A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs

Abstract

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.

Keywords: Detergent solubilization; G protein-coupled receptors; Membrane proteins; Mini-G; Native PAGE; Peptide agonist; Thermostability; hrCNE.

Figure 1.. Quantitative Assay Formats Overview.

Depiction of key steps of the quantitative hrCNE gel assay format corresponding to Procedure C-G and the Data analysis in the protocol.