Signalosome Profiling Reveals Allosteric Interactions Between G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) comprise the largest group of cell surface receptors, translating environmental signals into cellular responses via cognate G protein partners. Contrary to our initial understanding, most GPCRs do not function in living cells as monomers, but most likely dimers, or even larger arrays of receptors. Standard drug design approaches rely on the notion that drugs binding the two receptors in a given dimer likely function independently of one another. However, this view has been challenged by recent work showing that ligand binding at both receptors can modulate dimeric receptors via allosteric communication. While one receptor may actually be needed to drive signaling, the other acts to control or modulate these signals, without a direct signaling outcome itself. Based on the notion of allosteric modulation within homo- and heterodimers, we must now consider a broader examination of the effects of single- and dual-ligand stimulation in the context of tissues expressing multiple GPCRs. Here, we describe the use of a combination of calcium, cyclic adenosine monophosphate (cAMP), and mitogen-activated protein kinase (MAPK) signaling assays, and protein–protein interaction assays to characterize functional and physical interactions in a putative D2 dopamine receptor/oxytocin receptor heterodimer (D2R/OTR), in HEK 293 cells.