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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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Proximity ligation assay (PLA) shows that endogenous D1 and D2 receptors do not physically interact in the shell of the nucleus accumbens. (a) Whereas the non-specific nuclear background did not vary between conditions, the PLA signal (small dots) indicative of molecular proximity between D1 and D2 receptors was virtually absent in WT mice, as well as in D1 and D2 KO mice. Virally-mediated overexpression of D1 (D1OE) did not significantly increase PLA signal, whereas D2 (D2OE) receptor overexpression led to a small, but significant increase in PLA signal. Simultaneous overexpression of both receptors (D1D2OE) led to a very strong PLA signal, suggesting that the 2 receptors have the ability to interact if highly expressed in close proximity. (b) Quantification of the PLA signals was performed as described in Methods. N=4–8 (3–4 animals). * p<0.05; ** p<0.01. Scale bars=10 µm.
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Figure 6: Proximity ligation assay (PLA) shows that endogenous D1 and D2 receptors do not physically interact in the shell of the nucleus accumbens. (a) Whereas the non-specific nuclear background did not vary between conditions, the PLA signal (small dots) indicative of molecular proximity between D1 and D2 receptors was virtually absent in WT mice, as well as in D1 and D2 KO mice. Virally-mediated overexpression of D1 (D1OE) did not significantly increase PLA signal, whereas D2 (D2OE) receptor overexpression led to a small, but significant increase in PLA signal. Simultaneous overexpression of both receptors (D1D2OE) led to a very strong PLA signal, suggesting that the 2 receptors have the ability to interact if highly expressed in close proximity. (b) Quantification of the PLA signals was performed as described in Methods. N=4–8 (3–4 animals). * p<0.05; ** p<0.01. Scale bars=10 µm.

Mentions: We confirmed the lack of interaction of D1 and D2 receptors using a proximity-ligation assay (PLA) that allows for the detection of receptor complexes ex vivo47. PLA signals for this heteromer were virtually absent in the nucleus accumbens of WT, D1 receptor KO and D2 receptor KO mice (Figure 6). Overexpression of either D1 or D2 receptors by viral gene transfer led to a small increase in PLA signal, whereas overexpression of both receptors resulted in a very large increase in PLA signal (Figure 6). These data suggest that even in the small fraction of neurons that co-express D1 and D2 receptors, the receptors are segregated and do not physically interact. In contrast, when both receptors are dramatically overexpressed - either in HEK cells or in vivo - the receptors have the ability to interact.


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)

Proximity ligation assay (PLA) shows that endogenous D1 and D2 receptors do not physically interact in the shell of the nucleus accumbens. (a) Whereas the non-specific nuclear background did not vary between conditions, the PLA signal (small dots) indicative of molecular proximity between D1 and D2 receptors was virtually absent in WT mice, as well as in D1 and D2 KO mice. Virally-mediated overexpression of D1 (D1OE) did not significantly increase PLA signal, whereas D2 (D2OE) receptor overexpression led to a small, but significant increase in PLA signal. Simultaneous overexpression of both receptors (D1D2OE) led to a very strong PLA signal, suggesting that the 2 receptors have the ability to interact if highly expressed in close proximity. (b) Quantification of the PLA signals was performed as described in Methods. N=4–8 (3–4 animals). * p<0.05; ** p<0.01. Scale bars=10 µm.
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Related In: Results  -  Collection

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Figure 6: Proximity ligation assay (PLA) shows that endogenous D1 and D2 receptors do not physically interact in the shell of the nucleus accumbens. (a) Whereas the non-specific nuclear background did not vary between conditions, the PLA signal (small dots) indicative of molecular proximity between D1 and D2 receptors was virtually absent in WT mice, as well as in D1 and D2 KO mice. Virally-mediated overexpression of D1 (D1OE) did not significantly increase PLA signal, whereas D2 (D2OE) receptor overexpression led to a small, but significant increase in PLA signal. Simultaneous overexpression of both receptors (D1D2OE) led to a very strong PLA signal, suggesting that the 2 receptors have the ability to interact if highly expressed in close proximity. (b) Quantification of the PLA signals was performed as described in Methods. N=4–8 (3–4 animals). * p<0.05; ** p<0.01. Scale bars=10 µm.
Mentions: We confirmed the lack of interaction of D1 and D2 receptors using a proximity-ligation assay (PLA) that allows for the detection of receptor complexes ex vivo47. PLA signals for this heteromer were virtually absent in the nucleus accumbens of WT, D1 receptor KO and D2 receptor KO mice (Figure 6). Overexpression of either D1 or D2 receptors by viral gene transfer led to a small increase in PLA signal, whereas overexpression of both receptors resulted in a very large increase in PLA signal (Figure 6). These data suggest that even in the small fraction of neurons that co-express D1 and D2 receptors, the receptors are segregated and do not physically interact. In contrast, when both receptors are dramatically overexpressed - either in HEK cells or in vivo - the receptors have the ability to interact.

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