A model for receptor-peptide binding at the glucagon-like peptide-1 (GLP-1) receptor through the analysis of truncated ligands and receptors

Abstract

1. The receptor for glucagon-like peptide-1 (GLP-1) can be activated by both its physiological hormone and a peptide discovered in the venom of the Gila Monster, exendin-4, which shows promise as an antidiabetic agent. 2. Exendin-4 displays receptor-binding properties not observed for GLP-1. Firstly, exendin-4 can be truncated by up to eight residues at its N-terminus without a significant loss of affinity. Secondly, exendin-4 maintains high affinity for the isolated N-terminal domain of the receptor, suggesting that exendin-4 makes additional contacts with this domain of the receptor, which nullify the requirement for ligand-receptor interactions involving the extracellular loops and/or transmembrane helices of the receptor's core domain. 3. In order to further understand the nature of the receptor-peptide interaction, a variety of full length and truncated peptide analogues were used to quantify the contribution of each distinct region of exendin-4 and GLP-1 to receptor affinity. 4. Our data show that, for both exendin-4 and GLP-1, the primary interaction is between the putative helical region of the peptide and the extracellular N-terminal domain of the receptor. 5. However, we demonstrate that the contribution to receptor affinity provided by the N-terminal segment of GLP-1 is greater than that of exendin-4, while the C-terminal nine residue extension of exendin-4, absent in GLP-1, forms a compensatory interaction with the N-terminal domain of the receptor. 6. We describe a peptide-receptor binding model to account for these data.

Figure 1

Peptides and receptor constructs used in this work. (a) An alignment of the full-length and truncated peptides used in this study. In order to facilitate direct comparison with EX-4, the numbering of GLP-1 has been modified from the conventional system, so that its first residue is His-1. The three distinct regions of the peptides are highlighted by the bar below the sequences, such that the central helix is shown as white, the N-terminal region as black and the C-terminal region of EX-4 as hatched. This pictorial shorthand system will be used in Figures 2, 3, 4 and 5. All the peptides were C-terminally amidated. (b) Pictorial representation of the full-length (rGLP-1) and truncated (rNT-TM1) receptors used in this study. The extracellular N-terminal domain is shown by a shaded oval, while the TM helices are depicted as cylinders. (c) An alignment of three modified peptides based upon GLP-1 and EX-4. The modifications to the sequence are underlined. All the three peptides were C-terminally amidated.

Figure 2

Binding of the helical regions of EX-4 and GLP-1 to rGLP-1R and rNT-TM1. ¹²⁵I-exendin-4(9–39) competition-binding assays for (a) rGLP-1R and (b) rNT-TM1 with EX-4(9–30) and GLP-1(9–30). The figures are representative of one of at least three independent experiments to assess the affinity of the central helical region of EX-4 and GLP-1. pIC₅₀ values from Table 1 are given next to the symbol of each peptide.

Figure 3

Binding of N-terminal region of GLP-1 to rGLP-1R, and rNT-TM1. ¹²⁵I-exendin-4(9–39) competition-binding assays for (a) rGLP-1R and (b) rNT-TM1 with GLP-1(1–30). The figures are representative of one of at least three independent experiments to assess the affinity of the N-terminal region of GLP-1 by comparing GLP-1(1–30) with GLP-1(9–30). The dotted lines represent the GLP-1(9–30) curves from Figure 2. pIC₅₀ values from Table 1 are given next to the symbol of each peptide. The figure highlights the large improvement in affinity at rGLP-1R, resulting from the addition of the N-terminal region of GLP-1 in (a) (significantly different, P<0.0001). The difference in pIC₅₀ at rNT-TM1 (b) is not significant (P<0.05).

Figure 4

Binding of the N-terminal region of EX-4 to rGLP-1R and rNT-TM1. ¹²⁵I-exendin-4(9–39) competition-binding assays for (a) rGLP-1R and (b) rNT-TM1 with EX-4(1–30). The figures are representative of one of at least three independent experiments to assess the affinity of the N-terminal region of EX-4 by comparing EX-4(1–30) with EX-4(9–30). The dotted lines represent the EX-4(9–30) curves from Figure 2. pIC₅₀ values from Table 1 are given next to the symbol of each peptide. Panel (a) highlights the small improvement in affinity resulting from the addition of the N-terminal region of EX-4 (significantly different, P<0.01). This small enhancement of affinity is of the same magnitude and significance to that observed when comparing EX-4(1–39) with EX-4(9–39) (see Table 1).

Figure 5

Binding of the C-terminal region of EX-4 to rGLP-1R and rNT-TM1. ¹²⁵I-exendin-4(9–39) competition-binding assays for (a) rGLP-1R and (b) rNT-TM1 with EX-4(9–39). The figures are representative of one of at least three independent experiments to assess the affinity of the C-terminal region of EX-4 by comparing EX-4(9–39) with EX-4(9–30). The dotted lines represent the EX-4(9–30) curves from Figure 2. pIC₅₀ values from Table 1 are given next to the symbol of each peptide. The figure highlights the large improvement in affinity at both rGLP-1R and rNT-TM1 (significantly different, P<0.0001 and P<0.0005, respectively) resulting from the addition of the C-terminal region of EX-4. The enhancement of affinity is of a similar magnitude to that observed when comparing EX-4(1–39) with EX-4(1–30) (significantly different for both receptors, P<0.0001; see Table 1).

Figure 6

A model for peptide–receptor binding. (a) Schematic model for the H interaction between a putative groove on the N-terminal domain of the receptor and the conserved face of the helical region of the peptides. (b) Two schematic diagrams depicting the binding of GLP-1 (left) and EX-4 (right) to GLP-1R. The receptor is shown as consisting of two domains, the ‘N-terminal domain' and the ‘core domain'. The peptides are displayed between the circled symbols N and C, with their putative helical regions shown as cylinders. GLP-1 binding (left) involves an interaction H between its helical region and the receptor's N-terminal domain, which accounts for approximately 82% of the total binding energy of GLP-1 (see Discussion). The remaining binding energy comes from the interaction N^g between the N-terminal sequence of GLP-1 and the core domain. The interaction between EX-4 and GLP-1R (right) is also predominantly via the peptide's helical region and the receptor's N-terminal domain, with this H interaction accounting for approximately 79% of the total binding energy of EX-4. However, the N^ex only contributes 5% of the binding energy of the full-length receptor. In addition, EX-4 forms an additional Ex interaction via its C-terminal region and the N-terminal domain of the receptor. This Ex interaction accounts for approximately 16% of the total binding energy of EX-4. The magnitude of the N^g interaction is equivalent to that of the Ex interaction.