An accessible and generalizable in vitro luminescence assay for detecting GPCR activation

Abstract

G-protein coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here we describe the design of a generalizable and accessibleIn vitroGPCR splitNanoLuc ligandTriggeredReporter (IGNiTR), having broad and diverse applications. IGNiTR leverages the interaction between a conformationspecific binder and agonist-activated GPCR to reconstitute a split nanoluciferase. We have demonstrated IGNiTR with three Gs-coupled GPCRs and a Gi-coupled GPCR with three classes of conformation-specific binders: nanobodies, miniG proteins, and G-protein peptidomimetics. IGNiTR demonstrated binding efficacy and potency values of various Dopamine Receptor D1 (DRD1) ligands that agree well with reported values. IGNiTR also allows the use of a synthetic G protein peptidomimetic, providing easily standardized reagents for characterizing GPCRs and ligands. We demonstrated three applications of IGNiTR: 1) characterizing GPCR functionality during Nanodisc-based reconstitution process; 2) highthroughput screening of ligands against DRD1; 3) detection of opioids for in the field applications. Due to its convenience, accessibility and consistency, IGNiTR will find extensive applications in GPCR ligand detection, screening and GPCR characterization.

Protein-Based Tools for Studying Neuromodulation

“…49 We also showed its versatile applications, including high-throughput GPCR ligand screening, easy detection of opioids using a simple camera, and characterization of the functionality of extracted GPCRs. 49 IGNiTR is the first GPCR activity in vitro assay that can be easily scaled up and performed with only a one-step protocol. IGNiTR will find broad applications in highthroughput GPCR ligand screening and in vitro GPCR activity characterization.…”

“…50 Split NanoLuc fragments SmBiT and LgBiT are each fused to the conformation-specific binder and the GPCR of interest (Figure 4A). 49 Upon addition of GPCR agonists, the GPCR undergoes a conformational change and recruits the conformation-specific binder, reconstituting the split NanoLuc. We tested three classes of conformational specific binders: conformation specific nanobodies, miniG proteins, and G protein peptidomimetics.…”

“…We tested three classes of conformational specific binders: conformation specific nanobodies, miniG proteins, and G protein peptidomimetics. 49 G protein peptidomimetics were designed based on the α-5 helix of the G protein, with an unnatural amino acid, cyclohexylalanine (Cha), introduced in the penultimate position to enhance the peptide's interaction with the activated GPCR (Figure 4B,C). While nanobodies and miniG proteins can be coexpressed along with the GPCR of interest and provide a robust readout of the conformational change of GPCR activation, the use of G protein peptidomimetics enables the incorporation of unnatural amino acids for designing high affinity peptide binders.…”

“…IGNiTR is based on the use of conformation-specific binders for the activated GPCR conformational state and the proximity-dependent split NanoLuciferase (NanoLuc) . Split NanoLuc fragments SmBiT and LgBiT are each fused to the conformation-specific binder and the GPCR of interest (Figure A) . Upon addition of GPCR agonists, the GPCR undergoes a conformational change and recruits the conformation-specific binder, reconstituting the split NanoLuc.…”

“…Upon addition of GPCR agonists, the GPCR undergoes a conformational change and recruits the conformation-specific binder, reconstituting the split NanoLuc. We tested three classes of conformational specific binders: conformation specific nanobodies, miniG proteins, and G protein peptidomimetics . G protein peptidomimetics were designed based on the α-5 helix of the G protein, with an unnatural amino acid, cyclohexylalanine (Cha), introduced in the penultimate position to enhance the peptide’s interaction with the activated GPCR (Figure B,C).…”