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Dopamine Receptors: The Right Combination Unlocks Calcium Release

27 January 2007. Dopamine receptors come in five flavors (D1-D5), and until recently, they were regarded as five separate players, regulating adenylate cyclase signaling pathways in distinct ways. But the work of Susan George and colleagues at the University of Toronto, Ontario, Canada, cooks up a different recipe for dopamine receptor action.

Their newest paper, published online December 28, 2006, in PNAS, shows that hybrid oligomers of D1 and D2 dopamine receptors regulate calcium release and activation of the calmodulin-dependent kinase II α (CaMKIIα) in adult rat brain. Given the importance of this kinase in modulating synaptic plasticity, the results suggest that the novel D1/D2 partnership could help tune neuronal function in response to dopamine in adults. Understanding this wrinkle in dopamine action, and how it changes with age, may have some relevance for schizophrenia, drug abuse, and other disorders where dopamine signaling takes a central role (see SRF Current Hypothesis by Abi-Dargham).

Previously, George’s group has shown that the D1 and D2 receptors form hetero-oligomers in cells that have pharmacological and signaling properties distinct from either D1 or D2 alone (Lee et al., 2004; So et al., 2005). Rather than regulating adenylate cyclase, the combination receptor turns on phospholipase C, causing inositol phosphate production and internal calcium release.

In the new report, first author Asim Rashid leads the characterization of these hybrid receptors in the striatum in mouse brain. Just as in earlier cell-based experiments, the D1-D2 receptors were found to be coupled to phospholipase C activation via rapid stimulation of the Gq/11 signaling protein. The researchers show evidence that simultaneous activation of both D1 and D2 is required to see Gq/11 activation in membrane preparations from mouse striatum. Membranes from either D1 or D2 knockout mice did not support Gq/11 activation.

The pharmacology of the D2 receptor undergoes some changes when it meets up with D1, the researchers find. Two different agonists that are normally specific for D1 show binding to D2 when it is in the mixed complex. One feature of D2 that is not changed is the binding of the antipsychotic raclopride, which blocks the activation of Gq/11 by the mixed receptor.

Unexpectedly, the researchers found that the receptor complex is more abundant in adult mice (>8 months old) than in immature animals (3 months). Most studies on dopamine function use young rodents, which could explain the limited reports of functions for the D1-D2 receptors, the authors say.

In vivo studies demonstrate that the D1-D2 complex regulates calcium-dependent signals in adult rodents. Dosing rats with D1 and D2 agonists together resulted in activation of CaMKIIα in the nucleus accumbens, as measured by postmortem histochemistry. The activation required both D1 and D2, since either a D1 antagonist (SCH23390) or raclopride inhibited it, and neither D1 nor D2 knockout mice could support CaMKIIα activation.

Mixing and matching receptor subunits creates a novel dopaminergic signaling complex, which could play a role in schizophrenia, the authors conclude. The combinatorial receptor, they write, “is significant in that it provides a greater repertoire of signaling pathways by which dopamine can modulate neuronal function than would be possible by each of the five different dopamine receptor subtypes acting solely as separate units.” With both dopamine and calcium signaling implicated in schizophrenia, they conclude, their results provide “significant impetus to determine whether the D1-D2 receptor signaling complex is altered in neuropsychiatric decease.”—Pat McCaffrey.

Rashid AJ, So CH, Kong MM, Furtak T, El-Ghundi M, Cheng R, O'Dowd BF, George SR. D1-D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum. Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):654-9. Epub 2006 Dec 28. Abstract

Comments on News and Primary Papers
Comment by:  Christoph Kellendonk
Submitted 29 January 2007
Posted 30 January 2007
  I recommend the Primary Papers

The paper by Rashid et al. presents yet another interesting example of how dopamine D2 receptors may activate signaling pathways independent of the classical cAMP pathway, a finding that may have potential therapeutic implications. Most antipsychotic drugs that ameliorate positive symptoms antagonize D2 receptors, which may be also at the origin of many of the side effects associated with these medications. But, if antipsychotic action utilizes signaling pathways that are distinct from those responsible for the side effects we may have the chance to develop new compounds with higher specificity and reduced side effects. Observations such as those made in Rashid et al. are essential steps in this direction.

