The isolated N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor binds exendin peptides with much higher affinity than GLP-1.

Two fragments of the receptor for glucagon-like peptide-1 (GLP-1), each containing the N-terminal domain, were expressed and characterized in either bacterial or mammalian cells. The first fragment, rNT-TM1, included the N-terminal domain and first transmembrane helix and was stably expressed in the membrane of human embryonic kidney 293 cells. The second, 6H-rNT, consisted of only the N-terminal domain of the receptor fused with a polyhistidine tag at its N terminus. The latter fragment was expressed in Escherichia coli in the form of inclusion bodies from which the protein was subsequently purified and refolded in vitro. Although both receptor fragments displayed negligible (125)I-labeled GLP-1(7-36)amide-specific binding, they both displayed high affinity for the radiolabeled peptide antagonist (125)I-exendin-4(9-39). Competition binding studies demonstrated that the N-terminal domain of the GLP-1 receptor maintains high affinity for the agonist exendin-4 as well as the antagonists exendin-4(3-39) and exendin-4(9-39) whereas, in contrast, GLP-1 affinity was greatly reduced. This study shows that although the exendin antagonists are not dependent upon the extracellular loops and transmembrane helices for maintaining their normal high affinity binding, the endogenous agonist GLP-1 requires regions outside of the N-terminal domain. Hence, distinct structural features in exendin-4, between residues 9 and 39, provide additional affinity for the N-terminal domain of the receptor. These data are consistent with a model for the binding of peptide ligands to the GLP-1 receptor in which the central and C-terminal regions of the peptides bind to the N terminus of the receptor, whereas the N-terminal residues of peptide agonists interact with the extracellular loops and transmembrane helices.