Background

G protein-coupled receptors are a family of ~800 membrane-embedded receptors with critical physiologic roles in virtually every system of the human body and they are targeted by approximately one-third of FDA-approved therapeutics (Pierce et al., 2002; Sriram and Insel, 2018). Significant advances in biochemical, biophysical, and structural methods for the study of GPCRs have drastically improved our understanding of how an agonist-bound GPCR acts as a guanine nucleotide exchange factor, to activate intracellular heterotrimeric G proteins. Nonetheless, biochemical investigations of receptor-G protein coupling remain challenging due to the dynamic nature of the receptors and the complexity of their interactions with heterotrimeric G proteins, which are regulated by agonist binding to the GPCR, and guanine nucleotide binding to the G protein alpha subunit. Biochemical and biophysical methods that have been used to investigate this interaction include fluorescence- and nuclear magnetic resonance (NMR)-based methods (Yao et al., 2009; Gregorio et al., 2017; Huang et al., 2021). These methods are very powerful, but they are technically challenging and costly and require purified receptors in detergents or nanodiscs.

An alternative to working with G protein heterotrimers for biochemical studies is the use of “mini-G” proteins that were first developed as tools for GPCR structural studies (Carpenter and Tate, 2016; Nehmé et al., 2017). Mini-G proteins are minimal G protein alpha subunits that have enhanced stability in detergents and contain mutations that uncouple receptor binding from guanine nucleotide exchange, such that they trap GPCRs in an active state conformation, equivalent to that observed in structures of agonist-bound GPCRs in complex with nucleotide-free heterotrimer. Mini-G proteins are available for each of the four families of G protein alpha subunits. Biochemical assays for assessing mini-G interactions with GPCRs have been reported (Nehmé et al., 2017), including a fluorescent saturation binding assay using immobilized receptor, and an assay based on the fluorescence-detection size-exclusion chromatography (FSEC) technique (Kawate and Gouaux, 2006). These assays work with unpurified receptors in detergent-solubilized lysates and use GFP-tagged mini-G for detection.

We recently reported a novel biochemical assay for GPCR-mini-G coupling based on the high resolution clear native electrophoresis (hrCNE) technique, which is a membrane protein native-PAGE method compatible with fluorescently-labeled proteins (Wittig et al., 2007). We developed the assay during the course of our work to understand how calcitonin gene-related peptide and adrenomedullin peptide agonists and RAMP accessory proteins control G protein coupling of the class B GPCR calcitonin receptor-like receptor (CLR) (Roehrkasse et al., 2020). The method enables both rapid, cost-effective screening for detergent-stable GPCR-G protein complexes and quantitative studies of agonist-dependent receptor-mini-G coupling. It does not require a purified receptor. Instead, it uses an EGFP-tagged receptor transiently over-expressed in the mammalian HEK293S GnT1^–; cell line. In the screening format, adherent cell cultures are directly solubilized with detergent, subjected to a simple centrifugation step, and the supernatants are analyzed by native-PAGE with visualization of the receptor by in-gel fluorescence imaging. Exogenous addition of agonist peptides and purified mini-G yields a mobility shift indicative of complex formation. In the quantitative assay format, crude membrane preparations are used to improve reproducibility and increase throughput. In the first quantitative format, the agonist is varied in the presence of a constant excess of mini-G to generate a binding curve that measures the apparent affinity of the agonist for the mini-G-coupled receptor. In the second quantitative format, mini-G is varied in the presence of a constant receptor-saturating concentration of agonist to generate a binding curve that measures the apparent affinity of mini-G for the agonist-occupied receptor. This second format provides a partial measure of agonist efficacy (Roehrkasse et al., 2020).

The native PAGE method proved to be a relatively simple, inexpensive, and highly versatile assay, which allowed us to investigate the biochemistry of agonist and G protein interactions with CLR:RAMP complexes. The screening and quantitative assay formats are described in detail here. We have also found the method to be very useful in a thermostability format, which is not described here, but can be found in our original report (Roehrkasse et al., 2020). This assay should be applicable to other GPCRs and may be valuable for the biochemical study of other challenging membrane proteins and their binding partners.