Probing thermostability of detergent-solubilized CB2 receptor by parallel G protein–activation and ligand-binding assays
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 pha...
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Published in | The Journal of biological chemistry Vol. 295; no. 1; pp. 181 - 190 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A
Elsevier Inc
03.01.2020
American Society for Biochemistry and Molecular Biology |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | Edited by Henrik G. Dohlman Present address: University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.RA119.010696 |