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 cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR's use with three G scoupled GPCRs, two G i -coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proofof-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR's unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.

A cAMP-biosensor-based assay for measuring plasma arginine–vasopressin levels

“…Specifically, LC–MS/MS tends to yield lower values compared to RIA. Despite high correlations among RIA, the biosensor-based assay, and LC–MS/MS, their values do not perfectly align 21 , 22 . The discrepancies in values among these three methods may warrant examination in the future.…”

Protein-Based Tools for Studying Neuromodulation

“…(F) Dose–response curve of DRD1 IGNiTR with dopamine, fenoldopam, and SCH 23390. Reproduced with permission from ref . Copyright 2023 American Chemical Society.…”

“…Besides detecting neuromodulators in vivo, we have also developed an in vitro assay for detecting GPCR activation in cell lysates . Live cell-based assays have significantly advanced the screening and characterization of GPCR ligands. − However, there is a lack of in vitro GPCR assays that are convenient to use and have readily accessible reagents that can be prepared in large batches, frozen and stored.…”