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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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Related in: MedlinePlus

D1 and D2 receptors are co-expressed but do not colocalize in the shell of the nucleus accumbens. (a) Immunohistochemical detection of D2 (top) and D1 (bottom) in the shell of the nucleus accumbens in WT mice. Staining was virtually absent in D2 or D1 KO mice. (b) Co-staining of D1 and D2 receptors in the shell of the nucleus accumbens in mice revealed extremely limited colocalization. (c) Top-left: Low magnification confocal image of a horizontal section going through the striatum and nucleus accumbens in a double BAC Drd1a-tdTomato/Drd2-eGFP transgenic mouse. The Ds-Red (red) and eGFP (green) signals were enhanced using specific antibodies. Cells co-expressing active Drd1 and Drd2 promoters (yellow) were detectable in the shell (bottom-right) but not in the core (bottom-left) of the nucleus accumbens or in the dorsal striatum (CPu: top-right). (d) Zoom on tomato+ (D1) and eGFP+ (D2) dendritic areas in the shell of the nucleus accumbens after quadruple staining of DsRed, eGFP, D1 and D2. Top-left: tomato and eGFP signals; Top-right: yellow areas were detected using ImageJ software and identified with a mask (grey outline); Bottom-left: D1 (green) and D2 (red) receptors signals merged with the mask (grey). Bottom-right: D1 (red) and D2 (green) signals extracted within the mask area only showed no colocalization of D1 and D2 receptors. Scale bars=10 µm unless otherwise indicated.
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Figure 5: D1 and D2 receptors are co-expressed but do not colocalize in the shell of the nucleus accumbens. (a) Immunohistochemical detection of D2 (top) and D1 (bottom) in the shell of the nucleus accumbens in WT mice. Staining was virtually absent in D2 or D1 KO mice. (b) Co-staining of D1 and D2 receptors in the shell of the nucleus accumbens in mice revealed extremely limited colocalization. (c) Top-left: Low magnification confocal image of a horizontal section going through the striatum and nucleus accumbens in a double BAC Drd1a-tdTomato/Drd2-eGFP transgenic mouse. The Ds-Red (red) and eGFP (green) signals were enhanced using specific antibodies. Cells co-expressing active Drd1 and Drd2 promoters (yellow) were detectable in the shell (bottom-right) but not in the core (bottom-left) of the nucleus accumbens or in the dorsal striatum (CPu: top-right). (d) Zoom on tomato+ (D1) and eGFP+ (D2) dendritic areas in the shell of the nucleus accumbens after quadruple staining of DsRed, eGFP, D1 and D2. Top-left: tomato and eGFP signals; Top-right: yellow areas were detected using ImageJ software and identified with a mask (grey outline); Bottom-left: D1 (green) and D2 (red) receptors signals merged with the mask (grey). Bottom-right: D1 (red) and D2 (green) signals extracted within the mask area only showed no colocalization of D1 and D2 receptors. Scale bars=10 µm unless otherwise indicated.

Mentions: Even if the behavioral effects of SKF83959 are not mediated by D1/D2 receptor dimers, it is conceivable that such a complex nonetheless mediates differential signaling and represents a novel target for therapeutics. In fact, despite our inability to detect the activation of Gαq by D1/D2 receptors in HEK293 cells, previously using CODA-RET we did obtain support for functional selectivity in the Gαi pathway of a defined D1/D2 receptor heteromer5. This piqued our interest in this potential heteromer as a drug target and motivated us to pursue studies in ventral striatal brain slices where the receptors have been reported to be co-expressed. Coexpression of D1 and D2 receptors in the striatum and nucleus accumbens has been a contentious topic6. In situ hybridization and electron microscopy studies have generally supported the segregation of these receptors in distinct subpopulations of neurons7, 58, 59. However, modest colocalization as well as fluorescence resonance energy transfer (FRET) between D1 and D2 receptors have been observed in the shell of the nucleus accumbens using antibodies9, 10. Indirect measures using BAC transgenic GFP mice support co-expression of D1 and D2 receptor genes in a sub-population of neurons in the shell of the nucleus accumbens13, 60. In order to clarify whether D1 and D2 receptors colocalize in the shell of the nucleus accumbens, we took advantage of an anti-D2 receptor antibody that we previously generated and that shows high selectivity as confirmed by the lack of staining in D2 receptor-KO mice46, 47 (Figure 5a). We detected very limited colocalization of D1 and D2 receptors in the shell of the nucleus accumbens in WT mice (Figure 5b) or rats (Supplementary Figure 7), or in the ventral striatum of rhesus monkeys (Supplementary Figure 7). Because D1 and D2 receptor subcellular localization is mostly in the neuropil11, 46, 47, 59, 61, it is virtually impossible to visualize cell bodies that express – or not – specific dopamine receptors. In order to better identify cells that co-express both receptors we crossed two transgenic reporter lines, Drd1a-tdTomato45 and Drd2-eGFP44. As previously reported based on comparing expression in the individual reporter lines60, in the crossed lines we observed co-expression of both genes in a small fraction of cells restricted to the shell of the nucleus accumbens, but in only a handful of neurons within the NAc core or the dorsal striatum (Figure 5c).


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)

D1 and D2 receptors are co-expressed but do not colocalize in the shell of the nucleus accumbens. (a) Immunohistochemical detection of D2 (top) and D1 (bottom) in the shell of the nucleus accumbens in WT mice. Staining was virtually absent in D2 or D1 KO mice. (b) Co-staining of D1 and D2 receptors in the shell of the nucleus accumbens in mice revealed extremely limited colocalization. (c) Top-left: Low magnification confocal image of a horizontal section going through the striatum and nucleus accumbens in a double BAC Drd1a-tdTomato/Drd2-eGFP transgenic mouse. The Ds-Red (red) and eGFP (green) signals were enhanced using specific antibodies. Cells co-expressing active Drd1 and Drd2 promoters (yellow) were detectable in the shell (bottom-right) but not in the core (bottom-left) of the nucleus accumbens or in the dorsal striatum (CPu: top-right). (d) Zoom on tomato+ (D1) and eGFP+ (D2) dendritic areas in the shell of the nucleus accumbens after quadruple staining of DsRed, eGFP, D1 and D2. Top-left: tomato and eGFP signals; Top-right: yellow areas were detected using ImageJ software and identified with a mask (grey outline); Bottom-left: D1 (green) and D2 (red) receptors signals merged with the mask (grey). Bottom-right: D1 (red) and D2 (green) signals extracted within the mask area only showed no colocalization of D1 and D2 receptors. Scale bars=10 µm unless otherwise indicated.
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Figure 5: D1 and D2 receptors are co-expressed but do not colocalize in the shell of the nucleus accumbens. (a) Immunohistochemical detection of D2 (top) and D1 (bottom) in the shell of the nucleus accumbens in WT mice. Staining was virtually absent in D2 or D1 KO mice. (b) Co-staining of D1 and D2 receptors in the shell of the nucleus accumbens in mice revealed extremely limited colocalization. (c) Top-left: Low magnification confocal image of a horizontal section going through the striatum and nucleus accumbens in a double BAC Drd1a-tdTomato/Drd2-eGFP transgenic mouse. The Ds-Red (red) and eGFP (green) signals were enhanced using specific antibodies. Cells co-expressing active Drd1 and Drd2 promoters (yellow) were detectable in the shell (bottom-right) but not in the core (bottom-left) of the nucleus accumbens or in the dorsal striatum (CPu: top-right). (d) Zoom on tomato+ (D1) and eGFP+ (D2) dendritic areas in the shell of the nucleus accumbens after quadruple staining of DsRed, eGFP, D1 and D2. Top-left: tomato and eGFP signals; Top-right: yellow areas were detected using ImageJ software and identified with a mask (grey outline); Bottom-left: D1 (green) and D2 (red) receptors signals merged with the mask (grey). Bottom-right: D1 (red) and D2 (green) signals extracted within the mask area only showed no colocalization of D1 and D2 receptors. Scale bars=10 µm unless otherwise indicated.
Mentions: Even if the behavioral effects of SKF83959 are not mediated by D1/D2 receptor dimers, it is conceivable that such a complex nonetheless mediates differential signaling and represents a novel target for therapeutics. In fact, despite our inability to detect the activation of Gαq by D1/D2 receptors in HEK293 cells, previously using CODA-RET we did obtain support for functional selectivity in the Gαi pathway of a defined D1/D2 receptor heteromer5. This piqued our interest in this potential heteromer as a drug target and motivated us to pursue studies in ventral striatal brain slices where the receptors have been reported to be co-expressed. Coexpression of D1 and D2 receptors in the striatum and nucleus accumbens has been a contentious topic6. In situ hybridization and electron microscopy studies have generally supported the segregation of these receptors in distinct subpopulations of neurons7, 58, 59. However, modest colocalization as well as fluorescence resonance energy transfer (FRET) between D1 and D2 receptors have been observed in the shell of the nucleus accumbens using antibodies9, 10. Indirect measures using BAC transgenic GFP mice support co-expression of D1 and D2 receptor genes in a sub-population of neurons in the shell of the nucleus accumbens13, 60. In order to clarify whether D1 and D2 receptors colocalize in the shell of the nucleus accumbens, we took advantage of an anti-D2 receptor antibody that we previously generated and that shows high selectivity as confirmed by the lack of staining in D2 receptor-KO mice46, 47 (Figure 5a). We detected very limited colocalization of D1 and D2 receptors in the shell of the nucleus accumbens in WT mice (Figure 5b) or rats (Supplementary Figure 7), or in the ventral striatum of rhesus monkeys (Supplementary Figure 7). Because D1 and D2 receptor subcellular localization is mostly in the neuropil11, 46, 47, 59, 61, it is virtually impossible to visualize cell bodies that express – or not – specific dopamine receptors. In order to better identify cells that co-express both receptors we crossed two transgenic reporter lines, Drd1a-tdTomato45 and Drd2-eGFP44. As previously reported based on comparing expression in the individual reporter lines60, in the crossed lines we observed co-expression of both genes in a small fraction of cells restricted to the shell of the nucleus accumbens, but in only a handful of neurons within the NAc core or the dorsal striatum (Figure 5c).

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