Table 1.
Functional characterization of different ligands at the Y1 receptor
| at Y1R | Analytical data | Binding | GαqΔ6i4myr | Gi/o | arr-3 | ||
|---|---|---|---|---|---|---|---|
| Peptide | Mcalc/Da | Mobs [M+H]+ /Da | Purity/%a,b | Ki (nM) (pKi ± SEM) | EC50 (nM) (pEC50 ± SEM) | EC50 (nM) (pEC50 ± SEM) | EC50 (nM) (pEC50 ± SEM) |
| NPY | 4251.1 | 4252.1 | > 95a2,b2 | 0.6 (9.20 ± 0.20) | 0.6 (9.24 ± 0.05) | 0.1 (10.00 ± 0.08) | 2.9 (8.54 ± 0.07) |
| G34-NPY | 4180.1 | 4181.1 | > 95a1, b1 | 9.3 (8.03 ± 0.08) | 3.7 (8.43 ± 0.07) | 0.3 (9.56 ± 0.11) | 186 (6.73 ± 0.17) |
| F7,P34-NPY | 4253.1 | 4254.1 | > 95b1,b3 | 0.2 (9.62 ± 0.09) | 0.5 (9.33 ± 0.13) | 0.1 (9.88 ± 0.09) | 4.7 (8.33 ± 0.15) |
| K18-PEG20K-F7,P34-NPY | 25,326 | 25,347 | > 95b2,b3 | 3.3 (8.48 ± 0.07) | 2.0 (8.69 ± 0.07) | 0.2 (9.69 ± 0.14) | 107 (6.97 ± 0.22) |
Peptide identities were confirmed by MALDI-ToF mass spectrometry and purity of > 95% by RP-HPLC. Binding affinities were measured by competition of 75 pM 125I-PYY at membrane preparations of stably transfected HEK293-Y1R-eYFP cells, and the Ki was fit incorporating the Cheng-Prusoff-correction with a Kd of 203 ± 32 pM determined under the same experimental conditions. Functional data were obtained in transiently transfected HEK293 cells: G protein activation was determined downstream of a chimeric GαqΔ6i4myr by accumulation of inositol phosphates, and downstream of the native Gi/o proteins using a CRE-reporter gene system, respectively. Arr-3 recruitment was measured by BRET to an RLuc8-Arr3 fusion protein. Functional data (Ki/EC50) represent global fit of n ≥ 3 independent experiments conducted in technical triplicate, calculated molecular weight refers to the monoisotopic mass
aGradients: (a) 10–60% B in 30 min, (b) 20–70% B in 40 min
bRP-HPLC columns: (1) Phenomenex Proteo C18, 90 Å; (2) GraceVydac C18, 300 Å; (3) Phenomenex Aeris XB-C18