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. 2018 Feb 14;9:674. doi: 10.1038/s41467-017-02776-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Non-catechol D1R agonists fail to induce desensitization via impaired β-arrestin recruitment. a Schematic of agonist-stimulated desensitization of GPCRs. Prolonged activation reduces subsequent agonist-induced signaling. b Experimental design to test for agonist-stimulated D1R desensitization. c Agonist-induced (1 μM SKF-81297) cAMP elevation and percent desensitization of D1 receptor signaling in striatal neurons treated for 120 min with vehicle or 10 μM of the indicated agonists. One-way ANOVA revealed a significant effect of treatment on cAMP levels, F(9, 108) = 32.1, P < 0.001; post-hoc Dunnett’s comparisons indicated only catechol agonists had significantly different level of cAMP versus vehicle (***P < 0.001). Percent desensitization was significantly affected by treatment (one-way ANOVA F(8,99) = 24.2, P < 0.0001); Dunnett’s comparisons indicated significantly higher percent desensitization in all catechol groups versus vehicle (***P < 0.001), while non-catechol groups were not significantly different from vehicle. All data represent means ± s.e.m., n = 3 per group. d Percent D1 receptor desensitization after various times of agonist exposure. Two-way ANOVA revealed significant trends in time (linear F(1,59) = 44.2, P < 0.0001 and quadratic F(1,59) = 8.5, P < 0.005) and significant interaction effect (F(3,59) = 5.1, P < 0.003); pairwise Tukey post-hoc tests at time points 60,120, and 180 min indicated that catechol and non-catechol groups were significantly different (***P < 0.001, n = 4 per group; batches are independent from panel c). e Live cell total internal reflection fluorescence microscopy (TIRFM) imaging of β-arrestin-GFP recruitment to the plasma membrane of U2OS cells expressing human D1R. Images show β-arrestin-GFP at the plasma membrane of cells before and after exposure to the indicated agonists (1 μM). f Quantification of membrane β-arrestin-GFP puncta in the cells shown in e. g TIRFM images of β-arrestin-GFP at the plasma membrane in U2OS cells after 10 min of treatment with indicated agonists (1 μM). Scale bars, 10 μm. h Quantification of β-arrestin-GFP membrane recruitment via total signal per cell from ≥60 cells per group obtained across three independent experiments. One-way ANOVA revealed that treatment had a significant effect (F(9,33) = 130.9, P < 0.0001; Dunnett’s comparisons with dopamine group indicated significantly lower total signal in vehicle, PF-1119, PF-8294 (***P < 0.001) and PF-6142 (*P < 0.05), but not in PF-2334, and not for all catechol agonists groups. All data represent means ± s.e.m., n ≥ 3