Differential Structural Properties of GLP-1 and Exendin-4 Determine Their Relative Affinity for the GLP-1 Receptor N-Terminal Extracellular Domain

• Published in: Biochemistry (Easton) 2007-05, Vol.46 (19), p.5830-5840
• Main Authors: Runge, Steffen, Schimmer, Susann, Oschmann, Jan, Schiødt, Christine Bruun, Knudsen, Sanne Möller, Jeppesen, Claus Bekker, Madsen, Kjeld, Lau, Jesper, Thøgersen, Henning, Rudolph, Rainer
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Calorimetry, Differential Scanning; Circular Dichroism; Cricetinae; Glucagon-Like Peptide 1 - chemistry; Glucagon-Like Peptide 1 - metabolism; Glucagon-Like Peptide-1 Receptor; Hot Temperature; Humans; Ligands; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Receptors, Glucagon - metabolism; Sequence Alignment; Spectrometry, Fluorescence; Venoms - chemistry; Venoms - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi062309m

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.