Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Abstract

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo-and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.Membrane proteins such as tyrosine kinase, cytokine, or transforming growth factor receptors have been known for many years to form oligomers, and the link between their oligomerization and activity states has been well established (1). In contrast, G protein-coupled receptor (GPCR) 1 oligomerization has been documented only recently (2), and the relation between receptor activation and oligomerization is still poorly understood. Even the proportion between monomeric and oligomeric receptor species and the exact oligomerization state (dimer, trimer, tetramer, etc.) remains a matter of controversy. Currently, two models are proposed. In the first model GPCR are monomeric in their inactive state, and agonist activation induces the formation of receptor oligomers. This model is based on low basal and strong agonist-induced energy transfer signals observed in fluorescence and bioluminescence resonance energy transfer (FRET, BRET) experiments for the gonadotropin-releasing hormone (3), the somatostatin SSTR5 (4), and the SSTR5/dopamin D2R receptor oligomers (5). The second model proposes that GPCR are constitutively oligomerized and is supported by studies reporting high basal BRET or FRET signals for ␣-mating factor (6), ␤2-adrenergic (␤2AR) (7), tyrothropin-releasing hormone (8), ␦-opioid (9), type A cholecystokinin (10), and dopamine D2 receptors (11). Agonist-promoted increases in signals were observed in some of thes...

Constitutive dimerization of human serotonin 5‐HT4 receptors in living cells

“…To study receptor oligomerization in intact cells, we chose BRET, a technique that has been recently used to monitor protein-protein interaction [19,21]. The assay relies on the observation that the degree of physical proximity between molecules can be assessed in living cells by the level of energy transfer occurring between the energy donor RLuc and a fluorescent acceptor, YFP [23].…”

“…DNA sequencing was performed to verify the accuracy of all fusion constructs. The human MT 1 melatonin receptor fused in frame with either RLuc (MT 1 R-Rluc) or YFP (MT 1 R-YFP) have been previously described [21]. The b2AR fusion protein constructs b2AR-Rluc and b2AR-YFP have been described elsewhere [19].…”

“…BRET experiments were performed as previously described [21]. Briefly, 48 h after transfection, CHO or HEK 293 cells (3 · 10 6 cells/ 100 mm dish) expressing h5-HT 4 R fusion proteins were detached and washed with PBS.…”

Oligomeric potential of the M2 muscarinic cholinergic receptor

“…Whereas such strategies demonstrate that the receptor can be at least dimeric, they have contributed only indirectly to the determination of an explicit oligomeric size. The approach has been to compare the index of aggregation, measured at known levels of expression, with predictions based on the assumption that oligomers form randomly from the constituent monomers (Ayoub et al 2002;Park et al 2001;Mercier et al 2002). In the current study, three differently tagged forms of the M 2 muscarinic receptor were coexpressed in Sf9 cells in order to test directly for trimers or larger aggregates.…”

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes

“…FRET has been applied to the imaging of AKAP79 interactions with CaN catalytic subunit and PKA RII subunits, including data suggesting formation of the ternary complex of scaffold, CaN and PKA [51]. Neither FRET spectroscopy nor the more sensitive and advanced BRET 2 spectroscopy [52,75] has been applied to defining the distances between the scaffold and other molecules in the signalling complex. Furthermore, it will be very exciting to define the temporal, dynamic associations and distances between AKAP79/250, the β 2 AR and other members of the signalling complex.…”