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

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.

Detecting and measuring of GPCR signaling – comparison of human induced pluripotent stem cells and immortal cell lines

“…BRET is a powerful technique for investigating dynamic GPCR signaling in living cells. This technique enables the detection of changes in GPCR conformational states through the fusion of a donor luminescent enzyme and an acceptor fluorescent protein ( 28 , 63 ), GPCR activity ( 38 ), and GPCR regulation mechanisms ( 48 ). Four major BRET types ( 79 ) are currently used for GPCR research, although the field is rapidly expanding ( 80 ).…”

“…FRET assays have been used to detect the formation of heterodimers of A-family GPCRs, such as the oxytocin receptor (OTR) and the prostaglandin E2 receptor, (EP2) ( 4 ), which produces a flip effect on subsequent signal transduction. Intra- and intermolecular FRET sensors have been utilized for mGlu receptors, in conjunction with photo-uncaging, to investigate the stepwise activation mechanisms in class C GPCRs ( 185 ).Similarly, BRET has been used to detect dimerization of the A-family histamine H3 receptor (H3R) ( 28 ). It is important to note, however, that the decrease in distance between GPCRs may be due to receptor interference, rather than heterodimerization.…”

Chlorpromazine, an Inverse Agonist of D1R-Like, Differentially Targets Voltage-Gated Calcium Channel (CaV) Subtypes in mPFC Neurons

“…Studying the physiological impact of D1R-like and other GPCRs constitutive activity can be challenging due to the presence of endogenous ligands that simultaneously trigger agonist-evoked activity. In this regard, the use of ligands that reduce constitutive signaling -inverse agonists-to pharmacologically manipulate the level of constitutive activity in both native and recombinant systems has been a crucial strategy in the characterization of GPCR constitutive activity and its relevance in vivo [30][31][32][33][34][35][36]. Inverse agonists have been reported for the majority (~ 75%) of constitutively active GPCRs [29] and at least two of them are endogenous: the Agouti-related peptide (AgRP) for the melanocortin-4-receptor (MC4R) and the liver-expressed antimicrobial peptide 2 (LEAP-2) for the ghrelin receptor (GHSR) [37][38][39].…”

Chlorpromazine, an inverse agonist of D1R-like, differentially targets voltage-gated calcium channels (CaV) subtypes in mPFC neurons

“…Studying the physiological impact of D1R-like and other GPCRs constitutive activity can be challenging due to the presence of endogenous ligands that simultaneously trigger agonist-evoked activity. In this regard, the use of ligands that reduce constitutive signaling -inverse agonists-to pharmacologically manipulate the level of constitutive activity in both native and recombinant systems has been a crucial strategy in the characterization of GPCR constitutive activity and its relevance in vivo [30][31][32][33][34][35][36]. Inverse agonists have been reported for the majority (~ 75%) of constitutively active GPCRs [29] and at least two of them are endogenous: the Agouti-related peptide (AgRP) for the melanocortin-4-receptor (MC4R) and the liver-expressed antimicrobial peptide 2 (LEAP-2) for the ghrelin receptor (GHSR) [37][38][39].…”