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Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor. / Aiysha Thompson

Swansea University Author: Aiysha Thompson

Abstract

The binding of glucagon like peptide-1 (GLP-1] to its receptor, the GLP-1 receptor (GLP-IR), results in insulin secretion from pancreatic beta-cells. This makes the receptor an important drug target for type 2 diabetes. The GLP-IR is a family B G-protein coupled receptor (GPCR) and functions at the...

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Published: 2014
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa42586
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spelling 2018-08-29T15:40:00.1845984 v2 42586 2018-08-02 Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor. 3a285642c8f04630c27fd609fc8320e0 NULL Aiysha Thompson Aiysha Thompson true true 2018-08-02 The binding of glucagon like peptide-1 (GLP-1] to its receptor, the GLP-1 receptor (GLP-IR), results in insulin secretion from pancreatic beta-cells. This makes the receptor an important drug target for type 2 diabetes. The GLP-IR is a family B G-protein coupled receptor (GPCR) and functions at the cell surface by coupling to Galphas and Galphaq pathways and causing ERK phosphorylation. The objective of this study was to analyse trafficking, activity and internalisation of GLP-IR at the cellular and molecular level. The human GLP-IR (hGLP-1R) N-terminus is required for trafficking and maturation. This study demonstrated the importance of signal peptide (SP] cleavage, N-linked glycosylation and the hydrophobic region after the SP [HRASP] within the N-terminus of the hGLP-1R for cell surface expression. Due to difficulties in peptide drugs, orally active small molecule agonists of the GLP-IR are of high importance. Small molecule allosteric agonists, compounds 2 and B, were found to cause cAMP production similar to orthosteric GLP-1, but not intracellular Ca2+ accumulation, ERK phosphorylation or internalisation of the receptor. Compounds 2 and B binding to the GLP-IR inhibits GLP-1 internalisation, intracellular Ca2+ accumulation and ERK phosphorylation of the receptor. Agonist induced hGLP-1R internalisation is important for insulin secretion. Inhibition of the G?q pathway but not the Gas pathway reduced hGLP-1R internalisation. Consistent with this, the hGLP-1R T149M mutant and compounds 2 and B, which activate only the Gas pathway, failed to induce hGLP-1R internalisation. Chemical inhibitors of the Galphaq pathway significantly reduced agonist induced hGLP-1R internalisation and suppressed ERK phosphorylation demonstrating phosphorylated ERK acts downstream of the Galphaq pathway in hGLP-1R internalisation. Finally, distinct regions within the C-terminus of hGLP-1R required for its cell surface expression, activity and internalisation were identified. Residues 411- 418, 419-430 and 431-450 are essential for hGLP-1R cell surface expression, activity and internalisation, respectively. E-Thesis Molecular biology. 31 12 2014 2014-12-31 COLLEGE NANME Swansea University Medical School COLLEGE CODE Swansea University Doctoral Ph.D 2018-08-29T15:40:00.1845984 2018-08-02T16:24:29.7589956 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Aiysha Thompson NULL 1 0042586-02082018162506.pdf 10805344.pdf 2018-08-02T16:25:06.1400000 Output 22033397 application/pdf E-Thesis true 2018-08-02T16:25:06.1400000 false
title Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
spellingShingle Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
Aiysha Thompson
title_short Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
title_full Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
title_fullStr Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
title_full_unstemmed Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
title_sort Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor.
author_id_str_mv 3a285642c8f04630c27fd609fc8320e0
author_id_fullname_str_mv 3a285642c8f04630c27fd609fc8320e0_***_Aiysha Thompson
author Aiysha Thompson
author2 Aiysha Thompson
format E-Thesis
publishDate 2014
institution Swansea University
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
active_str 0
description The binding of glucagon like peptide-1 (GLP-1] to its receptor, the GLP-1 receptor (GLP-IR), results in insulin secretion from pancreatic beta-cells. This makes the receptor an important drug target for type 2 diabetes. The GLP-IR is a family B G-protein coupled receptor (GPCR) and functions at the cell surface by coupling to Galphas and Galphaq pathways and causing ERK phosphorylation. The objective of this study was to analyse trafficking, activity and internalisation of GLP-IR at the cellular and molecular level. The human GLP-IR (hGLP-1R) N-terminus is required for trafficking and maturation. This study demonstrated the importance of signal peptide (SP] cleavage, N-linked glycosylation and the hydrophobic region after the SP [HRASP] within the N-terminus of the hGLP-1R for cell surface expression. Due to difficulties in peptide drugs, orally active small molecule agonists of the GLP-IR are of high importance. Small molecule allosteric agonists, compounds 2 and B, were found to cause cAMP production similar to orthosteric GLP-1, but not intracellular Ca2+ accumulation, ERK phosphorylation or internalisation of the receptor. Compounds 2 and B binding to the GLP-IR inhibits GLP-1 internalisation, intracellular Ca2+ accumulation and ERK phosphorylation of the receptor. Agonist induced hGLP-1R internalisation is important for insulin secretion. Inhibition of the G?q pathway but not the Gas pathway reduced hGLP-1R internalisation. Consistent with this, the hGLP-1R T149M mutant and compounds 2 and B, which activate only the Gas pathway, failed to induce hGLP-1R internalisation. Chemical inhibitors of the Galphaq pathway significantly reduced agonist induced hGLP-1R internalisation and suppressed ERK phosphorylation demonstrating phosphorylated ERK acts downstream of the Galphaq pathway in hGLP-1R internalisation. Finally, distinct regions within the C-terminus of hGLP-1R required for its cell surface expression, activity and internalisation were identified. Residues 411- 418, 419-430 and 431-450 are essential for hGLP-1R cell surface expression, activity and internalisation, respectively.
published_date 2014-12-31T03:53:15Z
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score 11.017797

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