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 CB(2), 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 [(35)S]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 CB(2) thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB(2) variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB(1) 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.