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_s-coupled 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 provid

SUBMITTER: Miller RM 

PROVIDER: S-EPMC10588934 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature