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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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SKF83959-induced orofacial grooming (a, c, e, g) and locomotor activation (b, d, f, h) is absent in mice lacking the D1 receptor (a, b) but present in D2 receptor knockouts (c, d), Gαq  (e, f) and CaMKII (g, h) mutant mice. Asterisks denote significance (*p<0.05, **p<0.01, ***p<0.001) based on post-hoc Bonferroni multiple comparison tests following repeated measures ANOVA. The data contained in panel f from the Gαq  mice are a modified presentation of those published in42. Yellow bars = WT, Red bars = Mutant.
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Figure 4: SKF83959-induced orofacial grooming (a, c, e, g) and locomotor activation (b, d, f, h) is absent in mice lacking the D1 receptor (a, b) but present in D2 receptor knockouts (c, d), Gαq (e, f) and CaMKII (g, h) mutant mice. Asterisks denote significance (*p<0.05, **p<0.01, ***p<0.001) based on post-hoc Bonferroni multiple comparison tests following repeated measures ANOVA. The data contained in panel f from the Gαq mice are a modified presentation of those published in42. Yellow bars = WT, Red bars = Mutant.

Mentions: That pharmacological blockade of D1 or D2 receptors blunted the SKF83959-induced behaviors (data not shown) does not establish that the drug necessarily acts directly on these receptors. For example, D2 antagonism is well known to reduce locomotor activity, and it may prevent SKF83959-induced effects indirectly. In order to assess more directly which DA receptors are necessary for mediating SKF83959-induced signaling and behavior, we investigated the SKF83959-induced grooming and locomotor activity in a panel of DA receptor knockout mice. SKF83959 elicited a significant orofacial grooming response (Figure 4a; F(1,69)=23.6, p<0.001) in WT mice but not in D1 receptor knockout mice (post-hoc Bonferroni multiple comparison test; p<0.001). We also assessed SKF83959-induced locomotion (Figure 4b; F(1,34)=135.0, p<0.001). D1 receptor knockout mice were initially hyperactive compared to their WT littermates during the pre-injection baseline session (Figure 4b and Supplementary Figure 4). During the post-injection period, WT mice increased their locomotor activity in response to SKF83959 (Figure 4b, post-hoc Bonferroni multiple comparison test, p<0.001). The D1 mice, in contrast, did not respond to SKF83959 but continued to habituate to the chambers (Figure 4b and Supplementary Figure 4), resulting in a significant decrease in locomotor activity, as compared to pre-injection baseline (post-hoc Bonferroni multiple comparison test, p<0.001).


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)

SKF83959-induced orofacial grooming (a, c, e, g) and locomotor activation (b, d, f, h) is absent in mice lacking the D1 receptor (a, b) but present in D2 receptor knockouts (c, d), Gαq  (e, f) and CaMKII (g, h) mutant mice. Asterisks denote significance (*p<0.05, **p<0.01, ***p<0.001) based on post-hoc Bonferroni multiple comparison tests following repeated measures ANOVA. The data contained in panel f from the Gαq  mice are a modified presentation of those published in42. Yellow bars = WT, Red bars = Mutant.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: SKF83959-induced orofacial grooming (a, c, e, g) and locomotor activation (b, d, f, h) is absent in mice lacking the D1 receptor (a, b) but present in D2 receptor knockouts (c, d), Gαq (e, f) and CaMKII (g, h) mutant mice. Asterisks denote significance (*p<0.05, **p<0.01, ***p<0.001) based on post-hoc Bonferroni multiple comparison tests following repeated measures ANOVA. The data contained in panel f from the Gαq mice are a modified presentation of those published in42. Yellow bars = WT, Red bars = Mutant.
Mentions: That pharmacological blockade of D1 or D2 receptors blunted the SKF83959-induced behaviors (data not shown) does not establish that the drug necessarily acts directly on these receptors. For example, D2 antagonism is well known to reduce locomotor activity, and it may prevent SKF83959-induced effects indirectly. In order to assess more directly which DA receptors are necessary for mediating SKF83959-induced signaling and behavior, we investigated the SKF83959-induced grooming and locomotor activity in a panel of DA receptor knockout mice. SKF83959 elicited a significant orofacial grooming response (Figure 4a; F(1,69)=23.6, p<0.001) in WT mice but not in D1 receptor knockout mice (post-hoc Bonferroni multiple comparison test; p<0.001). We also assessed SKF83959-induced locomotion (Figure 4b; F(1,34)=135.0, p<0.001). D1 receptor knockout mice were initially hyperactive compared to their WT littermates during the pre-injection baseline session (Figure 4b and Supplementary Figure 4). During the post-injection period, WT mice increased their locomotor activity in response to SKF83959 (Figure 4b, post-hoc Bonferroni multiple comparison test, p<0.001). The D1 mice, in contrast, did not respond to SKF83959 but continued to habituate to the chambers (Figure 4b and Supplementary Figure 4), resulting in a significant decrease in locomotor activity, as compared to pre-injection baseline (post-hoc Bonferroni multiple comparison test, p<0.001).

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