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

Abstract

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically important family of cellsurface 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 crossregulation. 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.

Sphingosine 1‐phosphate but not Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes

“…Prior work has demonstrated the importance of AP1 for induction of BDNF transcription in neurons (Tuvikene, Pruunsild, Orav, Esvald, & Timmusk, 2016) and LIF in mammary epithelial cells (Levy et al, 2010); and EGR1 for induction of LIF, HBEGF, and PDGFB in mesenchymal stem cells (Kerpedjieva, Kim, Barbeau, & Tamama, 2012). We note that S1PR2 and S1PR3 have the capability to dimerize (Felce, MacRae, & Davis, 2019). Although not investigated in this study, it is possible that S1PR1/S1PR2 heterodimerization creates a signaling complex that drives sustained ERK1/2 signaling.…”

Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse

“…Both of these models would be consistent with our observations of greater CXCR4- (and GPCR-)dependence in naïve T cells, which are more reliant on both CD28 costimulation and stable synapse formation. CXCR4 is also known to interact with other GPCRs, such as CCR5 (Contento et al, 2008 ; Felce et al, 2019 ), and it is also possible that it is able to influence signaling from these receptors within in the IS. Regardless of mechanism, we observe a clear independence on ligation, and so it seems likely this can be regulated primarily by overall CXCR4 expression.…”

Experimental determination of the bioluminescence resonance energy transfer (BRET) Förster distances of NanoBRET and red-shifted BRET pairs

“…While BRET 2 has historically been applied in the evaluation of GPCR receptor signalling [ 36 ], NanoBRET (NCT) could take over the niche of BRET 2 in the study of macromolecular interactions due to its large Förster distance of 6.97 nm, just under that of BRET 2 . Unlike the shortcomings of BRET 2 , NanoBRET (NCT) exhibits bright and sustained bioluminescence together with an excellent spectral separation of 158 nm.…”