Limits...
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.

Show MeSH

Related in: MedlinePlus

Drug-induced conformational change of Gαq is not detected in D1 and/or D2L-expressing cells. (a) Drug-induced BRET change shown as ΔBRET (= BRET ratio with drug applied - basal BRET ratio) is detected between Gα and Gγ subunits. Note that due to the different positions of the donor sensors within the Gα subunits, activation of the heterotrimer is manifested as a decrease in BRET between the Gα donor and the Gγ acceptor for the Gαq and Gαι1 sensors but as an increase in BRET for the Gαs sensor. (b) M1R is shown as a positive control for Gαq activation (blue solid = ACh, blue open = ACh + pirenzepine, p < 0.001) whereas Gαq activation was not observed for either DA or SKF83959 in the D1R (red) or D2R (black). Conformational change of cognate G proteins by DA was induced and blocked by an agonist and an antagonist for (c) D1R (red, p < 0.001), (d) D2LR (black, p < 0.001). (e) Diagram showing the BRET configuration in (f). (f) Co-expression of D1 and D2L did not lead to Gαq activation after drug stimulation (red solid = DA, red open = SKF83959) whereas robust Gαq coupling to M1R was still seen after acetylcholine addition in the presence of D2LR (blue, p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492915&req=5

Figure 1: Drug-induced conformational change of Gαq is not detected in D1 and/or D2L-expressing cells. (a) Drug-induced BRET change shown as ΔBRET (= BRET ratio with drug applied - basal BRET ratio) is detected between Gα and Gγ subunits. Note that due to the different positions of the donor sensors within the Gα subunits, activation of the heterotrimer is manifested as a decrease in BRET between the Gα donor and the Gγ acceptor for the Gαq and Gαι1 sensors but as an increase in BRET for the Gαs sensor. (b) M1R is shown as a positive control for Gαq activation (blue solid = ACh, blue open = ACh + pirenzepine, p < 0.001) whereas Gαq activation was not observed for either DA or SKF83959 in the D1R (red) or D2R (black). Conformational change of cognate G proteins by DA was induced and blocked by an agonist and an antagonist for (c) D1R (red, p < 0.001), (d) D2LR (black, p < 0.001). (e) Diagram showing the BRET configuration in (f). (f) Co-expression of D1 and D2L did not lead to Gαq activation after drug stimulation (red solid = DA, red open = SKF83959) whereas robust Gαq coupling to M1R was still seen after acetylcholine addition in the presence of D2LR (blue, p < 0.001).

Mentions: Activation of G-proteins by specific DA receptor populations was systematically assessed using the BRET biosensor technique (Figure 1a). This approach allows measurement of G-protein activation based on a conformational change between the α and γ subunits (Figure 1a). The conformational changes assessed by the sensors have been previously demonstrated to accurately reflect activation of the G proteins38, 49.


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)

Drug-induced conformational change of Gαq is not detected in D1 and/or D2L-expressing cells. (a) Drug-induced BRET change shown as ΔBRET (= BRET ratio with drug applied - basal BRET ratio) is detected between Gα and Gγ subunits. Note that due to the different positions of the donor sensors within the Gα subunits, activation of the heterotrimer is manifested as a decrease in BRET between the Gα donor and the Gγ acceptor for the Gαq and Gαι1 sensors but as an increase in BRET for the Gαs sensor. (b) M1R is shown as a positive control for Gαq activation (blue solid = ACh, blue open = ACh + pirenzepine, p < 0.001) whereas Gαq activation was not observed for either DA or SKF83959 in the D1R (red) or D2R (black). Conformational change of cognate G proteins by DA was induced and blocked by an agonist and an antagonist for (c) D1R (red, p < 0.001), (d) D2LR (black, p < 0.001). (e) Diagram showing the BRET configuration in (f). (f) Co-expression of D1 and D2L did not lead to Gαq activation after drug stimulation (red solid = DA, red open = SKF83959) whereas robust Gαq coupling to M1R was still seen after acetylcholine addition in the presence of D2LR (blue, p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Drug-induced conformational change of Gαq is not detected in D1 and/or D2L-expressing cells. (a) Drug-induced BRET change shown as ΔBRET (= BRET ratio with drug applied - basal BRET ratio) is detected between Gα and Gγ subunits. Note that due to the different positions of the donor sensors within the Gα subunits, activation of the heterotrimer is manifested as a decrease in BRET between the Gα donor and the Gγ acceptor for the Gαq and Gαι1 sensors but as an increase in BRET for the Gαs sensor. (b) M1R is shown as a positive control for Gαq activation (blue solid = ACh, blue open = ACh + pirenzepine, p < 0.001) whereas Gαq activation was not observed for either DA or SKF83959 in the D1R (red) or D2R (black). Conformational change of cognate G proteins by DA was induced and blocked by an agonist and an antagonist for (c) D1R (red, p < 0.001), (d) D2LR (black, p < 0.001). (e) Diagram showing the BRET configuration in (f). (f) Co-expression of D1 and D2L did not lead to Gαq activation after drug stimulation (red solid = DA, red open = SKF83959) whereas robust Gαq coupling to M1R was still seen after acetylcholine addition in the presence of D2LR (blue, p < 0.001).
Mentions: Activation of G-proteins by specific DA receptor populations was systematically assessed using the BRET biosensor technique (Figure 1a). This approach allows measurement of G-protein activation based on a conformational change between the α and γ subunits (Figure 1a). The conformational changes assessed by the sensors have been previously demonstrated to accurately reflect activation of the G proteins38, 49.

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