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.

]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed ␦-opioid receptors and ␤ 2 -adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the ␦-opioid receptor-␤ 2 -adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human ␦-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.Recent studies have started to provide a significant body of evidence to support a concept of constitutive homo-oligomerization of a range of G protein-coupled receptors (GPCRs) 1 (1, 2).GPCRs for which such evidence exists include the ␤ 2 -adrenoreceptor (3, 4), the D 2 dopamine receptor (5), the M 3 muscarinic acetylcholine receptor (6), the V 2 vasopressin receptor (7), the ␦-opioid (8, 9) and -opioid receptors (9), the histamine H 2 receptor (10), and the CCR5 receptor (11). Furthermore, recent evidence has also indicated a requirement for the constitutive hetero-oligomerization of distinct GPCRs, such as between the GABA B R1 and GABA B R2 receptors, to generate a functional receptor expressed at the cell surface (12). Two important issues, however, remain contentious. The first of these is whether ligand occupancy alters the extent of GPCR oligomerization, and the second is the likely extent of GPCR hetero-oligomerization. In a range of reports, GPCR homo-oligomerization has been reported to be increased (3, 4, 11), decreased (8), or unaffected (6, 7) by the addition of receptor ligands. Similarly, in GPCR heterodimerization studies, interactions have been indicated to be unaffected (12), regulated (13), or almost entirely dependent upon (14) the addition of receptor agonists and hetero-oligomers have recently been reported to form between quite distinct (14), as well as between closely related (12, 13), GPCR sequences. The earliest studies on GPCR oligomerization relied on the capacity to co-immunoprecipitate co-expressed but differentially epitope-tagged forms of a GPCR (see Ref. 15 for review). Because of the hydrophobic nature of the seven trans-plasma membrane helices of GPCR family members, care must be taken, however, to exclude nonspecific interactions between GPCR pairs resulting from detergent dissolution of cellular membranes. More recent studies have employed vari...

The concept that GPCRs exist and potentially function as dimers and/or higher oligomers has progressed recently from hypothesis to being widely accepted. A range of techniques has contributed to this understanding, including co-immunoprecipitation and various forms of fluorescence and bioluminescence resonance energy transfer. Although co-immunoprecipitation studies indicate the capacity of a wide range of GPCRs to form hetero-dimers as well as homo-dimers, this approach is not well suited to examine selectivity of interactions. Both bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) have been applied to the detection of GPCR dimerisation in intact cells and BRET and FRET have been used to attempt to quantitate the fraction of GPCRs present as dimers. Following heterologous expression, a considerable fraction of many GPCRs is not fully processed and is trafficked to the proteasome or lysosome for destruction. A distinct limitation of both BRET and conventional FRET approaches is that both the energy donor and energy acceptor tags are inside the cell. Time-resolved FRET employing N-terminally epitope-tagged GPCRs has been used to allow detection only of dimers trafficked successfully to the cell surface. Reports indicating the appearance of distinct pharmacology and function following co-expression of two GPCRs are fascinating. Much remains to be examined, however, on the specificity and mechanisms of these interactions and to develop techniques to monitor the function only of hetero-dimers when the corresponding homo-dimers must also be present.

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human ␣ 1A-1 -adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the ␣ 1A-1 -adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the ␣ 1A-1 -adrenoceptor and the ␦ opioid peptide receptor. Only a fraction of the ␣ 1A-1 -adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of ␣ 1A-1 -adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human ␣ 1A-1-adrenoceptor with differences limited to the C-terminal tail. Each of the ␣ 1A-1 , ␣ 1A-2a , and ␣ 1A-3a -adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined ␣ 1L -adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the ␣ 1L -adrenoceptor.

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.