Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 Apr 15;11:8288. doi: 10.1038/s41598-021-87417-2

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Agonist-induced GRK2 recruitment, Ser³¹⁷/Thr³¹⁸ phosphorylation and β-arrestin2 recruitment. (A). HEK293 cells were transfected GRK2-Venus and hD_2LR-NLuc. GRK2 recruitment was measured by BRET 10 min after agonist addition at 37 °C. Data is presented as the increase in BRET ratio normalized to vehicle (0%) and the maximal effect of dopamine (100%). Data represents the mean ± SEM of 5 separate experiments performed in duplicate. (B) HA-hD_2LR expressing HEK293 cells were either stimulated with vehicle (solvent) or 10 μM of quinpirole (quin), dopamine (dop), pergolide (perg), ropinirole (rop), apomorphine (apo), cabergoline (cabergo), bromocriptine (bromo), terguride (terg), roxindole (roxin), aripiprazole (arip), MLS1547 (MLS) or UNC9994 (UNC) for 10 min at 37 °C. Lysates were immunoblotted with antibody to pSer³¹⁷/Thr³¹⁸ [5102]. Blots were stripped and reprobed for D₂R [5106] to confirm equal loading of the gel. Blots are representative, n = 3. (C) These Western blots were analyzed using densitometry to yield relative pSer³¹⁷/pThr³¹⁸ signals normalized to the corresponding total D₂R signal. Data represents the mean ± S.E.M. of three separate experiments. (D) Agonist-induced β-arrestin2 recruitment to the D₂R. HEK293 cells were transfected with hD_2LR-NLuc, GRK2 and YFP-β-arrestin2. β-arrestin2 recruitment was measured by BRET 10 min after agonist addition at 37 °C. Data is presented as the increase in BRET ratio normalized to vehicle (0%) and the maximal effect of dopamine (100%). Data represents the mean ± SEM of 5 separate experiments performed in duplicate. (E–G) Correlation of the effect of a saturating concentration of each agonist (10 μM) to stimulate Ser³¹⁷/Thr³¹⁸ phosphorylation maximal with the maximal effects of the various agonists in assays measuring GRK2 recruitment and β-arrestin2 recruitment determined. DA is marked in blue. The relationship between two variables was assessed using a two-tailed Spearman’s rank correlation allowing the calculation of the correlation coefficient, r_s. A P value of 0.05 was used as the cut-off for statistical significance and relationships depicted as trend lines.