Nasr, Elsayed Mohammed Nasr (2010) The binding and activation of the glucagon-like peptide-1 receptor by exendin-4. PhD thesis, University of Leeds.
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Background and purpose Exendin-4 (EX4) has the same physiological properties as glucagon-like peptide-1 (7-36)amide (GLP-1). EX4 has 50% identity with GLP-1, with an extra nine amino acids at its C-terminus. The two peptides mediate their functions through coupling to the glucagon like peptide-1 receptor (GLP-1R) with similar affinity and potency. Unlike N-terminally truncated GLP-1, (GLP-1(15-36)amide), the equivalently truncated EX4(9-39) binds GLP-1R without significant loss of affinity; furthermore, GLP-1(15-36) is a partial agonist while EX4(9-39) is an antagonist. Previous binding analysis of either N or C-terminally truncated EX4 at rGLP-1R suggested that the residues responsible for its extra affinity are at its C-terminus, EX4 residues 31-39. Crystal structures supported by mutagenesis showed similar interactions of both GLP-1 and EX4 at the isolated N-terminal domain of human GLP-1R (hGLP-1R-NTD) apart from a subtle hydrogen bond between Ser32 in EX4 and Glu68 in hGLP-1R-NTD. Experimental approach The affinities and activities of GLP-1, EX4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Computer models, molecular dynamics coupled with in silico mutagenesis, were used to model and interpret the data. Key results The membrane-tethered NTDs of hGLP-1R displayed similar affinity for GLP-1 and EX4 in contrast to previous studies using the soluble isolated domain. The selective high affinity at rGLP-1R and the rGLP-1R-like mutant hGLP-1R-Glu68Asp for EX4(9–39) over EX4(9–30) was due to Ser32 in the ligand. This selectivity was not observed with hGLP-1R and the hGLP-1R-like mutant rGLP-1R-Asp68Glu. Gly16-EX4(9–30) was an agonist for rGLP-1R and hGLP-1R-Glu68Asp but was an antagonist for hGLP-1R and rGLP-1R-Asp68Glu. Glu22-GLP-1(15-36) was a partial agonist for all tested receptors. Insertion of (EEEAVRL) of EX4 instead of their equivalent sequence in GLP-1(15-36) prevented its activity and did not enhance its affinity. Substitution of Ser32 in EX4 by similar hydrogen bond donor amino acids did not enhance EX4 affinity or potency. Conclusions and implications GLP-1 and EX4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of EX4 and Asp68 of rGLP-1R is responsible for the improved affinity of EX4 and can play a role in the antagonist/agonist switch of Gly16-EX4(9–30) at the rat receptor. The discovery of the novel antagonist/agonist switch suggests a new mechanism of activation by GLP-1 which does not require its extreme N-terminal residues.
|Item Type:||Thesis (PhD)|
|Department:||The University of Leeds > Faculty of Biological Sciences (Leeds)|
|Deposited By:||Ethos Import|
|Deposited On:||06 Oct 2011 14:54|
|Last Modified:||06 Oct 2011 14:57|
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