptations that contribute to altered behavioral sensitivity to psychostimulants that target the DAT.

Novel multifunctional dopamine D2/D3 receptors agonists with potential neuroprotection and anti-alpha synuclein protein aggregation properties.

Our ongoing drug development endeavor to design compounds for symptomatic and neuroprotective treatment of Parkinson's disease (PD) led us to carry out a structure activity relationship study based on dopamine agonists pramipexole and 5-OHDPAT. Our goal was to incorporate structural elements in these agonists in a way to preserve their agonist activity while producing inhibitory activity against aggregation of α-synuclein protein. In our design we appended various catechol and related phenol derivatives to the parent agonists via different linker lengths. Structural optimization led to development of several potent agonists among which (-)-8a, (-)-14 and (-)-20 exhibited potent neuroprotective properties in a cellular PD model involving neurotoxin 6-OHDA. The lead compounds (-)-8a and (-)-14 were able to modulate aggregation of α-synuclein protein efficiently. Finally, in an in vivo PD animal model, compound (-)-8a exhibited efficacious anti-parkinsonian effect.

Impact of disruption of secondary binding site S2 on dopamine transporter function.

The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 µM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm.

Efficacy of Hybrid Tetrahydrobenzo[d]thiazole Based Aryl Piperazines D-264 and D-301 at D2 and D3 Receptors.

In elucidating the role of pharmacodynamic efficacy at D3 receptors in therapeutic effectiveness of dopamine receptor agonists, the influence of study system must be understood. Here two compounds with D3 over D2 selectivity developed in our earlier work, D-264 and D-301, are compared in dopamine receptor-mediated G-protein activation in striatal regions of wild-type and D2 receptor knockout mice and in CHO cells expressing D2 or D3 receptors. In caudate-putamen of D2 knockout mice, D-301 was ~3-fold more efficacious than D-264 in activating G-proteins as assessed by [(35)S]GTPγS binding; in nucleus accumbens, D-301 stimulated G-protein activation whereas D-264 did not. In contrast, the two ligands exerted similar efficacy in both regions of wild-type mice, suggesting both ligands activate D2 receptors with similar efficacy. In D2 and D3 receptor-expressing CHO cells, D-264 and D-301 appeared to act in the [(35)S]GTPγS assay as full agonists because they produced maximal stimulation equal to dopamine. Competition for [(3)H]spiperone binding was then performed to determine Ki/EC50 ratios as an index of receptor reserve for each ligand. Action of D-301, but not D-264, showed receptor reserve in D3 but not in D2 receptor-expressing cells, whereas dopamine showed receptor reserve in both cell lines. Gαo1 is highly expressed in brain and is important in D2-like receptor-G protein coupling. Transfection of Gαo1 in D3- but not D2-expressing CHO cells led to receptor reserve for D-264 without altering receptor expression levels. D-301 and dopamine exhibited receptor reserve in D3-expressing cells both with and without transfection of Gαo1. Altogether, these results indicate that D-301 has greater intrinsic efficacy to activate D3 receptors than D-264, whereas the two compounds act on D2 receptors with similar intrinsic efficacy. These findings also suggest caution in interpreting Emax values from functional assays in receptor-transfected cell models without accounting for receptor reserve.