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

Chayma El Khamlichi,Laetitia Cobret,Jean-Michel Arrang,Séverine Morisset-Lopez

doi:10.3390/ijms221910638

Abstract

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.

Highlights

A large body of biochemical and biophysical evidence indicates that G-protein-coupled receptors (GPCRs) form dimers in heterologous systems and in native tissues [1,2,3,4,5,6]
bioluminescence resonance energy transfer (BRET) analysis, co-immunoprecipitation studies, and Western blot experiments confirm that H3 receptors exist under dimeric forms
In the present study, spontaneous dimerization was clearly observed for recombinant H3 receptor (H3 R) expressed in heterologous systems and for recombinant H3 Rs expressed in cerebral neurons as well as for native brain receptors

Summary

Introduction

A large body of biochemical and biophysical evidence indicates that G-protein-coupled receptors (GPCRs) form dimers in heterologous systems and in native tissues [1,2,3,4,5,6]. In some studies, ligands do not influence dimerization [20,21,22], whereas, in others, they induce important conformational changes in dimers, leading to allosteric interactions between the two protomers [23,24,25,26,27,28,29]. Inverse agonists decrease constitutive activity by promoting inactive conformations. Whether an alteration of this constitutive activity by inverse agonists leads to changes in the amounts and/or conformations of dimers remains unclear. Very few studies have addressed the putative relationship existing between constitutive activity and the dimerization of GPCRs. In cross-linking experiments performed on the dopamine D2 receptor, the homodimer interface in the inverse agonistbound conformation is consistent with the dimer of the inactive form of rhodopsin [25]

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Conclusion