1. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor
- Author
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Roger K. Sunahara, Yingying Cai, Elsa C. Y. Yan, Yang Yang, Yuting Liu, Brian T. DeVree, Kelly J. Culhane, and Shukla, Arun
- Subjects
0301 basic medicine ,Surfactants ,Cell Membranes ,Lipid Bilayers ,lcsh:Medicine ,Plasma protein binding ,Biochemistry ,Receptors, G-Protein-Coupled ,Cell Signaling ,Glucagon-Like Peptide 1 ,Receptors ,Nanotechnology ,Membrane Receptor Signaling ,5-HT5A receptor ,Receptor ,lcsh:Science ,Microscopy ,Multidisciplinary ,Physics ,Diabetes ,Condensed Matter Physics ,Lipids ,Cell biology ,Physical Sciences ,Engineering and Technology ,Cellular Structures and Organelles ,Lipoproteins, HDL ,Type 2 ,Research Article ,Signal Transduction ,Biotechnology ,Protein Binding ,Transmembrane Receptors ,HDL ,G protein ,General Science & Technology ,Lipoproteins ,1.1 Normal biological development and functioning ,Materials Science ,Detergents ,Material Properties ,Bioengineering ,Biology ,Electron ,Glucagon-Like Peptide-1 Receptor ,03 medical and health sciences ,G-Protein-Coupled ,Microscopy, Electron, Transmission ,Underpinning research ,Diabetes Mellitus ,Humans ,Hypoglycemic Agents ,Transmission ,Materials by Attribute ,Nanodisc ,Metabolic and endocrine ,G protein-coupled receptor ,G protein-coupled receptor kinase ,Venoms ,lcsh:R ,Biology and Life Sciences ,Proteins ,Membrane Proteins ,Reproducibility of Results ,Cell Biology ,Nanostructures ,G-Protein Signaling ,030104 developmental biology ,HEK293 Cells ,Diabetes Mellitus, Type 2 ,Membrane protein ,Bionanotechnology ,Anisotropy ,Exenatide ,Osteoporosis ,lcsh:Q ,G Protein Coupled Receptors ,Peptides - Abstract
Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
- Published
- 2017