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

Ramsay, D., Kellett, E., McVey, M. , Rees, S. and Milligan, G. (2002) 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. Biochemical Journal, 365, pp. 429-440. (doi: 10.1042/BJ20020251)

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). BRET2 (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 d-opioid receptor was confirmed using BRET2. Homo-oligomerization of the k-opioid receptor was observed using both BRET techniques. Both homo- and hetero-oligomers, containing both d- and k-opioid receptors, were unaffected by the presence of receptor ligands. BRET detection of opioid receptor homo- and hetero-oligomers required expression of 50000–100000 copies of the receptor energy acceptor construct per cell. The effectiveness of d–k-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 b2-adrenoceptor was also assessed. Although such interactions were detected, at least 250000 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 k-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 100000 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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Milligan, Professor Graeme and McVey, Dr Mary
• Authors: Ramsay, D., Kellett, E., McVey, M., Rees, S., and Milligan, G.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences; College of Medical Veterinary and Life Sciences > School of Life Sciences
• Journal Name: Biochemical Journal
• Publisher: Portland Press Ltd.
• ISSN: 0264-6021
• ISSN (Online): 1470-8728
• Published Online: 24 April 2002