TABLE 1

Effects of human GLP-1R 149 or 333 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. pIC50 values were then corrected for radioligand occupancy using the radioligand dissociation constant for each mutant, allowing determination of ligand affinity (Ki). 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). For specific [125I]exendin(9–39) binding, data are expressed as a maximum of specific [125I]exendin(9–39) binding at the wild-type human GLP-1R. Cell surface expression was determined through antibody detection of the N-terminal c-myc epitope label, with data expressed as a maximum of wild-type human GLP-1R expression. All values are expressed as mean ± S.E.M. of three to four independent experiments, conducted in duplicate. Data were analyzed with one-way analysis of variance and Dunnett’s post-test.

Binding (pKi)Cell-Surface ExpressionSpecific [125I]Exendin(9–39) Binding
GLP-1(7–36)NH2Exendin-4OxyntomodulinExendin(9–39)a
%Wild-type
Wild-type (T149, S333)9.1 ± 0.19.7 ± 0.18.1 ± 0.18.0 ± 0.0100 ± 6100 ± 14
M149b6.5 ± 0.2*7.9 ± 0.2*5.5 ± 0.2*NA47 ± 9*NA
A1496.8 ± 0.1*7.8 ± 0.1*6.2 ± 0.1*8.0 ± 0.0110 ± 7113 ± 30
C1497.6 ± 0.1*8.4 ± 0.1*6.9 ± 0.1*8.0 ± 0.0156 ± 6*218 ± 29**
F1496.4 ± 0.1*7.5 ± 0.1*5.6 ± 0.1*8.0 ± 0.094 ± 690 ± 23
I1496.8 ± 0.1*7.8 ± 0.1*6.0 ± 0.2*8.0 ± 0.099 ± 5154 ± 20
S1498.3 ± 0.1*9.4 ± 0.07.6 ± 0.1*8.0 ± 0.088 ± 7125 ± 24
V1496.7 ± 0.1*7.8 ± 0.1*6.2 ± 0.1*8.0 ± 0.072 ± 4*139 ± 26
Y1496.8 ± 0.1*7.5 ± 0.1*6.0 ± 0.1*8.0 ± 0.053 ± 5*87 ± 15
C333b9.0 ± 0.19.6 ± 0.18.0 ± 0.1NA51 ± 6*NA
A3339.1 ± 0.19.6 ± 0.18.0 ± 0.18.0 ± 0.0122 ± 9124 ± 15
V3339.2 ± 0.19.8 ± 0.18.4 ± 0.18.0 ± 0.086 ± 5101 ± 26

  • NA, data not experimentally determined in Koole et al., 2011.

  • ↵a Equivalent at one significant figure.

  • ↵b Data obtained from Koole et al., 2011. Reported values for binding are pIC50.

  • ↵* Statistically significant at P < 0.05, one-way analysis of variance and Dunnett’s post-test in comparison with wild-type control; **P = 0.11.