View all comments by Christoph KellendonkComment by:  Eleanor Simpson
Submitted 29 January 2007
Posted 30 January 2007
  I recommend the Primary Papers

This is a very exciting paper. The concept of D1 and D2 cellular coexpression had been debated for a long time; with limited antibodies for these receptors available, investigators had found conflicting results, dependent on the method of detection used.

The authors recently described the existence of D1-D2 hetero-oligomers. Here they elucidate a possible function of such a complex. The authors begin with a very thorough biochemical characterization in HEK cells stably expressing either D1, D2, or both receptors, concluding that SKF83959 is a specific agonist for Gq/11 coupled D1-D2 receptor hetero-oligomers. By using striatal membrane preparations from wild-type, D1 mutant, or D2 mutant mice, the authors identify a D1-D2 Gq11 complex in the brains of mature mice.

The authors conclude by suggesting that D1-D2 receptor signaling may be altered in neuropsychiatric disease and that this should be explored. This may be a little premature, and perhaps some more fundamental characterization of this newly discovered complex should first be undertaken. The increase in GTPgS incorporation by 100 uM dopamine is modest compared to the increase observed with 100 uM SKF+Quin treatment. Since none of these experiments are under in vivo physiological conditions, it would be reassuring to see that this modest DA response is also blocked by SCH or RAC.

The fact that the D1-D2 Gq/11 complex was detected in 8-month-old mice but not 3-month-old mice is fascinating and begs the questions, when do these complexes form? How and why do they form? Both RT-PCR and primary culture experiments suggest that at least a fraction of neurons in the striatum coexpress D1 and D2 receptors in young adult mice. Does the number of coexpressing neurons increase with age? Or does hetero-oligomer coupling to Gq/11 increase with age? There is evidence that D1 receptor-Gs protein coupling is reduced in very old rats (Sugawa et al., 1996). Is the appearance of D1-D2 Gq/11 complexes in the striatum relevant to brain maturation, or does it relate to a decline in DA signaling efficiency?

View all comments by Eleanor Simpson

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Related News: The New "Inverted U”—Cellular Basis for Dopamine Response Pinpointed

Comment by:  Andreas Meyer-Lindenberg
Submitted 8 February 2007
Posted 8 February 2007

This fascinating paper contributes to our mechanistic understanding of a fundamental nonlinearity governing the response of prefrontal neurons during working memory to dopaminergic stimulation: the “inverted U” response curve (Goldman-Rakic et al., 2000), which proposes that an optimum range of dopaminergic stimulation exists, and that either too little or too much dopamine impairs tuning, or the relationship between task-relevant (“signal”) and task-irrelevant (“noise”) firing of these neurons. On the level of behavior, this is predicted to result in impaired working memory performance outside the optimum middle range, and this has been confirmed in a variety of species. This is a topic of high relevance for schizophrenia where prefrontal dysfunction and related cognitive deficits, and dopaminergic dysregulation, have long been in the center of research interest (Weinberger et al., 2001), and may be linked (Meyer-Lindenberg et al., 2002). In particular, evidence for abnormally decreased dopamine levels in prefrontal cortex would predict that patients with schizophrenia are positioned to the left of the optimum. This line of thought has recently received impetus from genetic studies on COMT, the major enzyme catabolizing dopamine in prefrontal cortex (Tunbridge et al., 2004). Neuroimaging studies have shown that genetic variants with high COMT activity are positioned to the left, those with lower activity nearer the optimum of the inverted U curve, and that this position predicts nonlinear response to amphetamine stimulation (Mattay et al., 2003), as well as interactions between dopamine synthesis and prefrontal response (Meyer-Lindenberg et al., 2005). Variants with sub- (Egan et al., 2001; Nicodemus et al., 2007) or superoptimal (Gothelf et al., 2005) stimulation were associated with schizophrenia risk. Task-related and task-unrelated prefrontal function reacted in opposite ways to genetic variation in dopamine synthesis, suggesting a tuning mechanism (Meyer-Lindenberg et al., 2005). Recently, interacting genetic variants in COMT have also been found to affect prefrontal cortex function in an inverted U fashion (Meyer-Lindenberg et al., 2006).

