Homo- and hetero-oligomeric interactions between G-protein-coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences

Abstract

Homo- and hetero-oligomerization of G-protein-coupled receptors (GPCRs) were examined in HEK-293 cells using two variants of bioluminescence resonance energy transfer (BRET). BRET(2) (a variant of BRET) offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared with traditional BRET. Previously recorded homo-oligomerization of the human delta-opioid receptor was confirmed using BRET(2). Homo-oligomerization of the kappa-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both delta- and kappa-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50,000-100,000 copies of the receptor energy acceptor construct per cell. The effectiveness of delta-kappa-opioid receptor hetero-oligomer formation was as great as for homomeric interactions. The capacity of the two opioid receptors to form oligomeric complexes with the beta(2)-adrenoceptor was also assessed. Although such interactions were detected, at least 250,000 copies per cell of the energy acceptor were required. Requirement for high levels of receptor expression was equally pronounced in attempts to measure hetero-oligomer formation between the kappa-opioid receptor and the thyrotropin-releasing hormone receptor-1. These studies indicate that constitutively formed homo- and hetero-oligomers of opioid receptor subtypes can be detected in living cells containing less than 100,000 copies of the receptors. However, although hetero-oligomeric interactions between certain less closely related GPCRs can be detected, they appear to be of lower affinity than homo- or hetero-oligomers containing closely related sequences. Interactions recorded between certain GPCR family members in heterologous expression systems are likely to be artefacts of extreme levels of overexpression.

Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

“…We have also demonstrated homo-oligomerization of the TRHR1 using a biophysical method, bioluminescence resonance energy transfer (BRET) (30); however, it is not known if TRHR2 undergoes homo-oligomerization, nor if TRHR subtypes have the potential to hetero-oligomerize. BRET represents a powerful technique to study protein-protein interactions in living cells and has been used to provide evidence for homo and heterodimerization of other GPCRs (4,(32)(33)(34)(35)(36)(37). In the present study, we show that TRHR subtypes could form both homo-oligomers and heterooligomers in living mammalian cells.…”

G-Protein–Coupled Receptor Mas Is a Physiological Antagonist of the Angiotensin II Type 1 Receptor

“…The potential for such direct interactions between Mas and the AT 1 receptor was thus investigated with various forms of BRET. 19,24,25 eYFP was appended to the C-terminal tail of the AT 1 receptor from which the stop codon was eliminated. When this construct was expressed transiently in HEK293 cells, fluorescence microscopy demonstrated it to be present largely at the cell surface (data not shown).…”

“…The production of thyrotropin-releasing hormone (TRH) receptor-1-eYFP and demonstration of the improved resolution of energy transfer signal from the emission spectrum of Renilla luciferase that is produced by the use of a variant of the bioluminescence resonance energy transfer (BRET 2 ) have been described elsewhere. 19 …”

Cys-27 Variant of Human δ-Opioid Receptor Modulates Maturation and Cell Surface Delivery of Phe-27 Variant via Heteromerization

“…Refs. [7][8][9][10][11][12][13][14][15][16]. These approaches utilizing heterologous expression systems have been recently complemented by in vivo studies.…”