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: | Online |
| Language: | English |
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Public Library of Science
2014
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973576/ |
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pubmed-3973576 |
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pubmed-39735762014-04-04 A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor Jun, Lucy S. Showalter, Aaron D. Ali, Nosher Dai, Feihan Ma, Wenzhen Coskun, Tamer Ficorilli, James V. Wheeler, Michael B. Michael, M. Dodson Sloop, Kyle W. Research Article 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. Public Library of Science 2014-04-02 /pmc/articles/PMC3973576/ /pubmed/24695667 http://dx.doi.org/10.1371/journal.pone.0093746 Text en © 2014 Jun et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Jun, Lucy S. Showalter, Aaron D. Ali, Nosher Dai, Feihan Ma, Wenzhen Coskun, Tamer Ficorilli, James V. Wheeler, Michael B. Michael, M. Dodson Sloop, Kyle W. |
| spellingShingle |
Jun, Lucy S. Showalter, Aaron D. Ali, Nosher Dai, Feihan Ma, Wenzhen Coskun, Tamer Ficorilli, James V. Wheeler, Michael B. Michael, M. Dodson Sloop, Kyle W. A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor |
| author_facet |
Jun, Lucy S. Showalter, Aaron D. Ali, Nosher Dai, Feihan Ma, Wenzhen Coskun, Tamer Ficorilli, James V. Wheeler, Michael B. Michael, M. Dodson Sloop, Kyle W. |
| author_sort |
Jun, Lucy S. |
| title |
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_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_sort |
novel humanized glp-1 receptor model enables both affinity purification and cre-loxp deletion of the receptor |
| 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. |
| publisher |
Public Library of Science |
| publishDate |
2014 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973576/ |
| _version_ |
1612073427057770496 |