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

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• 作者：Steffen Runge ; Susann Schimmer ; Jan Oschmann ; Christine Bruun Schi//pubs.acs.org/images/gifchars/oslash.gif" border="0">dt ; Sanne M&ouml ; ller Knudsen ; Claus Bekker Jeppesen ; Kjeld Madse
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：May 15, 2007
• 年：2007
• 卷：46
• 期：19
• 页码：5830 - 5840
• 全文大小：494K
• 年卷期：v.46,no.19(May 15, 2007)
• ISSN：1520-4995

文摘

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with establishedpotential 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-terminalextracellular 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 waspreviously 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 structuralproperties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracercompetition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinityof full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution andthe conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroismand fluorescence spectroscopy. We found a correlation between the helical content of the free ligandsand their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical atleast in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superiorhelicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explainedalmost 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 interactionbetween Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.