Table 1.
Effects of human GLP-1R TM1/N-terminal mutants on peptide ligand binding and cell-surface expression
Binding data were analyzed using a three-parameter logistic equation and normalized to the maximal binding of the radiolabeled antagonist 125I-exendin-4(9–39) and the nonspecific binding in the presence of 1 μm exendin-4(9–39). pKi is the negative logarithm of peptide affinity. All the values for binding are mean ± S.E. of three independent experiments, conducted in triplicate. Cell-surface expression was accessed through ELISA detecting the N-terminal c-Myc epitope label on the receptor. Mutant data are compared with the wildtype human GLP-1R expression and shown as percentage. The data for cell-surface expression are mean ± S.E. of four to six independent experiments, conducted in duplicate. One-way ANOVA and Dunnett's post-test were used to determine statistical differences (*, p < 0.05). ND, not determined.
| Whole-cell competition radioligand binding (pKi) |
Cell-surface expression (% wildtype) | ||||
|---|---|---|---|---|---|
| GLP-1(7–36)-NH2 | Exendin-4 | Oxyntomodulin | Exendin(9–39) | ||
| Wildtype | 8.12 ± 0.06 | 9.31 ± 0.06 | 7.52 ± 0.08 | 7.85 ± 0.05 | 100 ± 7 |
| R24A | 8.10 ± 0.08 | 9.54 ± 0.13 | 7.69 ± 0.08 | 7.74 ± 0.06 | 130 ± 8 |
| P25A | 8.17 ± 0.09 | 9.56 ± 0.12 | 7.80 ± 0.08 | 7.71 ± 0.06 | 107 ± 15 |
| Q26A | 7.96 ± 0.09 | 9.23 ± 0.11 | 7.56 ± 0.10 | 7.72 ± 0.07 | 115 ± 13 |
| G27A | ND | ND | ND | ND | 28 ± 8* |
| A28G | 7.67 ± 0.08* | 8.89 ± 0.11* | 7.07 ± 0.08* | 7.61 ± 0.06 | 106 ± 15 |
| T29A | ND | ND | ND | ND | 16 ± 5* |
| V30A | 8.16 ± 0.09 | 9.31 ± 0.11 | 7.72 ± 0.08 | 7.77 ± 0.08 | 108 ± 8 |
| E128A | ND | ND | ND | ND | 6 ± 2* |
| S129A | 8.10 ± 0.11 | 9.94 ± 0.10 | 7.71 ± 0.15 | 7.79 ± 0.10 | 83 ± 6 |
| K130A | 8.12 ± 0.08 | 9.25 ± 0.07 | 7.52 ± 0.12 | 7.64 ± 0.09 | 51 ± 8* |
| R131A | 8.28 ± 0.12 | 9.37 ± 0.09 | 7.72 ± 0.14 | 7.78 ± 0.09 | 114 ± 15 |
| G132A | ND | ND | ND | ND | 80 ± 6 |
| E133A | 8.16 ± 0.07 | 9.45 ± 0.10 | 7.70 ± 0.09 | 7.84 ± 0.10 | 97 ± 12 |
| R134A | 8.12 ± 0.09 | 9.14 ± 0.08 | 7.60 ± 0.12 | 7.65 ± 0.11 | 118 ± 4 |
| S135A | 8.35 ± 0.09 | 9.40 ± 0.10 | 7.79 ± 0.09 | 7.82 ± 0.09 | 60 ± 14* |
| S136A | 7.84 ± 0.11 | 9.29 ± 0.07 | 7.39 ± 0.13 | 7.59 ± 0.15 | 123 ± 15 |
| P137A | 7.18 ± 0.13* | 8.54 ± 0.09* | 6.94 ± 0.12* | 6.98 ± 0.18* | 102 ± 14 |
| E138A | 8.22 ± 0.07 | 9.19 ± 0.10 | 7.21 ± 0.07 | 7.87 ± 0.07 | 164 ± 13* |
| E139A | ND | ND | ND | ND | 37 ± 8* |
| Q140A | 7.84 ± 0.15 | 8.65 ± 0.13* | 7.68 ± 0.21 | 8.15 ± 0.16 | 37 ± 6* |
| L141A | 7.50 ± 0.06 | 8.53 ± 0.06* | 6.17 ± 0.28* | 7.17 ± 0.06* | 136 ± 8 |
| L142A | 7.89 ± 0.07 | 9.23 ± 0.07 | 6.89 ± 0.10* | 7.84 ± 0.07 | 94 ± 11 |
| F143A | 8.19 ± 0.06 | 9.33 ± 0.09 | 7.35 ± 0.09 | 7.65 ± 0.06 | 102 ± 11 |
| L144A | 7.94 ± 0.15 | 9.41 ± 0.17 | 7.15 ± 0.19 | 8.03 ± 0.15 | 25 ± 6* |
| Y145A | 8.25 ± 0.09 | 9.51 ± 0.09 | 7.38 ± 0.10 | 7.92 ± 0.07 | 88 ± 8 |
| I146A | 8.07 ± 0.09 | 9.32 ± 0.12 | 7.57 ± 0.14 | 7.91 ± 0.09 | 50 ± 8* |
| I147A | 7.78 ± 0.05 | 9.00 ± 0.08 | 7.26 ± 0.08 | 7.67 ± 0.08 | 108 ± 18 |
| Y148A | 6.79 ± 0.15* | 8.06 ± 0.06* | 6.26 ± 0.25* | 8.09 ± 0.08 | 67 ± 8 |