Effects of modifications at position 36 of NPY on the binding affinity and activation of different pathways
Binding affinities were determined in displacement binding experiments (compare with Table 1). Activation was measured by ligand-dependent increase of the BRET ratio for the respective effector (Gαi1, GαΔ6qi4myr, arrestin3) at saturating F/L ratio. Stimulation with 1Nal36-NPY, but not 2Nal36-NPY, largely compensated for the signaling deficits of W6.48 mutants, and was able to increase maximum arrestin3 recruitment. Both results from the classic sigmoidal (logistic) fit and the transduction coefficient from the operational model of agonism are given, with the Hill slope set to unity for all analyses. Values are given as the mean (95% CI) corrected for multiple comparisons. Significance levels of logIC50 and log(τ/KA), respectively, were evaluated by two-way analysis of variance and Bonferroni’s post-test for each mutant relative to the WT Y2R treated with the same peptide.
| Displacement Binding (60 pM 125I-PYY) | Ligand-Dependent BRET | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| logIC50 | x-fold over WT/NPY | Gαi1 | GαΔ6qi4myr | Arrestin3 | |||||||||
| logEC50 | x-fold over WT/NPY | log (τ/KA) | logEC50 | x-fold over WT/NPY | log (τ/KA) | logEC50 | x-fold over WT/NPY | netBRET | % WT | log (τ/KA) | |||
| NPY | |||||||||||||
| WT Y2R | −8.59 (8.74–8.44) | (1) | −8.50 (8.91–8.10) | (1) | 8.39 (8.07–8.72) | −7.42 (7.87–6.98) | (1) | 7.33 (6.99–7.67) | −7.12 (7.21–7.02) | (1) | 0.51 (0.48–0.53) | (100) | 7.15 (7.08–7.21) |
| W6.48Y | −8.75 (8.98–8.53)ns | 1 | −7.99 (8.50–7.48) | 5 | 7.95 (7.45–8.45)ns | −6.79 (7.44–6.14) | 4 | 6.81 (6.39–7.23)* | −7.08 (7.23–6.92) | 1 | 0.28 (0.25–0.30) | 55 | 6.80 (6.69–6.91)* |
| W6.48H | −8.37 (8.50–8.25)* | 1 | −7.11 (−7.57 to 6.65) | 30 | 7.17 (6.71–7.63)*** | >−5.70a | >50 | 5.49 (4.63–6.36)*** | −6.01 (6.14–5.89) | 13 | 0.25 (0.23–0.28) | 49 | 5.72 (5.59–5.84)*** |
| W6.48T | −8.62 (8.76–8.48)ns | 1 | −7.35 (7.71–6.98) | 18 | 7.43 (7.07–7.80)*** | −6.14 (6.72–5.55) | 20 | 6.04 (5.56–6.53)*** | −6.44 (6.54–6.33) | 5 | 0.26 (0.24–0.27) | 51 | 6.13 (6.02–6.25)*** |
| 1Nal36-NPY | |||||||||||||
| WT Y2R | −8.41 (8.66–8.16) | 1 | −8.18 (9.11–7.25) | 2 | 8.05 (7.42–8.67) | −6.93 (7.46–6.41) | 3 | 6.88 (6.46–7.30) | −7.08 (7.25–6.92) | 1 | 0.46 (0.42–0.50) | 90 | 7.02 (6.90–7.13) |
| W6.48Y | −8.58 (8.84–8.32)ns | 1 | −7.91 (8.86–6.95) | 5 | 7.84 (7.18–8.49)ns | −6.74 (7.38–6.09) | 5 | 7.11 (6.71–7.52)ns | −7.41 (7.57–7.25) | 0.5 | 0.26 (0.24–0.28) | 51 | 7.10 (6.89–7.31)ns |
| W6.48H | −9.45 (9.61–9.29)*** | 0.1 | −7.79 (8.66–6.93) | 6 | 7.64 (6.88–8.39)ns | −6.47 (7.01–5.92) | 9 | 6.24 (5.71–6.78)** | −7.00 (7.14–6.86) | 1 | 0.36 (0.33–0.39) | 71 | 6.85 (6.70–7.00)ns |
| W6.48T | −9.22 (9.43–9.01)*** | 0.2 | −8.05 (8.81–7.30) | 3 | 8.04 (7.32–8.76)ns | −6.56 (7.18–5.94) | 8 | 6.57 (6.12–7.02)ns | −7.18 (7.28–7.09) | 1 | 0.39 (0.37–0.40) | 76 | 7.06 (6.92–7.20)ns |
| 2Nal36-NPY | |||||||||||||
| WT Y2R | −8.10 (8.39–7.82) | 2 | −8.13 (8.84–7.42) | 4 | 8.23 (7.75–8.71) | −6.74 (7.41–6.07) | 5 | 6.63 (6.18–7.09) | −7.02 (7.16–6.89) | 1 | 0.41 (0.38–0.44) | 80 | 6.90 (6.75–7.05) |
| W6.48Y | −8.28 (8.52–8.04)ns | 2 | −7.98 (8.81–7.16) | 5 | 8.01 (7.53–8.49)ns | −6.53 (7.07–6.00) | 8 | 6.59 (6.19–6.99)ns | −7.00 (7.16–6.83) | 1 | 0.26 (0.24–0.28) | 51 | 6.67 (6.44–6.90)ns |
| W6.48H | −8.14 (8.41–7.87)ns | 3 | −7.09 (7.52–6.65) | 38 | 6.97 (6.48–7.47)*** | >−6.00a | >25 | 5.92 (5.20–6.65)** | −6.30 (6.46–6.15) | 7 | 0.17 (0.15–0.18) | 33 | 5.78 (5.39–6.17)*** |
| W6.48T | −8.11 (8.47–7.76)ns | 3 | −7.16 (7.81–6.51) | 26 | 7.08 (6.54–7.63)*** | >−6.00a | >25 | 5.70 (4.92–6.49)*** | −6.60 (6.95–6.25 | 3 | 0.06 (0.05–0.07) | 12 | 5.73 (4.66–6.79)*** |