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. 2020 Feb 3;5(6):2648–2659. doi: 10.1021/acsomega.9b03146

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Copyright © 2020 American Chemical Society

This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.

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ß-arrestin recruitment upon activation of WT and DRY mutants of AT1_AR in HEK293TN cells. (A, B) Confocal images depicting subcellular localization of WT AT1_AR-mCh (upper panels) coexpressed together with ßarr1-mTQ2 (A, lower panel) or ßarr2-mTQ2 (B, lower panel) in HEK293TN cells. From left to right: subcellular localization before, 125 s after, and 500 s after stimulation with 1 μM AngII. The last (right) panel: the merged image of AT1AR-mCh (set to magenta) and ßarr1-mTQ2 (A) or ßarr2-mTQ2 (B) (set to green) localization 125 and 500 s after AngII stimulation, where the shared localization is depicted in white. Pearson’s coefficient (R) of colocalization between ß-arrestin and Lck was calculated for each of the three time points. Arrowheads point to the AngII-induced localization of ß-arrestin at or near the plasma membrane (A) or in the intracellular compartment (B). The size of the images is 60 μm × 60 μm. (C, D) Confocal images depicting subcellular localization of AT1AR DRY-mCh (upper panels) coexpressed together with ßarr1-mTQ2 (C, lower panel) or ßarr2-mTQ2 (D, lower panel) in HEK293TN cells. From left to right: subcellular localization before, 125 s after, and 500 s after stimulation with 1 μM AngII. The last (right) panel: the merged image of AT1AR DRY-mCh (set to magenta) and ßarr1-mTQ2 (C) or ßarr2-mTQ2 (D) (set to green) localization 125 and 500 s after AngII stimulation, where the shared localization is depicted in white. The size of the images is 60 μm × 60 μm. (E) Dotplot showing AngII-induced relocation of ß-arrestin to the plasma membrane. WT AT1_AR-mCh or AT1AR DRY-mCh was coexpressed together with ßarr1-mTQ2 or ßarr2-mTQ2 in HEK293TN cells. The cytosolic fraction of ß-arrestin was measured at 250 s after stimulation with 1 μM AngII and normalized to the total ß-arrestin content prior to stimulation; centerlines show the median. (F) Line plot showing colocalization of ß-arrestin1 and AT1AR at the plasma membrane. Fluorescence intensity of ß-arrestin (blue trace), AT1_AR (red trace), and Lck (black trace) was measured along the line shown in the inset. The line was drawn through the ß-arrestin puncta indicated with the arrowhead in panel (A). (G) Line plot showing colocalization of ß-arrestin1 and AT1_AR in the endosomal compartment. Fluorescence intensity of ß-arrestin (blue trace) and AT1_AR (red trace) was measured along the line shown in the inset. The line was drawn through the ß-arrestin puncta indicated with the arrowhead in panel (B).