Differential Structural Properties of GLP-1 and Exendin-4 Determine Their Relative Affinity for the GLP-1 Receptor N-Terminal Extracellular Domain
| Autor: | Susann Schimmer, Henning Thøgersen, Steffen Runge, Sanne Möller Knudsen, Jesper Lau, Rainer Rudolph, Kjeld Madsen, Jan Oschmann, Claus Bekker Jeppesen, Christine Bruun Schiødt |
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| Rok vydání: | 2007 |
| Předmět: |
Protein Denaturation
Protein Folding Circular dichroism Hot Temperature Molecular Sequence Data Ligands Biochemistry Glucagon-Like Peptide-1 Receptor Protein structure Glucagon-Like Peptide 1 Cricetinae Receptors Glucagon Extracellular Animals Humans Potency Amino Acid Sequence Receptor Peptide sequence Calorimetry Differential Scanning Venoms Chemistry Circular Dichroism Transmembrane protein Protein Structure Tertiary Spectrometry Fluorescence Biophysics Exenatide Protein folding Peptides Sequence Alignment |
| Zdroj: | Biochemistry. 46:5830-5840 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4. |
| Databáze: | OpenAIRE |
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