Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.
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 unexpl...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5469476?pdf=render |
| _version_ | 1811197349252300800 |
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| author | Yingying Cai Yuting Liu Kelly J Culhane Brian T DeVree Yang Yang Roger K Sunahara Elsa C Y Yan |
| author_facet | Yingying Cai Yuting Liu Kelly J Culhane Brian T DeVree Yang Yang Roger K Sunahara Elsa C Y Yan |
| author_sort | Yingying Cai |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T01:13:11Z |
| format | Article |
| id | doaj.art-41e14cb32d78488fb6603b14de6663b9 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T01:13:11Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-41e14cb32d78488fb6603b14de6663b92022-12-22T03:54:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01126e017956810.1371/journal.pone.0179568Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.Yingying CaiYuting LiuKelly J CulhaneBrian T DeVreeYang YangRoger K SunaharaElsa C Y YanFamily 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.http://europepmc.org/articles/PMC5469476?pdf=render |
| spellingShingle | Yingying Cai Yuting Liu Kelly J Culhane Brian T DeVree Yang Yang Roger K Sunahara Elsa C Y Yan Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. PLoS ONE |
| title | Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. |
| title_full | Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. |
| title_fullStr | Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. |
| title_full_unstemmed | Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. |
| title_short | Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. |
| title_sort | purification of family b g protein coupled receptors using nanodiscs application to human glucagon like peptide 1 receptor |
| url | http://europepmc.org/articles/PMC5469476?pdf=render |
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