Quantitative Assessment of β1- and β2-Adrenergic Receptor Homo- and Heterodimerization by Bioluminescence Resonance Energy Transfer

Abstract

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of ␤ 1 -and ␤ 2 -adrenergic receptor homo-and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. ␤ 1 -and ␤ 2 -adrenergic receptors were found to have similar propensity to engage in homo-and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo-and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo-and heterodimers between the closely related ␤ 1 -and ␤ 2 -adrenergic receptors is determined by their relative levels of expression. G protein-coupled receptors (GPCRs)1 represent the largest family of transmembrane receptors involved in cell signaling. In the past few years, many studies indicated that GPCR dimerization can occur between two identical receptors (homodimerization), between two different receptor subtypes of the same family, or even between receptors that are only distantly related (heterodimerization) (for a review, see Refs. 1 and 2). In most instances, co-immunoprecipitation was used as the primary experimental evidence supporting the existence of such dimers. More recently, however, light resonance energy transfer techniques such as fluorescence and bioluminescence resonance energy transfer (FRET and BRET) were also used. These "non-invasive" proximity-based assays confirmed that GPCR dimerization does not represent biochemical artifacts due to receptor solubilization and can occur in living cells. They have been used to demonstrate homodimerization of the ␤ 2 -adrenergic (3), the yeast alpha mating factor (4), the SST5 somatostatin (5), the gonadotropin releasing hormone (6), the luteinizing hormone (7), the ␦-opioid (8), the thyrotropin-releasing hormone (9), the cholecystokinin (10), and the melatonin (11) receptors as well as heterodimerization between somatostatin receptor subtypes (5), somatostatin and dopamine receptors (12), melatonin receptor subtypes (11), and opioid receptor subtypes (13).An advantage of BRET and FRET over co-immunoprecipitation approaches lies in the more quantitative nature of the assay. However, relatively few studies exploited this quantitative potential for the study of GPCR dimerization. For the melatonin receptors, Ayoub et al. (11) recently used BRET competition assays to determine that the transfer of energy resulted from the formation of dimers and not of higher order oligomers. They also showed that...

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.…”

“…The basic structural unit appears to be a tetramer, but the basic functional unit is unknown. The oligomeric size of the melatonin receptor and the b 2 adrenergic receptor has been inferred from studies in which the association was monitored by measuring the transfer of energy from a bioluminescent tag (Ayoub et al 2002;Mercier et al 2002). The effects of varying the ratio of donor and acceptor or of an unlabeled competitor and the donor-acceptor pair led in each case to the conclusion that the receptor exists as a dimer.…”

“…Although the distribution of receptors among different states of aggregation is static in solution, and oligomers are not an artifact of solubilization, the origin of monomers and their prevalence in the membrane remains unclear (Park and Wells 2003). It has been suggested on the basis of resonance energy transfer and biotinylation that most if not all b 2 adrenergic and A 2A adenosine receptors are oligomeric in the plasma membrane (Mercier et al 2002;Canals et al 2004). If that is so for the M 2 muscarinic receptor, the monomers identified in crude extracts and after purification may arise in part from the disaggregation of oligomers during solubilization.…”

C5a Receptor Oligomerization

“…Heterodimerization of opioid receptors and ␦ results in novel ligand binding and function, proving that GPCR oligomerization has physiologic relevance (12). More recently, energy transfer studies, using either fluorescence (FRET) or bioluminescence (BRET), have also shown the proximity of monomers of many members of the rhodopsin family of GPCRs, including ␤ 2 -adrenergic receptor (13), somatostatin receptors (14), opioid receptors (15), and the C5a receptor (57).…”

G Protein-Coupled Receptor Heteromer Identification Technology: Identification and Profiling of GPCR Heteromers

“…This is important as ligand dependency allows discrimination between a signal because of genuine physical proximity of receptors as opposed to their random collision because of overexpression in recombinant systems. Furthermore, this assay has advantages over BRET saturation assays, 23 which are laborious and not ligand dependent, rendering them unsuitable for even secondary screening. 6 …”