Extended Data Table 4.
IP accumulation assays of wild-type (WT) and mutant Y1Rs for NPY/NPY analogues
| a. IP accumulation assays of complementary mutagenesis between NPY/NPY analogues and WT and mutant Y1Rs
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|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Peptides* | WT | Q 1203.32H | I293ECL3N | N299732A | N2836.55A | D2876.59A‖ | ||||||||||||
| EC50 (nM)† (pEC50 ± SEM) | X-fold over WT‡ | n§ | EC50 (nM) (PEC50 ± SEM) | X-fold over WT | n | EC50 (nM) (PEC50 ± SEM) | X-fold over WT | n | EC50 (nM) (PEC50 ± SEM) | X-fold over WT | n | EC50 (nM) (PEC50 ± SEM) | X-fold over WT | n | EC50 (nM) (PEC50 ± SEM) | X-fold over WT | n | |
| NPY | 1.5 (8.83 ± 0.02) | 1 | 51 | 39 (7.41 ±0.11) | 26 | 3 | 169 (6.77 ± 0.08) | 113 | 8 | 91 (7.04 ±0.09) | 61 | 5 | 1,053 (5.98 ±0.13) | 702 | 6 | 1,384 (5.86 ±0.09) | 461 | 3 |
| [N30]NPY | 289 (6.54 ± 0.06) | 1 | 9 | 1 | / | / | 3116; 1274 | 11; 4 | 2 | / | / | / | / | / | / | / | / | / |
| [A33]NPY | 5,395 (5.27 ± 0.07) | 1 | 3 | / | / | / | / | / | / | 5,306 (5.28 ± 0.07) | 1 | 3 | / | / | / | / | / | / |
| [A35]NPY | >10,000 | 1 | 3 | / | / | / | / | / | / | / | / | / | / | / | / | >4,900 | >5 | 3 |
| [A36]NPY | 1,378 (5.86 ± 0.06) | 1 | 6 | / | / | / | / | / | i | / | / | / | nd | nd | 3 | / | / | / |
| NPY-tyramide | 68 (7.17 ±0.11) | 1 | 9 | nd | nd | 3 | / | i | i | / | / | / | / | i | / | / | / | / |
| b. IP accumulation assays of WT Y1R for NPY/NPY analogues
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|---|---|---|---|---|---|---|---|
| NPY | Ac-NPY | [A1]NPY | [A2]NPY | [A1,A2]NPY | NPY(3-36) | NPY(13-36) | |
| EC50 (nM) (pEC50 ± SEM) | 1.5 (8.83 ± 0.02) | 0.5; 3.0 | 6.5; 2.4 | 7.3 (8.14 ±0.11) | 8.0 (8.10 ±0.06) | 83 (7.08 ± 0.09) | 477;744 |
| X-fold over NPY | 1 | 1; 2 | 4; 2 | 5 | 5 | 55 | 318; 496 |
| n | 51 | 2 | 2 | 4 | 3 | 3 | 2 |
| c. NPY-induced IP accumulation assays of WT and mutant Y1Rs
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|---|---|---|---|---|---|---|---|---|---|---|
| WT | F184ECL2A | F184ECL2N | V187ECL2N | L189ECL2N | Y192ECL2S | V197ECL2A | V197ECL2N | F199ECL2N | F202ECL2N | |
| EC50 (nM) (pEC50 ± SEM) | 1.5 (8.83 ± 0.02) | 18.7 (7.73 ± 0.08) | 23 (7.64 ±0.10) | 1.9 (8.72 ± 0.08) | 1.9 (8.73 ± 0.16) | 3.8 (8.42 ± 0.11) | 1.9 (8.71 ± 0.13) | 188 (6.73 ± 0.11) | 3.4 (8.47 ± 0.11) | 1.2 (8.93 ± 0.15) |
| X-fold over WT | 1 | 13 | 15 | 1 | 1 | 2.5 | 1 | 125 | 2 | 1 |
| n | 51 | 7 | 14 | 3 | 3 | 3 | 5 | 7 | 3 | 3 |
Peptides were synthesized following the methods described in peptide synthesis section of Methods.
EC50 were determined using GraphPad Prism 5.0. All curves were normalized to the top and bottom values of the Y1R/NPY curve. Nonlinear regression (curve fit) was performed for normalized response in all assays. All data are shown as means from at least three independent experiments (n>2) or results of two individual experiments (n=2) each performed in technical duplicate.
The EC50 shifts were determined by EC50(mutant) / EC50(WT), set Hill slope to 1. For the wild-type receptor x-fold is set to 1. Lower EC50 shift of NPY analogue/mutant compared to NPY/mutant was interpreted as no further loss of function and a direct interaction between both positions.
Sample size n, the number of independent experiments performed in technical duplicate.
Data are from reference 27.
nd, not determined up to 10−4 M agonist concentration; /, not tested.