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. 2020 Mar 30;18:49. doi: 10.1186/s12964-020-00537-6

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© The Author(s) 2020

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Sustained inhibition of the adenylyl cylase (AC) by Y₂R stimulation can be abolished by PTX-treatment. a Concentration-response curves of transiently transfected HEK293-hY₂R were measured by cAMP–accumulation assay without first stimulation (w/o; black line) and after stimulation with 10 nM, 100 nM and 1 μM NPY, respectively (brown, red, olive-green). AC inhibition caused by Gα_i activity was measured in the presence of 5 μM forskolin. The maximum cAMP response is reduced in stimulated cells. b Data were verified by testing native Gα_i-signaling with SMS-KAN cells that endogenously express Y₂R. We observed the same reduction of the maximal inducible adenylyl cyclase response induced by forskolin in stimulated cells (olive-green). Data were validated with c) an internalization deficient mutant S³⁷⁴T³⁷⁶T³⁷⁹D-Y₂R. d Concentration-response curves of transiently co-transfected HEK293-hY₂R/hY₁R were measured by cAMP–accumulation assay. Cells without first stimulation (NPY, black line; F⁷, P³⁴-NPY, orange line; Ahx^5–24-NPY, blue line) or with first stimulation with 1 μM F⁷, P³⁴-NPY, a selective Y₁R peptide (FP), followed by stimulation with Ahx^5–24-NPY, a selective Y₂R agonist (dashed blue line) exhibited no significant loss in AC-activity. First stimulation with 1 μM Ahx^5–24-NPY, followed by stimulation with F⁷, P³⁴-NPY (FP, dashed orange line) revealed an obvious inhibition of AC-activity. Data were normalized to Ahx^5–24-NPY as control curve (100%, blue line). e Concentration-response curves of transiently co-transfected HEK293-hY₂R with melanocortin 1 receptor were measured by cAMP–accumulation assay and had only slight impact on Gα_s-signaling, as the inhibitory effect of Y₂R is still present after stimulation with 1 μM NPY (left panel) but is completely abolished in presence of PTX and thus preventing Gα_i- signaling (right panel). f cAMP-accumulation assay of transiently transfected HEK293-hY₂R treated with varying concentrations of pertussis toxin (PTX) caused inhibition of AC-activity compared to control curve (black line). g AC-activity is rescued comparing ± PTX- treated cells after first stimulation with different NPY concentration and reached again the cAMP level of control (w/o). Experiments represent data n ≥ 3; significance was determined by one-way ANOVA, Tukey post test, ns: not significant, ***: P < 0.0001, **: P < 0.001