TABLE 2

Effects of human GLP-1 receptor mutation on peptide ligand binding and cell-surface expression

Binding data were analyzed using a three-parameter logistic equation as defined in eq. (1) to obtain pIC50 values. Data were normalized to maximum 125I-exendin(9-39) binding in the absence of ligand, with nonspecific binding measured in the presence of 1-μM exendin(9-39). Cell-surface expression (Bmax) was determined through homologous competition binding with [125I]exendin(9-39), and data are expressed as a maximum of specific [125I]exendin(9-39) binding at the wild-type human GLP-1R. All values are expressed as mean ± S.E.M. of three to seven independent experiments, conducted in duplicate. Data were analyzed with one-way analysis of variance and Dunnett’s post-test.

Binding (pIC50)Cell-Surface Expression ELISA (% Wild-Type)Cell-Surface Expression (Bmax) (% Wild-Type)
GLP-1 (7-36)NH2Exendin-4OxyntomodulinExendin(9-39)
Wild-Typea8.7 ± 0.19.0 ± 0.07.3 ± 0.18.1 ± 0.1100 ± 1100 ± 3
R2.60190Aa7.4 ± 0.1*7.3 ± 0.1*7.6 ± 0.27.6 ± 0.1*53 ± 3*44 ± 2*
N3.43240Aa8.2 ± 0.1*8.7 ± 0.17.4 ± 0.18.3 ± 0.186 ± 392 ± 2
Q7.49394Aa8.6 ± 0.18.8 ± 0.17.3 ± 0.28.2 ± 0.1103 ± 3111 ± 2
N3.43240A/Q7.49394A8.0 ± 0.1*8.1 ± 0.1*7.4 ± 0.18.0 ± 0.165 ± 4*71 ± 4*
N3.43240Q8.3 ± 0.18.8 ± 0.17.6 ± 0.18.3 ± 0.184 ± 680 ± 7*
Q7.49394N8.7 ± 0.18.8 ± 0.17.4 ± 0.18.2 ± 0.198 ± 793 ± 5
N3.43240Q/Q7.49394N8.4 ± 0.18.6 ± 0.1*7.3 ± 0.07.7 ± 0.1*88 ± 1080 ± 7*
E6.53364A7.3 ± 0.1*7.5 ± 0.1*7.1 ± 0.27.5 ± 0.1*41 ± 4*51 ± 6*
H6.52363Aa7.3 ± 0.1*7.5 ± 0.1*6.5 ± 0.1*7.4 ± 0.1*59 ± 4*53 ± 2*
  • * Statistically significant at p < 0.05, one-way analysis of variance and Dunnett’s post-test in comparison with wild-type control. For easy comparison between effects of different ligands and mutants, these are also highlighted in bold.

  • a Data obtained from Wootten et al. (2013a).