A seminal contribution to the cellular mechanisms of the inverted U curve is the paper by Williams (one of the authors of the current study) and Goldman-Rakic in Nature 1995 (Williams and Goldman-Rakic, 1995). In this work, dopamine D1 receptor antagonists were used and shown to increase prefrontal cell activity in low levels, whereas high levels inhibited firing. This implicated a mechanism related to D1 receptors and suggested that the neurons studied were to the right of the optimum on the inverted U curve, that is, their dopamine stimulation was excessive. The present study, from Amy Arnsten’s lab at Yale, further defines the cellular mechanisms underlying the inverted U curve in recordings from PFC neurons of awake behaving monkeys exposed to various levels of stimulation by a dopamine 1 receptor agonist. A spatial working memory paradigm was used, enabling the determination of the degree to which the neurons were tuned by comparing the firing rate to stimuli in the preferred spatial stimulus direction (“signal”) to the firing rate to nonpreferred stimuli (“noise”). The authors recorded both from neurons that were highly tuned (supposedly receiving optimum stimulation) and neurons that were less tuned. As would be predicted from the model, highly tuned neurons did not improve, or worsened, during stimulation, while weakly tuned neurons became more focused in their activity profile. It is not quite clear to me why the previous paper (Williams and Goldman-Rakic, 1995) found neurons that were predominantly to the right of the optimum, while this work identified neurons using a similar paradigm that were either to the left or near the optimum. Perhaps it is because Williams and Goldman-Rakic (Williams and Goldman-Rakic, 1995) screened neurons for a response to the D1 antagonist first. In both studies, extracellular dopamine was not actually measured, meaning that the state of basal stimulation can only be inferred indirectly from the response to the iontophoresed agonist or antagonist. Importantly, the effect of D1 stimulation was always suppressive; effects on tuning were due to the fact that the reduction in response to the signal and the noise were different in extent, such that for weakly tuned neurons and low levels of D1 stimulation, the noise firing was more suppressed than that of the signal, resulting in increased signal to noise. In a second set of pharmacological experiments, which included validation in a rat working memory model, the authors show that these effects are cAMP, but not PKC-dependent, suggesting a preferential cellular mechanism through Gs-proteins, which might be useful for exploration of more specific drug targets.

This work has interesting implications for our understanding of prefrontal function in schizophrenia. Since dopamine stimulation was found to be almost exclusively suppressive, cortical dopamine depletion in schizophrenia would be predicted to lead to relatively increased, but inefficient (untuned) cortical cognitive response, as has indeed been observed (Callicott et al., 2000). However, it is an open question precisely how cortical physiology assessed by imaging relates to these cellular events. The data by Arnsten suggest that each patch of prefrontal cortex will contain a population of neurons at various states of tuning that will respond differently to drug-induced or cognitively related changes in extracellular dopamine, with some improving, some decreasing their tuning. Depending on whether imaging signals and tasks are more sensitive to overall firing rate, or to specific signal-to-noise properties, the resulting blood flow change might be quite different. Perhaps this contributes to some of the puzzling discrepancies between hypo- and hyperactivation both being observed in comparable tasks and regions of prefrontal cortex in schizophrenia.


1. Goldman-Rakic PS, Muly EC 3rd, Williams GV. D(1) receptors in prefrontal cells and circuits. Brain Res Brain Res Rev. 2000 Mar;31(2-3):295-301. Review. No abstract available. Abstract

2. Weinberger DR, Egan MF, Bertolino A, Callicott JH, Mattay VS, Lipska BK, Berman KF, Goldberg TE. Prefrontal neurons and the genetics of schizophrenia. Biol Psychiatry. 2001 Dec 1;50(11):825-44. Review. Abstract

3. Meyer-Lindenberg A, Miletich RS, Kohn PD, Esposito G, Carson RE, Quarantelli M, Weinberger DR, Berman KF. Reduced prefrontal activity predicts exaggerated striatal dopaminergic function in schizophrenia. Nat Neurosci. 2002 Mar;5(3):267-71. Abstract

4. Tunbridge EM, Bannerman DM, Sharp T, Harrison PJ. Catechol-o-methyltransferase inhibition improves set-shifting performance and elevates stimulated dopamine release in the rat prefrontal cortex. J Neurosci. 2004 Jun 9;24(23):5331-5. Abstract

