Abstract
A large number of GPCRs are potentially valuable drug targets but remain understudied. Many of these lack well-validated activating ligands and are considered "orphan" receptors, and G protein coupling profiles have not been defined for many orphan GPCRs. Here we asked if constitutive receptor activity can be used to determine G protein coupling profiles of orphan GPCRs. We monitored nucleotide-sensitive interactions between 48 understudied orphan GPCRs and five G proteins (240 combinations) using bioluminescence resonance energy transfer (BRET). No receptor ligands were used, but GDP was used as a common G protein ligand to disrupt receptor-G protein complexes. Constitutive BRET between the same receptors and β-arrestins was also measured. We found sufficient GDP-sensitive BRET to generate G protein coupling profiles for 22 of the 48 receptors we studied. Altogether we identified 48 coupled receptor-G protein pairs, many of which have not been described previously. We conclude that receptor-G protein complexes that form spontaneously in the absence of guanine nucleotides can be used to profile G protein coupling of constitutively-active GPCRs. This approach may prove useful for studying G protein coupling of other GPCRs for which activating ligands are not available.
Highlights
G protein-coupled receptors (GPCRs) are the targets of a large fraction of clinically-useful drugs, and efforts to develop new drugs targeting GPCRs are ongoing [1]
In a recent study we found that many GPCRs would spontaneously form complexes with cognate G proteins in the absence of guanine nucleotides, and these complexes were disrupted by the addition of GDP [14]
We have previously monitored direct interactions between GPCRs and G proteins using bioluminescence resonance energy transfer (BRET) between receptors fused to Renilla luciferase no added ligand
Summary
G protein-coupled receptors (GPCRs) are the targets of a large fraction of clinically-useful drugs, and efforts to develop new drugs targeting GPCRs are ongoing [1]. Because each of the four G protein families (Gs/olf, Gi/o, Gq/11, and G12/13) activates different downstream effectors, GPCR-G protein coupling profiles have traditionally been determined using second messenger assays, most commonly those that measure intracellular cyclic AMP (cAMP) and calcium. These assays are robust and quite sensitive, crosstalk between pathways can complicate interpretation, and comparable second messenger assays are not available for all four families. These results may facilitate efforts aimed at understanding the physiological roles these receptors, and at discovering and validating new drugs acting at GPCRs
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