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Evidence against dopamine D1/D2 receptor heteromers.

Frederick AL, Yano H, Trifilieff P, Vishwasrao HD, Biezonski D, Mészáros J, Urizar E, Sibley DR, Kellendonk C, Sonntag KC, Graham DL, Colbran RJ, Stanwood GD, Javitch JA - Mol. Psychiatry (2015)

Bottom Line: We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors.Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes.These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Graduate Program, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT
Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation, because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer, ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout (KO) mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq KO mice, as well as in knock-in mice expressing a mutant Ala(286)-CaMKIIα that cannot autophosphorylate to become active. Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

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Neither DA nor SKF 83959 induces calcium mobilization in stable cell lines expressing D1 and/or D2 receptors. Using the FLIPR5 calcium assay system, intracellular calcium levels were measured every 2 seconds and plotted against time. Ligands (ACh = 10 µM acetylcholine, vehicle, DA = 10 µM dopamine, 10 µM SKF83959, 10 µM U73122, or 10 µM pirenzepine) were added at 20 secs, indicated by an arrow, in (a) D1R, (b) D2R, and (c) D1R/D2R stable cells. Calcium level is shown in percentage normalized to 10 µM acetylcholine (ACh). Traces are representatives of n = 3 experiments.
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Figure 3: Neither DA nor SKF 83959 induces calcium mobilization in stable cell lines expressing D1 and/or D2 receptors. Using the FLIPR5 calcium assay system, intracellular calcium levels were measured every 2 seconds and plotted against time. Ligands (ACh = 10 µM acetylcholine, vehicle, DA = 10 µM dopamine, 10 µM SKF83959, 10 µM U73122, or 10 µM pirenzepine) were added at 20 secs, indicated by an arrow, in (a) D1R, (b) D2R, and (c) D1R/D2R stable cells. Calcium level is shown in percentage normalized to 10 µM acetylcholine (ACh). Traces are representatives of n = 3 experiments.

Mentions: At the functional level, D1/D2 heteromer-dependent activation of Gαq/11 has been proposed to lead to the recruitment of calcium signaling28, 52. We therefore tested whether, despite the absence of Gαq or Gα11 recruitment, co-expression of D1 and D2 receptors leads to calcium mobilization (Figure 3). Neither D1 (Figure 3a) nor D2S activation (Figure 3b) in the respective stably transfected cells caused calcium mobilization. We next created dual D1/D2S stable cells but still did not observe achange in intracellular calcium in response to D1 and D2 co-stimulation with either DA or SKF83959 (Figure 3c). In contrast, acetylcholine potently stimulated calcium via endogenously expressed muscarinic M3 receptors (Figure 3a, b, c).


Evidence against dopamine D1/D2 receptor heteromers.

Frederick AL, Yano H, Trifilieff P, Vishwasrao HD, Biezonski D, Mészáros J, Urizar E, Sibley DR, Kellendonk C, Sonntag KC, Graham DL, Colbran RJ, Stanwood GD, Javitch JA - Mol. Psychiatry (2015)

Neither DA nor SKF 83959 induces calcium mobilization in stable cell lines expressing D1 and/or D2 receptors. Using the FLIPR5 calcium assay system, intracellular calcium levels were measured every 2 seconds and plotted against time. Ligands (ACh = 10 µM acetylcholine, vehicle, DA = 10 µM dopamine, 10 µM SKF83959, 10 µM U73122, or 10 µM pirenzepine) were added at 20 secs, indicated by an arrow, in (a) D1R, (b) D2R, and (c) D1R/D2R stable cells. Calcium level is shown in percentage normalized to 10 µM acetylcholine (ACh). Traces are representatives of n = 3 experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492915&req=5

Figure 3: Neither DA nor SKF 83959 induces calcium mobilization in stable cell lines expressing D1 and/or D2 receptors. Using the FLIPR5 calcium assay system, intracellular calcium levels were measured every 2 seconds and plotted against time. Ligands (ACh = 10 µM acetylcholine, vehicle, DA = 10 µM dopamine, 10 µM SKF83959, 10 µM U73122, or 10 µM pirenzepine) were added at 20 secs, indicated by an arrow, in (a) D1R, (b) D2R, and (c) D1R/D2R stable cells. Calcium level is shown in percentage normalized to 10 µM acetylcholine (ACh). Traces are representatives of n = 3 experiments.
Mentions: At the functional level, D1/D2 heteromer-dependent activation of Gαq/11 has been proposed to lead to the recruitment of calcium signaling28, 52. We therefore tested whether, despite the absence of Gαq or Gα11 recruitment, co-expression of D1 and D2 receptors leads to calcium mobilization (Figure 3). Neither D1 (Figure 3a) nor D2S activation (Figure 3b) in the respective stably transfected cells caused calcium mobilization. We next created dual D1/D2S stable cells but still did not observe achange in intracellular calcium in response to D1 and D2 co-stimulation with either DA or SKF83959 (Figure 3c). In contrast, acetylcholine potently stimulated calcium via endogenously expressed muscarinic M3 receptors (Figure 3a, b, c).

Bottom Line: We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors.Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes.These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Graduate Program, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT
Hetero-oligomers of G-protein-coupled receptors have become the subject of intense investigation, because their purported potential to manifest signaling and pharmacological properties that differ from the component receptors makes them highly attractive for the development of more selective pharmacological treatments. In particular, dopamine D1 and D2 receptors have been proposed to form hetero-oligomers that couple to Gαq proteins, and SKF83959 has been proposed to act as a biased agonist that selectively engages these receptor complexes to activate Gαq and thus phospholipase C. D1/D2 heteromers have been proposed as relevant to the pathophysiology and treatment of depression and schizophrenia. We used in vitro bioluminescence resonance energy transfer, ex vivo analyses of receptor localization and proximity in brain slices, and behavioral assays in mice to characterize signaling from these putative dimers/oligomers. We were unable to detect Gαq or Gα11 protein coupling to homomers or heteromers of D1 or D2 receptors using a variety of biosensors. SKF83959-induced locomotor and grooming behaviors were eliminated in D1 receptor knockout (KO) mice, verifying a key role for D1-like receptor activation. In contrast, SKF83959-induced motor responses were intact in D2 receptor and Gαq KO mice, as well as in knock-in mice expressing a mutant Ala(286)-CaMKIIα that cannot autophosphorylate to become active. Moreover, we found that, in the shell of the nucleus accumbens, even in neurons in which D1 and D2 receptor promoters are both active, the receptor proteins are segregated and do not form complexes. These data are not compatible with SKF83959 signaling through Gαq or through a D1/D2 heteromer and challenge the existence of such a signaling complex in the adult animals that we used for our studies.

Show MeSH
Related in: MedlinePlus