5. Mattay VS, Goldberg TE, Fera F, Hariri AR, Tessitore A, Egan MF, Kolachana B, Callicott JH, Weinberger DR. Catechol O-methyltransferase val158-met genotype and individual variation in the brain response to amphetamine. Proc Natl Acad Sci U S A. 2003 May 13;100(10):6186-91. Epub 2003 Apr 25. Abstract

6. Meyer-Lindenberg A, Kohn PD, Kolachana B, Kippenhan S, McInerney-Leo A, Nussbaum R, Weinberger DR, Berman KF. Midbrain dopamine and prefrontal function in humans: interaction and modulation by COMT genotype. Nat Neurosci. 2005 May;8(5):594-6. Epub 2005 Apr 10. Abstract

7. Egan MF, Goldberg TE, Kolachana BS, Callicott JH, Mazzanti CM, Straub RE, Goldman D, Weinberger DR. Effect of COMT Val108/158 Met genotype on frontal lobe function and risk for schizophrenia. Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6917-22. Epub 2001 May 29. Abstract

8. Nicodemus KK, Kolachana BS, Vakkalanka R, Straub RE, Giegling I, Egan MF, Rujescu D, Weinberger DR. Evidence for statistical epistasis between catechol-O-methyltransferase (COMT) and polymorphisms in RGS4, G72 (DAOA), GRM3, and DISC1: influence on risk of schizophrenia. Hum Genet. 2007 Feb;120(6):889-906. Epub 2006 Sep 28. Abstract

9. Gothelf D, Eliez S, Thompson T, Hinard C, Penniman L, Feinstein C, Kwon H, Jin S, Jo B, Antonarakis SE, Morris MA, Reiss AL. COMT genotype predicts longitudinal cognitive decline and psychosis in 22q11.2 deletion syndrome. Nat Neurosci. 2005 Nov;8(11):1500-2. Epub 2005 Oct 23. Abstract

10. Meyer-Lindenberg A, Nichols T, Callicott JH, Ding J, Kolachana B, Buckholtz J, Mattay VS, Egan M, Weinberger DR. Impact of complex genetic variation in COMT on human brain function. Mol Psychiatry. 2006 Sep;11(9):867-77, 797. Epub 2006 Jun 20. Abstract

11. Williams GV, Goldman-Rakic PS. Modulation of memory fields by dopamine D1 receptors in prefrontal cortex. Nature. 1995 Aug 17;376(6541):572-5. Abstract

12. Callicott JH, Bertolino A, Mattay VS, Langheim FJ, Duyn J, Coppola R, Goldberg TE, Weinberger DR. Physiological dysfunction of the dorsolateral prefrontal cortex in schizophrenia revisited. Cereb Cortex. 2000 Nov;10(11):1078-92. Abstract

View all comments by Andreas Meyer-Lindenberg

Related News: The New "Inverted U”—Cellular Basis for Dopamine Response Pinpointed

Comment by:  Terry Goldberg
Submitted 6 April 2007
Posted 6 April 2007

In this landmark study, Arnsten and colleagues used a full dopamine agonist in awake behaving monkeys to make key points about the inverted U response at the cellular level and how this maps to the behavioral level. There were a number of surprises. The first was that stimulation of the D1 receptor had consistently suppressive effects on neuronal firing during delays in a working memory task. The second was that when responses were optimized, suppressive effects differentially affected non-preferred directional neurons, rather than preferred direction neurons. Thus, it appeared that noise was reduced rather than signal amplified. Too much D1 stimulation resulted in suppression of both classes of neurons.

The implications of this work are important because it suggests that there is a neurobiological algorithm at work that can reliably produce this unexpected physiological pattern (perhaps as the authors suggest on the basis of baseline activity). It remains to be elucidated whether the D1 receptor effects are mediated by glutamatergic neurons or GABA interneurons, or both. There is another layer of complexity to the story. As Arnsten and colleagues note, possible excitatory influences of D1 stimulation may not have been observed because endogenous dopamine had already triggered this process. It is unclear if D2 receptors in the cortex have a role in shaping or terminating this activity.

Last, it is tempting to speculate about the implications of these findings for other types of tasks that engage prefrontal cortex in humans. What does tuning mean in the context of tasks like the N Back which demands updating, the ID/ED test from the CANTAB, which involves suppression of salient distractors at early set shifting stages, or a task which demands heavy doses of cognitive control like the flanker task, all of which have been shown to be sensitive to manipulations of the dopamine system (Goldberg et al., 2003; Jazbec et al., 2007; Diaz-Asper et al., in press; Blasi et al., 2005)?

