Internalization dissociates β₂-adrenergic receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β₂-adrenergic receptors (β₂ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β₂ARs between subcellular compartments. BRET between β₂ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β₂ARs and endosome markers increases. Energy transfer between β₂ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β₂ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β₂ARs associate transiently with each other in the plasma membrane, or that β₂AR dimers or oligomers are actively disrupted during internalization.