Extended Data Table 3.
Binding of Y1R antagonists and agonists to membrane preparations from Sf9 cells expressing wild-type and mutant Y1Rs
| a. Binding of antagonists to wild-type and mutant Y1Rs
| ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Y1R mutants | Kd (nM)* |
Ki (nM)†
|
||||||||||
| [3H]-UR-MK299 | n‡ | BMS-193885 | n | BIBP3226 | n | BIBO3304 | n | UR-MK136 | n | UR-MK289 | n | |
| Wild type | 0.17 ± 0.03 | 3 | 22 ± 6 | 3 | 2.4; 3.1 | 2 | 1.6 ± 0.3 | 3 | 2.8; 4.0 | 2 | 25; 28 | 2 |
| Crystallization construct | 0.33 ± 0.06 | 3 | 38 ± 2 | 4 | / | / | / | / | ||||
| C1213.33A | 1.4; 2.4 | 2 | / | / | / | / | / | |||||
| I1243.36A | 7.0; 8.0 | 2 | 9,500 ± 1,700 | 3 | 15 ± 5 | 3 | 11 ± 2 | 3 | 12 ± 2 | 3 | 80 ± 16 | 3 |
| I1243.36F | 1.3; 1.9 | 2 | / | / | / | / | / | |||||
| F1734.60A | 9.1 ± 2.2 | 4 | 590 ± 220 | 3 | 68; 84 | 2 | 120 ± 17 | 4 | 88; 110 | 2 | 660 ± 110§ | 5 |
| F1734.60W | 0.31; 0.32 | 2 | 110; 130 | 2 | 15; 26 | 2 | 13 ± 4 | 3 | 3.6; 4.3 | 2 | 6.8; 9.2 | 2 |
| T2125.39A | 0.12; 0.18 | 2 | 150; 150 | 2 | 13; 12 | 2 | 2.5 ± 0.3 | 3 | 5.7; 7.5 | 2 | 18; 21 | 2 |
| L2155.42G | 4.2; 5.6 | 2 | 9.6 ± 1.9 | 4 | 43; 23 | 2 | 47 ± 4 | 3 | 11; 8.4 | 2 | 29; 30 | 2 |
| Q2195.46A | 4.1; 5.1 | 2 | 0.50 ± 0.07 | 4 | 35; 53 | 2 | 6.2 ± 0.7 | 3 | 16 ± 4 | 3 | 13 ± 4 | 3 |
| Q2195.46V | 5.1 ± 1.2 | 3 | / | / | / | / | / | |||||
| W2766.48A | >500 | 3 | / | / | / | / | / | |||||
| L2796.51A | 1.0; 1.1 | 2 | 160; 220 | 2 | 110; 110 | 2 | 13 ± 2 | 3 | 120; 150 | 2 | 320 ± 40 | 3 |
| T2806.52A | 0.16 ± 0.04 | 3 | 7,300 ± 1,300 | 3 | 32 ± 7 | 3 | 2.6 ± 0.4 | 3 | 4.2 ± 1.1 | 3 | 8.2 ± 4.2 | 3 |
| N2836.55A | >500; >500 | 2 | / | / | / | / | / | |||||
| D2876.59A | >500; >500 | 2 | / | / | / | / | / | |||||
| F3027.35A | >500; >500 | 2 | / | / | / | / | / | |||||
| b. Binding of NPYs to wild-type Y1R
| ||||||
|---|---|---|---|---|---|---|
| Kd (nM)* |
Ki (nM)†
|
|||||
| Y1R | [3H]-UR-MK299 | n | Human NPY | n | Porcine NPY | n |
| Wild type | 0.89; 1.1 | 2 | 4.1; 4.2 | 2 | 2.8 ± 0.4 | 4 |
Dissociation constant determined by saturation binding at Sf9 membranes (receptor expression was confirmed by western blot analysis) using a sodium-containing buffer (a) or a sodium-free buffer (b) (Note: the sodium-free buffer was used for the determination of agonist binding affinity because porcine NPY exhibited approx. 10-fold higher affinity in the sodium-free buffer compared to the sodium-containing buffer (data not shown)).
Dissociation constant determined by competition binding with [3H]-UR-MK299 at Sf9 membranes using a sodium-containing buffer (a) or a sodium-free buffer (b).
Sample size, the number of independent experiments performed in technical triplicate. If n>2, data are shown as means ± s.e.m. If n=2, results of two individual experiments are shown.
The lower curve plateau of the four-parameter logistic fit, amounting to 17±3% of specifically bound [3H]-UR-MK299 (mean ± s.e.m. from five independent experiments), was different from zero (P < 0.005, one-sample one-tailed t-test), which is indicative of a non-competitive mechanism.
/: not tested.