Fig. 4: Y2R NT mutations differentially modulate G protein activation and recruitment of arrestin-3. | Nature Communications

Fig. 4: Y2R NT mutations differentially modulate G protein activation and recruitment of arrestin-3.

From: Transient ligand contacts of the intrinsically disordered N-terminus of neuropeptide Y2 receptor regulate arrestin-3 recruitment

Fig. 4

a Close-up view to membrane proximal Y2R NT from cryo-EM (PDB 7X9B) highlighting interactions of D42-S43-T44NT. b Live-cell fluorescence microscopy shows that Y2R NT variants are transported to the plasma membrane like the wild-type. Y2R-eYFP variants are shown in yellow, cell nuclei are stained by H33342 and shown in blue, scale bar equals 10 µm. All pictures were acquired with identical light exposure and picture processing. c Quantification of cellular receptor expression is based on eYFP fluorescence in a plate reader, normalized to wild-type Y2R. d Activity of Y2R variants towards Gi1 proteins as measured by a direct BRET assay (Gi1-CASE) 10 min after ligand stimulation. All variants show full activation; D42A-S43A-T44A has the strongest shift in potency, while all others show mild or no effects. e Kinetics of Gi1 activation after stimulation with 1 nM NPY is overall similar for most variants except Δ2–41 and D42A-S43A-T44A, which have a tendency for faster apparent rate constants (kobs). 95% CI of wild-type Y2R kobs is given as a gray rectangle for comparison. f Recruitment of arrestin-3 to Y2R variants as measured by BRET 10 min after NPY stimulation. D42A-S43A-T44A and Δ2–41 variants reduce recruitment of arrestin-3 to the receptor. Neutral (in green) and charge-inverted (in blue) variants of the acidic patch in Y2R NT lead to distinct behavior, with only charge reversal impairing arrestin-3 recruitment. g Kinetic analysis of arrestin-3 recruitment after stimulation with 100 nM/ 1 µM NPY. Charge reversal in acidic patches 1 and 2 leads to faster initial recruitment, but also early signal decay. The bar plot shows quantification of the initial rate of arrestin-3 recruitment to receptor variants. Apparent rate constants (kobs) are plotted in comparison to wild-type Y2R-eYFP and its 95% CI (gray rectangle). * P < 0.05, ** P < 0.01 in one-way ANOVA, with Dunnett’s post-hoc test corrected for multiple comparisons against wild-type Y2R. For the color legend, please see panel (e). Data in (c–g) are the mean ± SEM of n = 5 (c), n = 4-9 (d), n = 5-6 (e), n = 3 (f), n = 3-4 (g) independent experiments, each performed in technical triplicate.

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