BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The his...

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Bibliographic Details
Published in:International journal of molecular sciences 2021-10, Vol.22 (19), p.10638
Main Authors: El Khamlichi, Chayma, Cobret, Laetitia, Arrang, Jean-Michel, Morisset-Lopez, Séverine
Format: Article
Language:English
Subjects:
Allosteric properties
Antibodies
Bioluminescence
Biosynthesis
Dimerization
Dopamine
Energy
Energy transfer
Experiments
G protein-coupled receptors
Histamine
Histamine H3 receptors
Inverse agonists
Life Sciences
Ligands
Neurons
Physiology
Proteins
Regression analysis
Western blotting
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Summary:G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms221910638