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...

In the last four to five years, the view that G protein-coupled receptors (GPCRs) function as monomeric proteins has been challenged by numerous studies, which suggests that GPCRs exist as dimers or even higher-structure oligomers. Recently, biophysical methods based on luminescence and fluorescence energy transfer have confirmed the existence of such oligomeric complexes in living cells. Although no consensus exists on the role of receptor dimerization, converging evidence suggests potential roles in various aspects of receptor biogenesis and function. In several cases, receptors appear to fold as constitutive dimers early after biosynthesis, whereas ligand-promoted dimerization at the cell surface has been proposed for others. The reports of heterodimerization between receptor subtypes suggest a potential level of receptor complexity that could account for previously unexpected pharmacological diversities. In addition to fundamentally changing our views on the structure and activation processes of GPCRs, the concept of homo- and heterodimerization could have dramatic impacts on drug development and screening.

No abstract

Since the unexpected discovery of the antipsychotic activity of chlorpromazine, a variety of therapeutic agents have been developed for the treatment of schizophrenia. Despite differences in their activities at various neurotransmitter systems, all clinically effective antipsychotics share the ability to interact with D2 class dopamine receptors (D2R). D2R mediate their physiological effects via both G protein-dependent and independent (␤-arrestin 2-dependent) signaling, but the role of these D2R-mediated signaling events in the actions of antipsychotics remains unclear. We demonstrate here that while different classes of antipsychotics have complex pharmacological profiles at G protein-dependent D2R long isoform (D2 LR) signaling, they share the common property of antagonizing dopamine-mediated interaction of D2 LR with ␤-arrestin 2. Using two cellular assays based on a bioluminescence resonance energy transfer (BRET) approach, we demonstrate that a series of antipsychotics including haloperidol, clozapine, aripiprazole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone all potently antagonize the ␤-arrestin 2 recruitment to D2 LR induced by quinpirole. However, these antipsychotics have various effects on D2 LR mediated Gi/o protein activation ranging from inverse to partial agonists and antagonists with highly variable efficacies and potencies at quinpirole-induced cAMP inhibition. These results suggest that the different classes of clinically effective antipsychotics share a common molecular mechanism involving inhibition of D2 LR/␤-arrestin 2 mediated signaling. Thus, selective targeting of D2LR/␤-arrestin 2 interaction and related signaling pathways may provide new opportunities for antipsychotic development.BRET ͉ schizophrenia ͉ signaling ͉ functional selectivity