A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor.
Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation effo...
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| Format: | Article |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0093746&type=printable |
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| author | Lucy S Jun Aaron D Showalter Nosher Ali Feihan Dai Wenzhen Ma Tamer Coskun James V Ficorilli Michael B Wheeler M Dodson Michael Kyle W Sloop |
| author_facet | Lucy S Jun Aaron D Showalter Nosher Ali Feihan Dai Wenzhen Ma Tamer Coskun James V Ficorilli Michael B Wheeler M Dodson Michael Kyle W Sloop |
| author_sort | Lucy S Jun |
| collection | DOAJ |
| description | Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r-/- animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner proteins, and testing of novel therapeutic agents targeting the hGLP-1R. |
| format | Article |
| id | doaj-art-578e71df589f4d7ab60826be5886a3bc |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-578e71df589f4d7ab60826be5886a3bc2025-08-20T03:00:32ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0194e9374610.1371/journal.pone.0093746A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor.Lucy S JunAaron D ShowalterNosher AliFeihan DaiWenzhen MaTamer CoskunJames V FicorilliMichael B WheelerM Dodson MichaelKyle W SloopClass B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r-/- animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner proteins, and testing of novel therapeutic agents targeting the hGLP-1R.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0093746&type=printable |
| spellingShingle | Lucy S Jun Aaron D Showalter Nosher Ali Feihan Dai Wenzhen Ma Tamer Coskun James V Ficorilli Michael B Wheeler M Dodson Michael Kyle W Sloop A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. PLoS ONE |
| title | A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. |
| title_full | A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. |
| title_fullStr | A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. |
| title_full_unstemmed | A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. |
| title_short | A novel humanized GLP-1 receptor model enables both affinity purification and Cre-LoxP deletion of the receptor. |
| title_sort | novel humanized glp 1 receptor model enables both affinity purification and cre loxp deletion of the receptor |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0093746&type=printable |
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