Table 3.
Effects of human GLP-1R TM1/N-terminal mutants on agonist-mediated [Ca2+]i mobilization
[Ca2+]i mobilization data were analyzed using a three-parameter logistic equation to determine pEC50 and Emax values. pEC50 is the negative logarithm of the molar concentration of agonist that induced half the maximal response. Emax for mutants is expressed as a percentage of wildtype. Logτc is the operational efficacy value (determined via the Black and Leff operational model (24)), corrected for cell-surface expression of GLP-1R. All values for cAMP accumulation 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.
| Agonist-mediated intracellular calcium mobilization |
||||||
|---|---|---|---|---|---|---|
| GLP-1(7–36)-NH2 |
Exendin-4 |
|||||
| pEC50 | E max | Logτc | pEC50 | E max | Logτc | |
| Wildtype | 7.83 ± 0.04 | 100.00 ± 1.11 | −0.22 ± 0.03 | 8.45 ± 0.04 | 100.00 ± 1.35 | −0.22 ± 0.02 |
| R24A | 7.60 ± 0.27 | 179.60 ± 20.27* | 0.19 ± 0.10* | 8.25 ± 0.12 | 130.40 ± 5.76* | −0.12 ± 0.05 |
| P25A | 7.89 ± 0.17 | 151.30 ± 11.07* | 0.09 ± 0.09 | 8.07 ± 0.14 | 120.50 ± 6.80* | −0.11 ± 0.05 |
| Q26A | 8.14 ± 0.29 | 146.40 ± 16.50* | 0.02 ± 0.09 | 8.31 ± 0.12 | 104.50 ± 4.87 | −0.24 ± 0.05 |
| G27A | ND | 16.77 ± 4.36* | ND | ND | ND | ND |
| A28G | 7.86 ± 0.14 | 96.66 ± 5.98 | −0.27 ± 0.10 | 8.45 ± 0.19 | 165.90 ± 11.23* | 0.21 ± 0.09 |
| T29A | ND | ND | ND | ND | ND | ND |
| V30A | 7.93 ± 0.24 | 70.75 ± 7.07* | 0.17 ± 0.07 | 8.31 ± 0.24 | 111.50 ± 9.64 | −0.17 ± 0.06 |
| E128A | ND | ND | ND | ND | ND | ND |
| S129A | 7.83 ± 0.12 | 101.30 ± 5.03 | −0.13 ± 0.07 | 8.47 ± 0.19 | 80.73 ± 5.52 | −0.27 ± 0.06 |
| K130A | 7.83 ± 0.18 | 52.81 ± 4.09* | −0.32 ± 0.13 | 7.89 ± 0.17 | 59.33 ± 4.30* | −0.25 ± 0.11 |
| R131A | 7.76 ± 0.25 | 133.80 ± 14.52* | −0.05 ± 0.11 | 8.50 ± 0.24 | 98.00 ± 8.13 | −0.28 ± 0.07 |
| G132A | 7.79 ± 0.20 | 58.45 ± 4.75* | −0.47 ± 0.13 | 8.14 ± 0.23 | 76.96 ± 7.21* | −0.29 ± 0.07 |
| E133A | 7.84 ± 0.18 | 89.51 ± 6.77 | −0.28 ± 0.10 | 8.30 ± 0.21 | 84.37 ± 6.62 | −0.31 ± 0.06 |
| R134A | 8.15 ± 0.32 | 79.99 ± 9.90 | −0.44 ± 0.09 | 8.36 ± 0.19 | 84.59 ± 5.60 | −0.40 ± 0.06 |
| S135A | 7.99 ± 0.20 | 48.35 ± 3.85* | −0.44 ± 0.15 | 8.71 ± 0.22 | 34.19 ± 2.48* | −0.61 ± 0.11* |
| S136A | 7.81 ± 0.18 | 61.68 ± 4.72* | −0.61 ± 0.12* | 8.56 ± 0.16 | 106.70 ± 5.75 | −0.26 ± 0.06 |
| P137A | 7.35 ± 0.25 | 124.00 ± 14.18 | 0.08 ± 0.12 | 8.13 ± 0.10 | 126.00 ± 5.21 | −0.04 ± 0.08 |
| E138A | 7.56 ± 0.13 | 209.20 ± 11.92* | 0.35 ± 0.10* | 8.27 ± 0.20 | 253.40 ± 18.09* | 1.13 ± 0.27* |
| E139A | 7.94 ± 0.20 | 42.50 ± 3.51* | −0.30 ± 0.11 | 7.94 ± 0.21* | 58.47 ± 4.87* | −0.12 ± 0.08 |
| Q140A | 8.89 ± 0.30* | 20.06 ± 2.38* | −0.83 ± 0.14* | 7.49 ± 0.42* | 34.38 ± 4.54* | −0.41 ± 0.17 |
| L141A | 7.49 ± 0.25 | 101.00 ± 11.56 | −0.34 ± 0.11 | 7.79 ± 0.18 | 80.46 ± 6.34 | −0.49 ± 0.09 |
| L142A | 8.74 ± 0.32* | 25.97 ± 2.71* | −0.95 ± 0.17* | 8.77 ± 0.23 | 33.56 ± 2.55* | −0.82 ± 0.11* |
| F143A | 7.65 ± 0.12 | 127.20 ± 7.06 | −0.05 ± 0.11 | 8.48 ± 0.08 | 140.70 ± 3.98* | 0.05 ± 0.05 |
| L144A | ND | ND | ND | ND | ND | ND |
| Y145A | 6.76 ± 0.20* | 108.50 ± 10.35 | −0.12 ± 0.12 | 8.10 ± 0.25 | 94.96 ± 9.30 | −0.19 ± 0.09 |
| I146A | 7.97 ± 0.19 | 60.52 ± 4.60* | −0.23 ± 0.10 | 8.80 ± 0.28 | 52.91 ± 5.09* | −0.30 ± 0.07 |
| I147A | 7.60 ± 0.22 | 44.28 ± 4.42* | −0.73 ± 0.15* | 8.50 ± 0.28 | 66.87 ± 6.33* | −0.51 ± 0.07 |
| Y148A | 7.99 ± 0.25 | 40.42 ± 3.79* | −0.59 ± 0.14 | 8.30 ± 0.21 | 18.91 ± 1.50* | −0.95 ± 0.22* |