title: Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.
abstract: 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.
authors: Department of Structure and Biophysical Chemistry, Novo Nordisk, DK-2880 Bagsvaerd, Denmark. sffr@novonordisk.com
authors: Steffen
authors: S
authors: Runge
authors: Susann
authors: S
authors: Schimmer
authors: Jan
authors: J
authors: Oschmann
authors: Christine Bruun
authors: CB
authors: Schiødt
authors: Sanne Möller
authors: SM
authors: Knudsen
authors: Claus Bekker
authors: CB
authors: Jeppesen
authors: Kjeld
authors: K
authors: Madsen
authors: Jesper
authors: J
authors: Lau
authors: Henning
authors: H
authors: Thøgersen
authors: Rainer
authors: R
authors: Rudolph
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006224:Cricetinae; D000077270:Exenatide; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry* / Q000378:metabolism*; D000067757:Glucagon-Like Peptide-1 Receptor; D006358:Hot Temperature; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D018027:Receptors, Glucagon / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014688:Venoms / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D010455:Peptides; D018027:Receptors, Glucagon; D014688:Venoms; D052216:Glucagon-Like Peptide 1; D000077270:Exenatide