View all comments by Terry Goldberg

Related News: Coupling Between D1 and D2 Receptors Implicated in Depression

Comment by:  Christoph Kellendonk
Submitted 14 December 2010
Posted 14 December 2010

Heterodimerization between D1 and D2 receptors is a recently discovered, novel mechanism by which D1 and D2 receptors activate Gq-mediated signaling in the brain. Although first met with skepticism, evidence for the existence of functional D1/D2 heterodimers under physiological conditions has become more and more convincing.

Heterodimerization between D1 and D2 receptors is linked to their coexpression in the same cell. The localization of D1 and D2 receptors has been extensively studied in the striatum. After D1 and D2 receptors were cloned 20 years ago, in situ hybridization studies suggested that there are two main populations of neurons in the striatum: one population that predominantly expresses D1 receptors and projects mono-synaptically to the substantia nigra (called the striato-nigral or direct pathway), and the other population that expresses D2 receptors and projects over several synapses to the substantia nigra (called the striato-pallidal or indirect pathway).

When these studies were followed up using single cell PCR and immunohistochemistry (IHC) using antibodies against D1 and D2 receptors, the percentage of neurons coexpressing both receptors increased to 15-30 percent, or even 100 percent for some IHC studies. One argument for the inconsistency between the early in situ hybridization studies and the newer studies had been that in situ hybridization may not be as sensitive as single cell PCR or IHC. However, the main problem with IHC is that different antibodies were used in different studies, and it is known that not all available antibodies against D2 receptors are really specific.

Recently, mice that express green fluorescent protein under the control of either the D1 or the D2 promoter have been developed that allow for selective labeling of D1- and D2-positive MSNs, respectively. The findings with D1- and D2-GFP mice are more in line with the original in situ hybridization studies showing relatively low overlap of expression in the dorsal striatum (5-7 percent of MSNs) and higher overlap in the ventral striatum (around 20 percent).

The problem of antibody specificity has also been a problem for studying heterodimers in the striatum. Therefore, in a recent study, Susan George's laboratory at the University of Toronto took great effort in testing the specificity of their antibodies (Perreault et al., 2010). One important control they included was knockout mice in which the D1 or the D2 receptor gene was inactivated. Immunohistochemistry for D1 and D2 did not show any signal in these mice, indicating specificity of the employed antibodies. Moreover, colocalization studies with these antibodies showed a degree of overlap that was comparable to what had been observed in the classical in situ hybridization studies and the recent studies using D1- and D2-GFP mice. Last, the authors used FRET technology and demonstrated coexpression at a spatial resolution that supports a direct interaction between both receptors in vivo.

The laboratory of Fang Liu, also at the University of Toronto, has now found that heterodimerization may be increased under pathological conditions. Using immunoprecipitation experiments, Pei et al. found increased coupling between D1Rs and D2Rs in the striatum and the cortex of patients with major depression (Pei et al., 2010). Perreault et al. found increased affinity for SKF83959, a heterodimer specific dopamine receptor agonist in the globus pallidus of patients with schizophrenia (Perreault et al., 2010). Since the globus pallidus is the main output structure of the indirect pathway of the striatum, it raises the question whether the degree of D1 and D2 receptor coexpression may be increased under pathological conditions

Obviously, both postmortem findings will need replication using higher subject numbers. Due to the confounding effects of postmortem tissue analysis and medication, PET imaging studies could greatly benefit this analysis. Imaging could be done earlier in the disease process and under drug-naïve conditions. The challenge here may be the development of appropriate tracers that are both suitable for PET imaging and specific for detecting heterodimers.

That SKF83959 selectively activates heterodimers raises the possibility for the development of selective antagonists. If increased heterodimers are indeed involved in the pathophysiology of depression and schizophrenia, they may be good targets for treating negative symptoms such as anhedonia and avolition that are associated with both disorders. They may also help against psychosis, though we would then expect that D1 receptor antagonists, which block heterodimer-mediated signaling, would be effective antipsychotics.


