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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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Defined receptor dimer pairs do not recruit Gαq after drug stimulation. Using the CODA-RET approach, a receptor dimer pair is defined by complementation of the luminescent protein Rluc8. (a) Diagram showing the BRET configuration. (b, c) The complemented M1RL1-M1RL2 and 5HT2ARL1-5HT2ARL2 pairs are shown as positive controls for Gαq coupling (solid = agonist, open = agonist + antagonist, p < 0.001). In contrast, the complemented DA receptor dimer pairs shown (d) D2SR-D1R, (e) D2SR-D5R, (f) D2LR-D1R, (g) D2LR-D5R] failed to reveal drug-induced Gαq recruitment (red solid = DA, red open = SKF83959 + quinpirole).
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Figure 2: Defined receptor dimer pairs do not recruit Gαq after drug stimulation. Using the CODA-RET approach, a receptor dimer pair is defined by complementation of the luminescent protein Rluc8. (a) Diagram showing the BRET configuration. (b, c) The complemented M1RL1-M1RL2 and 5HT2ARL1-5HT2ARL2 pairs are shown as positive controls for Gαq coupling (solid = agonist, open = agonist + antagonist, p < 0.001). In contrast, the complemented DA receptor dimer pairs shown (d) D2SR-D1R, (e) D2SR-D5R, (f) D2LR-D1R, (g) D2LR-D5R] failed to reveal drug-induced Gαq recruitment (red solid = DA, red open = SKF83959 + quinpirole).

Mentions: When two receptors are co-expressed, it is not possible to differentiate signaling between individual protomers, homomers, or heteromers. Therefore we used the CODA-RET configuration5 (Figure 2a), to measure Gαq-coupling specifically from the defined heterodimer. In this method, split luciferase is fused to the C terminus of two receptors, and luminescence only results from complementation of the luciferase due to association of the receptors bearing the halves. Thus, BRET between receptor and an acceptor-tagged Gα is indicative of signaling of the defined dimer through a defined G protein5. Homodimer complementation of the prototypical Gαq-coupled M1 receptor (Figure 2b) or 5HT2A receptor (Figure 2c) showed efficient agonist-stimulated Gαq-coupling and antagonist blockade. This pair of positive controls showed that complemented luciferase does not interfere with Gαq-coupling, at least in the case of these homodimers. In contrast, DA stimulation alone or SKF83959 and quinpirole co-stimulation failed to induce Gαq-coupling of complemented D1-D2S receptors (Figure 2d), D2S-D5 receptors (Figure 2e), D1-D2L receptors (Figure 2f), or D2L-D5 receptors (Figure 2g). Despite its better sensitivity, the BRET2 configuration also failed to detect drug-induced recruitment of Gαq to either individual (Supplementary Table 1) or complemented DA receptors in any combination (Supplementary Table 1). We showed previously using CODA-RET that D1 and D2 receptors can interact in HEK cells and that signaling of the D1/D2 heteromer to Gαi is differentially altered for the agonist NPA relative to DA5. Thus, although these receptors can interact and modulate one another in HEK cells, our new data demonstrate that in these cells D1/D2 receptor heteromers are unable to recruit Gαq in response to DA, SKF83959, or combined quinpirole and SKF83959.


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)

Defined receptor dimer pairs do not recruit Gαq after drug stimulation. Using the CODA-RET approach, a receptor dimer pair is defined by complementation of the luminescent protein Rluc8. (a) Diagram showing the BRET configuration. (b, c) The complemented M1RL1-M1RL2 and 5HT2ARL1-5HT2ARL2 pairs are shown as positive controls for Gαq coupling (solid = agonist, open = agonist + antagonist, p < 0.001). In contrast, the complemented DA receptor dimer pairs shown (d) D2SR-D1R, (e) D2SR-D5R, (f) D2LR-D1R, (g) D2LR-D5R] failed to reveal drug-induced Gαq recruitment (red solid = DA, red open = SKF83959 + quinpirole).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Defined receptor dimer pairs do not recruit Gαq after drug stimulation. Using the CODA-RET approach, a receptor dimer pair is defined by complementation of the luminescent protein Rluc8. (a) Diagram showing the BRET configuration. (b, c) The complemented M1RL1-M1RL2 and 5HT2ARL1-5HT2ARL2 pairs are shown as positive controls for Gαq coupling (solid = agonist, open = agonist + antagonist, p < 0.001). In contrast, the complemented DA receptor dimer pairs shown (d) D2SR-D1R, (e) D2SR-D5R, (f) D2LR-D1R, (g) D2LR-D5R] failed to reveal drug-induced Gαq recruitment (red solid = DA, red open = SKF83959 + quinpirole).
Mentions: When two receptors are co-expressed, it is not possible to differentiate signaling between individual protomers, homomers, or heteromers. Therefore we used the CODA-RET configuration5 (Figure 2a), to measure Gαq-coupling specifically from the defined heterodimer. In this method, split luciferase is fused to the C terminus of two receptors, and luminescence only results from complementation of the luciferase due to association of the receptors bearing the halves. Thus, BRET between receptor and an acceptor-tagged Gα is indicative of signaling of the defined dimer through a defined G protein5. Homodimer complementation of the prototypical Gαq-coupled M1 receptor (Figure 2b) or 5HT2A receptor (Figure 2c) showed efficient agonist-stimulated Gαq-coupling and antagonist blockade. This pair of positive controls showed that complemented luciferase does not interfere with Gαq-coupling, at least in the case of these homodimers. In contrast, DA stimulation alone or SKF83959 and quinpirole co-stimulation failed to induce Gαq-coupling of complemented D1-D2S receptors (Figure 2d), D2S-D5 receptors (Figure 2e), D1-D2L receptors (Figure 2f), or D2L-D5 receptors (Figure 2g). Despite its better sensitivity, the BRET2 configuration also failed to detect drug-induced recruitment of Gαq to either individual (Supplementary Table 1) or complemented DA receptors in any combination (Supplementary Table 1). We showed previously using CODA-RET that D1 and D2 receptors can interact in HEK cells and that signaling of the D1/D2 heteromer to Gαi is differentially altered for the agonist NPA relative to DA5. Thus, although these receptors can interact and modulate one another in HEK cells, our new data demonstrate that in these cells D1/D2 receptor heteromers are unable to recruit Gαq in response to DA, SKF83959, or combined quinpirole and SKF83959.

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