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Abstract
G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [35S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.
Highlights
CB2 is a class A Gprotein coupled receptor whose activity is critical to immunoregulatory and anti-inflammatory processes
We report a higher thermostability of the constitutively active T3.46I CB2 variant solubilized in detergent micelles relative to the wild-type receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation
The expression levels of these proteins in E. coli membranes were analyzed by Western blot; ligand-binding properties – by a competition binding assay with radiolabeled [3H]CP-55,940; the G protein activation properties – by guanine exchange factor (GEF) assay on E. coli membranes containing recombinantly expressed receptor variants
Summary
CB2 is a class A (rhodopsin-family) Gprotein coupled receptor whose activity is critical to immunoregulatory and anti-inflammatory processes. We further demonstrate that Façade-TEG improves CB2 thermostability relative to other detergents, promotes monodisperse CB2 reconstitution, and tolerates very high CB2 concentration (vide infra), It was reported that the mutations T3.46I or T3.46A of CB1 produce constitutively active and inactive receptor variants respectively.
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