Class A G protein-coupled receptors assemble into functional higher-order hetero-oligomers.

Although class A seven-transmembrane-helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC) and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3), α1a -adrenoceptor (α1a -AR), and arginine vasopressin receptor 1A (AVPR1A) form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.