Perreault ML, Hasbi A, Alijaniaram M, Fan T, Varghese G, Fletcher PJ, Seeman P, O'Dowd BF, George SR The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia. J Biol Chem 285:36625-36634. Abstract

Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ, Nobrega JN, Liu F Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med 16:1393-1395. Abstract

View all comments by Christoph Kellendonk

Related News: Coupling Between D1 and D2 Receptors Implicated in Depression

Comment by:  Jeremy Seamans
Submitted 23 December 2010
Posted 23 December 2010

Christoph nicely summarized key aspects of the paper in the context of the relevant literature. In addition, I feel the paper makes an important contribution because it draws attention to a signaling mechanism that may help explain some of the more contentious effects of dopamine.

View all comments by Jeremy Seamans

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Susan George
Submitted 15 January 2015
Posted 15 January 2015

The paper by Frederick et al. is presented as definitive evidence against the existence of the dopamine D1-D2 receptor heteromer and attempts to convey the impression that all the conclusions from the work conducted in my laboratory or those of other scientific colleagues who have worked on this complex are wrong.

This paper simply cannot be accepted at face value as definitive, as we and others have extremely robust evidence of D1 and D2 receptors forming complexes, not just in heterologously expressing HEK cells, but also in rat brain and in human brain (Lee et al., 2004; Pei et al., 2010). If the receptors were not within a complex, they would not co-immunoprecipitate with each other. Furthermore, direct receptor-receptor interaction has been shown in cultured rat striatal neurons and rat nucleus accumbens in situ by confocal FRET and FRET photobleaching (Hasbi et al., 2009; Perreault et al., 2010) and from human striatal tissue by GST pulldown and human prefrontal cortex by co-immunoprecipitation (Pei et al., 2010). These facts are overwhelming proof that the D1 and D2 receptors directly interact and exist within a complex in brain.

With respect to the calcium signal, the paper contradicts recent data from one of the co-authors, Dr. Sibley, who previously reported, "When cells were transfected with D1R and D2LR or D1R with D2SR, a clear dose-dependent activation of calcium mobilization was observed in response to DA," with the conclusion that "These data suggest that expression and activation of both the D1R and D2R are essential for coupling to calcium mobilization and signaling" (Chun et al., 2013). We and others have obtained a calcium signal from cell lines expressing sub-picomolar densities of both receptors (Rashid et al., 2007) and we have demonstrated a calcium signal by D1-D2 heteromer activation using nanomolar concentrations of DA and other agonists in early postnatal cultured striatal neurons (Hasbi et al., 2009; Verma et al., 2010).

Regarding the involvement of Gq in the signaling of this complex, we showed this by selectively immunoprecipitating 35S-GTPγS-labeled Gq from striatum (Rashid et al., 2007) and by use of a selective Gq inhibitor in cultured striatal neurons (Hasbi et al., 2009). We have never attributed the Gq pathway as being involved in the behavioral effects of SKF 83959 such as locomotion or grooming, nor have we suggested that the only mechanism by which D1-D2 heteromer signals is through Gq, so the parts of the paper showing lack of involvement of Gq or CaMKII in the behavioral studies are not necessarily surprising. We have evidence for other signaling pathways activated by the heteromer, and it remains to be analyzed what the precise mechanism for these behavioral effects might be.

Our data show that the highest expression of the D1-D2 complex in rat striatum is in nucleus accumbens shell, where we estimate 20-30 percent of medium spiny neurons expressing the D1 receptor also express the D2 receptor. Of these neurons, approximately 90 percent had evidence for heteromers by confocal FRET. In contrast, in dorsal striatum, only 5-6 percent of D1 neurons also expressed the D2 receptor, and only 20 percent of these neurons had evidence for heteromer formation. Furthermore, these neurons had a distinctive and unique phenotype in expressing both of the neuropeptides that are considered exclusive markers of D1- or D2-receptor-expressing medium spiny neurons, namely dynorphin and enkephalin. Our numbers from rat striatum are consistent with what has been reported for D1 and D2 receptor colocalization in BAC transgenic mice by numerous groups, ranging about 17 percent in nucleus accumbens (Bertran-Gonzalez et al., 2008; Matamales et al., 2009). A detailed study of subregions of the nucleus accumbens in these mice found D1 and D2 receptor colocalization in the medial bundle region of nucleus accumbens to be as high as 38 percent (Gangarossa et al., 2013). Thus, the paper by Frederick et al. reporting colocalization of D1 and D2 receptors in the nucleus accumbens shell of BAC-Tg mice of 2-5 percent is distinctly out of the range compared to what other groups have reported.

