Constraints on GPCR Heterodimerization Revealed by the Type-4 Induced-Association BRET Assay

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically important family of cell-surface receptors encoded by the human genome. In many instances, the distinct signaling behavior of certain GPCRs has been explained in terms of the formation of heteromers with, for example, distinct signaling properties and allosteric cross-regulation. Confirmation of this has, however, been limited by the paucity of reliable methods for probing heteromeric GPCR interactions in situ. The most widely used assays for GPCR stoichiometry, based on resonance energy transfer, are unsuited to reporting heteromeric interactions. Here, we describe a targeted bioluminescence resonance energy transfer (BRET) assay, called type-4 BRET, which detects both homo- and heteromeric interactions using induced multimerization of protomers within such complexes, at constant expression. Using type-4 BRET assays, we investigate heterodimerization among known GPCR homodimers: the CXC chemokine receptor 4 and sphingosine-1-phosphate receptors. We observe that CXC chemokine receptor 4 and sphingosine-1-phosphate receptors can form heterodimers with GPCRs from their immediate subfamilies but not with more distantly related receptors. We also show that heterodimerization appears to disrupt homodimeric interactions, suggesting the sharing of interfaces. Broadly, these observations indicate that heterodimerization results from the divergence of homodimeric receptors and will therefore likely be restricted to closely related homodimeric GPCRs.