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.
Highlights
Gprotein-coupled receptors (GPCRs) constitute the largest family of membrane proteins that detect extracellular stimuli and activate intracellular signal transduction pathways
There appears to be a band at approximately ~100 kDa representing the dimer of glucagon-like peptide-1 receptor (GLP1R) [43], further indicating the expression of the receptor in the HEK293 cells stably transfected with the GLP1R gene
The MSP1E3D1 protein generates nanodiscs in a diameter of ~15 nm [34], which is suitable for the incorporation of GLP1R and family B GPCRs in general
Summary
Gprotein-coupled receptors (GPCRs) constitute the largest family of membrane proteins that detect extracellular stimuli and activate intracellular signal transduction pathways. All GPCRs share a common seven-transmembrane topology, and are often classified into five main sub-. Purification of family B G protein-coupled receptors using nanodiscs. Innovator Award DP2-GM114830 (to support YY and to maintain the TEM facility). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
