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Cross-Talk between G Protein-Coupled Receptors
Cross-Talk between G Protein-Coupled Receptors
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antagonists will be discussed. It is expected that further characterization of the functional role and biology of TAARs and identification of their endogenous and exogenous ligands will eventually promote these receptors as an attractive class of targets to correct monoaminergic processes that could be dysfunctional in a host of disorders of brain and periphery.
Cross-Talk between G Protein-Coupled Receptors: Challenges of Distinguishing Upstream from Downstream Mechanisms Jonathan Javitch College of Physicians and Surgeons, Columbia University, USA
A major obstacle to understanding the functional importance of dimerization between class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control, and we have used this approach to study human dopamine D2 receptor function. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our new methodology also makes possible the characterization of signaling from a defined heterodimer unit. We have also developed a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we have applied the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-npropylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a GPCR protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling. The existence of GPCR dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and
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THEME D Catecholamine Receptors and Catecholaminergic Signaling
biophysical assays, and these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology. Nonetheless, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. We have adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.
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antagonists will be discussed. It is expected that further characterization of the functional role and biology of TAARs and identification of their endogenous and exogenous ligands will eventually promote these receptors as an attractive class of targets to correct monoaminergic processes that could be dysfunctional in a host of disorders of brain and periphery.
Cross-Talk between G Protein-Coupled Receptors: Challenges of Distinguishing Upstream from Downstream Mechanisms Jonathan Javitch College of Physicians and Surgeons, Columbia University, USA
A major obstacle to understanding the functional importance of dimerization between class A G protein-coupled receptors (GPCRs) has been the methodological limitation in achieving control of the identity of the components comprising the signaling unit. We have developed a functional complementation assay that enables such control, and we have used this approach to study human dopamine D2 receptor function. The minimal signaling unit, two receptors and a single G protein, is maximally activated by agonist binding to a single protomer, which suggests an asymmetrical activated dimer. Inverse agonist binding to the second protomer enhances signaling, whereas agonist binding to the second protomer blunts signaling. Ligand-independent constitutive activation of the second protomer also inhibits signaling. Thus, GPCR dimer function can be modulated by the activity state of the second protomer, which for a heterodimer may be altered in pathological states. Our new methodology also makes possible the characterization of signaling from a defined heterodimer unit. We have also developed a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we have applied the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-npropylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a GPCR protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling. The existence of GPCR dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and
94
THEME D Catecholamine Receptors and Catecholaminergic Signaling
biophysical assays, and these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology. Nonetheless, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. We have adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.