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. 2019 Nov 8;9:16312. doi: 10.1038/s41598-019-52540-8

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© The Author(s) 2019

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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Ca²⁺ release following 5-HT_2AR activation and BRET saturation assay to confirm specific interactions. Neither in (a) HT22 nor in (b) HEK293T quantitative differences in intracellular calcium could be detected in co-expressing cells upon stimulation with the 5-HT_2AR agonist DOI. The amount of released calcium is depicted as a ratio of Fura-2, excited at 340 nm in the calcium bound and at 380 nm in the unbound state. Activation or inibition of the D₂R protomer did not alter [Ca²⁺]_i. For each treatment, all differences between 5-HT_2AR and 5-HT_2AR-D₂R cells were non-significant. Data were analyzed with one-way ANOVA and Tukey’s multiple comparisons test presented as mean ± SEM, n = 6, performed in hexaplicates. (c) Saturated BRET titration curve for increasing concentrations of D_2LR-mVenus as BRET acceptor and constant amounts of 5-HT_2AR-Rluc8 as a donor. Close proximity of donor and acceptor molecule exerts in energy transfer after enzymatic conversion of the substrate coelenterazine-h indicating a direct interaction between both receptors. (d) Comparable positive BRET signals detectable for swapped donor-acceptor pair. Pooled data, performed in HEK293T cells, presented as mean ± SEM, n = 3.