Detection of beta(2)-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

Heptahelical receptors that interact with heterotrimeric G proteins represe nt the largest family of proteins involved in signal transduction across bi ological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the ex istence of G protein-coupled receptor (GPCR) dimers at the surface of livin g cells is still lacking. Here, using bioluminescence resonance energy tran sfer (BRET), as a protein-protein interaction assay in whole cells, we unam biguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR ) forms constitutive homodimers when expressed in HEK-293 cells. Receptor s timulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to th e BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers a t the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at th e cell surface was confirmed further by the observation that BS3, a cell-im permeable cross-linking agent increased BRET between beta(2)AR molecules. T he selectivity of the constitutive interaction was documented by demonstrat ing that no BRET occurred between the beta(2)AR and two other unrelated GPC R. In contrast, the well characterized agonist-dependent interaction betwee n the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functiona l dimers in vivo and that BRET-based assays can be used to study both const itutive and hormone-promoted selective protein-protein interactions.