Further, the PLA technique used in the paper is another method by which protein complexes may be detected in situ. It is also based on immunohistochemistry, and the signals generated critically depend on the performance and affinities of the antibodies used. Moreover, it seems unwarranted to disregard the D1 and D2 receptors co-expressed on the neuronal cell bodies and only analyze presence of heteromer in the neuropil, as there is precedent for important extrasynaptic functions that can be regulated by receptors from the cell soma. We did demonstrate D1-D2 heteromer colocalized with synaptophysin in the dendritic field, indicating presynaptic localization in intrastriatal projections (Perreault et al., 2010). Regardless of the number of nucleus accumbens neurons expressing the D1-D2 receptor heteromer, it is still the functional importance that needs to be analyzed. There is no question of the immense importance of interneurons that only comprise 1-2 percent of neurons in striatum, in spite of the very low number.

We have used SKF 83959 to activate the D1-D2 receptor complex in our studies, fully cognizant of the fact that this atypical agonist is an agonist at the D5 receptor, has little ability to activate D1 receptor mediated adenylyl cyclase, and has several other unwanted cross-reactivities. However, in cultured striatal neurons, where there is no D5 receptor, the use of D1 and D2 receptor antagonists has ascertained the fidelity of the responses we were studying. To transition our studies into the whole animal, we have delineated the specific residues mediating a critical interaction interface between D1 and D2 receptors (two amino acids in each receptor), and have generated a specific peptide based on the D1 receptor sequence and relied on the use of this peptide in vivo to dissect out specific heteromer mediated functions. We have some very exciting results emerging regarding the physiological functions and certain pathophysiological roles of the D1-D2 heteromer, so stay tuned. The chapter has not ended.


Lee I, Skinner MA, Guo HB, Sujan A, Pierce M. Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression. J Biol Chem. 2004 Dec 17; 279(51):53007-14. Abstract

Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ, Nobrega JN, Liu F. Uncoupling the dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med. 2010 Dec; 16(12):1393-5. Abstract

Hasbi A, Fan T, Alijaniaram M, Nguyen T, Perreault ML, O'Dowd BF, George SR. Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth. Proc Natl Acad Sci U S A. 2009 Dec 15; 106(50):21377-82. Abstract

Perreault ML, Hasbi A, Alijaniaram M, Fan T, Varghese G, Fletcher PJ, Seeman P, O'Dowd BF, George SR. The dopamine D1-D2 receptor heteromer localizes in dynorphin/enkephalin neurons: increased high affinity state following amphetamine and in schizophrenia. J Biol Chem. 2010 Nov 19; 285(47):36625-34. Abstract

Chun LS, Free RB, Doyle TB, Huang XP, Rankin ML, Sibley DR. D1-D2 dopamine receptor synergy promotes calcium signaling via multiple mechanisms. Mol Pharmacol. 2013 Aug; 84(2):190-200. Abstract

Rashid AJ, So CH, Kong MM, Furtak T, El-Ghundi M, Cheng R, O'Dowd BF, George SR. D1-D2 dopamine receptor heterooligomers with unique pharmacology are coupled to rapid activation of Gq/11 in the striatum. Proc Natl Acad Sci U S A. 2007 Jan 9; 104(2):654-9. Abstract

Verma V, Hasbi A, O'Dowd BF, George SR. Dopamine D1-D2 receptor Heteromer-mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2. J Biol Chem. 2010 Nov 5; 285(45):35092-103. Abstract

Bertran-Gonzalez J, Bosch C, Maroteaux M, Matamales M, Herve, Valjent E, Girault JA. Opposing patterns of signaling activation in dopamine D1 and D2 receptor-expressing striatal neurons in response to cocaine and haloperidol. J Neurosci. 2008 May 28; 28(22):5671-85. Abstract

Matamales M, Bertran-Gonzalez J, Salomon L, Degos B, Deniau JM, Valjent E, Herve, Girault JA. Striatal medium-sized spiny neurons: identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice. PLoS One. 2009; 4(3):e4770. Abstract

