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Cellular trafficking and functional characterisation of the human glucagon like peptide-1 receptor. / Aiysha Thompson
Swansea University Author: Aiysha, Thompson
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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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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.
Swansea University Medical School