The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Abstract

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.

Fig. 1.

Static in vitro insulin secretion. RIN 1046–38 cells were incubated with glucose (5 mM) in the absence or presence peptides (10 nM) for 1 h at 37 °C. The amount of insulin secreted into the medium was then determined. Data are the mean ± S.E.M. of two or three experiments performed in triplicate.

Fig. 2.

Displacement of [¹²⁵I]GLP-1 binding to CHO/GLP-1R cells with the analogs of GLP-1, GLP-1 Gly⁸, and truncated analogs of Ex-4. [¹²⁵I]GLP-1 binding to intact CHO/GLP-1R cells was competed with various concentrations of the peptides shown. The data are normalized to maximum values obtained in the presence of 10 nM of the respective peptides. The data points represent the mean of three to five experiments performed in triplicate. The IC₅₀ value for each GLP-1 analog is shown in Table 2. B₀, maximum binding in the absence of cold peptide.

Fig. 3.

cAMP dose response curves for selected peptides. Intracellular cAMP levels were measured in CHO/GLP-1R cells after treatment with the indicated concentrations of the peptides for 30 min at 37 °C. Ex-4 is compared with the truncated analog Ex (1–30) (A). GLP-1 and GLP-1 Gly⁸ [GG] are compared with their respective analogs in which the PSSGAPPPS sequence has been added (B). The data are normalized to maximum values obtained in each experiment in the presence of the 10 nM of the individual peptide. The data points represent the mean of three experiments performed in triplicate.