Binding properties of WT and mutant Y2R
Results of equilibrium as well as kinetic experiments are presented. W6.48 mutants displayed similar binding characteristics compared with WT. A large difference between KD and Ki values in quasi-homologous binding experiments suggested the presence of two affinity states, which was corroborated by biphasic properties in koff experiments. The presence of GTPγS shifted the population of these states, suggesting the high-affinity state to be G protein dependent. All values are given as the mean (95% CI) corrected for multiple comparisons. Significance levels of KD and logIC50, respectively, relative to WT Y2R were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test. Data for percentage of fast phase with or without GTPγS were analyzed by two-way ANOVA and compared with the values in the absence of GTPγS by Bonferroni’s post-test.
| Saturation Binding 125I-PYY | Displacement Binding (60 pM 125I-PYY/ NPY) | Dissociation Rate koff | ||||||
|---|---|---|---|---|---|---|---|---|
| KD/pM | Bmax/fmol mg−1 | logIC50 | logKi | koff − fast/min−1 | koff − slow/min−1 | % fast | % fast + 100 µM GTPγS | |
| WT Y2R | 50 (7–93) | 4329 (2802–5856) | −8.59 (8.74–8.44) | −8.93 (9.08–8.78) | 0.41 (0.11–0.71) | 0.0070 (0.0005–0.0135) | 35 (23–46) | 60 (42–78)* |
| W6.48Y | 57 (0–120)ns | 2614 (1386–3842) | −8.75 (8.98–8.53)* | −9.06 (9.29–8.84) | 0.34 (0–0.75) | 0.0066 (0–0.0160) | 34 (12–56) | 56 (26–86)ns |
| W6.48H | 40 (0–80)ns | 2827 (1741–3913) | −8.37 (8.50–8.25)* | −8.77 (8.89–8.64) | 0.18 (0–0.36) | 0.0061 (0.0008–0.0115) | 15 (0–33) | 52 (10–94)** |
| W6.48T | 59 (10–107)ns | 3421 (2173–4669) | −8.62 (8.76–8.48)ns | −8.93 (9.07–8.78) | 0.10 (0–0.20) | 0.0052 (0–0.0148) | 28 (0–58) | 77 (45–100)*** |