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

James H Felce,Alasdair Macrae,Simon J Davis

doi:10.1016/j.bpj.2018.09.034

Abstract

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.

Highlights

Comprising >700 proteins, G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors encoded by the human genome and are of great pharmacological importance
The most powerful tool for examining receptor stoichiometry, that of single-molecule imaging, has yet to be used to demonstrate GPCR heterodimerization, even though it is largely responsible for the present consensus that homodimerization is generally transient and the dominant GPCR stoichiometry is that of monomers [8]
In the type-4 bioluminescence resonance energy transfer (RET) (BRET) assay, putative interaction partners are coexpressed in the same cells: protein A in the form of a Rluc- and green fluorescent protein (GFP)-tagged BRET pair and protein B fused to FK506-binding protein (FKBP) (Fig. 1 A)

Summary

Introduction

Comprising >700 proteins, G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors encoded by the human genome and are of great pharmacological importance. The widely used BRET saturation assay involves the Biophysical Journal 116, 31–41, January 8, 2019 31 measurement of BRET efficiency (BRETeff) at a range of acceptor concentrations and constant donor density, but the interpretation of this assay is complicated by the varying levels of nonspecific RET resulting from changes in acceptor density alone [7] This is problematic in the case of heterodimerization because differences in expression rates or subcellular distribution of receptor subunits increases the likelihood of data deviating from pseudolinearity and the risk of reporting false dimers. Singlemolecule spectroscopic techniques, such as fluorescence cross-correlation spectroscopy, have reported heterodimerization in a number of cases [9], but these approaches have yet to become routine

Methods

Results

Conclusion