Gangarossa G, Espallergues J, Mailly P, De Bundel D, de Kerchove d'Exaerde A, Herve D, Girault JA, Valjent E, Krieger P. Spatial distribution of D1R- and D2R-expressing medium-sized spiny neurons differs along the rostro-caudal axis of the mouse dorsal striatum. Front Neural Circuits. 2013; 7():124. Abstract

View all comments by Susan George

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Richard Mailman (Disclosure)
Submitted 16 January 2015
Posted 20 January 2015
  I recommend the Primary Papers

Dr. George's response ignores two papers recently published by my group. In Lee et al. (2014a), we offered experimental evidence challenging much of the foundation on which the D1/D2 heterodimer hypothesis rests. Again in Lee et al. (2014b), we critically reviewed the relevant literature. Although this was some time prior to the current controversy, we concluded: "The ... hypothesis [of] a critical role for D1-D2 heterodimers .... is the one of the greatest of current interest because of the novel underlying molecular mechanism....

Although it was based originally on the D1-PLC mechanism and the use of SKF-83959, it has now taken on a life of its own because of the hypothesized unique role of D1-D2 heterodimers in subsets of co-expressing striatal neurons (Perreault et al., 2012; 2014). We have reviewed the available data that we feel can be construed as weakening this hypothesis. Above and beyond the experimental issues (including our failure to be able to see evidence of PLC signaling in D1-D2-co-transfected HEK293 cells [Lee et al., 2014a]), there is the question of the degree to which D1-D2-co-localization occurs in the mature nervous system. Thus, our view is that [this] hypothesis is also false."

We urge those who still feel that D1-D2 dimers are important to read our review and then consider the elegant aforementioned studies of Javitch, Stanwood, and their colleagues. Together, the weight of the evidence suggests strongly that this hypothesis should be given a long overdue retirement.


Lee SM, Kant A, Blake D, Murthy V, Boyd K, Wyrick SJ, Mailman RB. SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C. Neuropharmacology. 2014a Jun 12. S0028-3908(14)00218-4. Abstract

Lee SM, Yang Y, Mailman RB. (2014) Dopamine D1 receptor signaling: Does GQ-phospholipase C actually play a role? J Pharmacol Exp Ther. 2014b Jul 22. Abstract

Perreault ML, Fan T, Alijaniaram M, O'Dowd BF, George SR. Dopamine D1-D2 receptor heteromer in dual phenotype GABA/glutamate-coexpressing striatal medium spiny neurons: regulation of BDNF, GAD67 and VGLUT1/2. PLoS One. 2012; 7(3):e33348. Abstract

Perreault ML, Hasbi A, O'Dowd BF, George SR. Heteromeric dopamine receptor signaling complexes: emerging neurobiology and disease relevance. Neuropsychopharmacology. 2014 Jan; 39(1):156-68. Abstract Paper ID:

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Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Philip Seeman (Disclosure)
Submitted 16 January 2015
Posted 20 January 2015
  I recommend the Primary Papers

Numerous papers cited by the other authors in this discussion have already confirmed that D1 and D2 receptors can be immunoprecipitated together from human brain, indicating that an interaction can occur between these two receptors. The extent of immunoprecipitation may vary depending on the brain region. The single paper by Frederick et al. is out of line and does not reflect the true situation.

View all comments by Philip Seeman

Related News: Elusive Hybrid Dopamine Receptor Stirs Controversy

Comment by:  Bryan Roth, SRF Advisor
Submitted 22 January 2015
Posted 22 January 2015

How might this controversy be settled?

As the original in vitro signaling assays were key to the genesis of the model, I would recommend that independent labs attempt to replicate the in vitro findings—ideally using cell lines supplied by both groups. As one of the key original findings was an apparent Ca++ mobilization signal, this should be the easiest to quantify and replicate.

Going forward, what would be needed would be selective and potent agonists for the D1/D2 complex with minimal off-target actions. The test agent SKF 83959 has always been problematic, as it has quite high potency at Gq-coupled serotonin receptors and other GPCRs—a point made by others (see Professor Mailman's recent review: Lee et al., 2014). Assuming D1/D2 heteromer selective compounds could be identified, they would be exceedingly illuminating for the field.


Lee SM, Yang Y, Mailman RB. Dopamine D1 receptor signaling: does GaQ-phospholipase C actually play a role? J Pharmacol Exp Ther. 2014 Oct; 351(1):9-17. Abstract

View all comments by Bryan Roth