Fig. 6: Gs-coupled β₂AR drives IP₃ formation, IP₁ accumulation, and PIP₂ depletion after Gq priming.

a Representative Iso-induced Ca²⁺ traces and their quantification in the absence or presence of 50 µM of the IP₃R antagonist 2-APB in naive HEK293 cells after ATP priming. b Exemplary label-free whole cell activation profiles, based on detection of dynamic mass redistribution (DMR) in response to ATP-stimulated Gq-coupled P2Y receptors in untreated (w/o), 2-APB-treated (50 µM), and FR-treated (1 µM) HEK293 cells, and corresponding quantification. c BRET-based real-time IP₃ detection following a two consecutive addition protocol. Cartoon illustrating the IP₃ intramolecular BRET biosensor principle⁶⁵. In IP₃-free conditions, energy donor Renilla luciferase (Rluc) and energy acceptor Venus, each fused to the IP₃R ligand binding ___domain (LBD) are in close proximity (high BRET state). Binding of an IP₃ molecule triggers donor:acceptor separation, resulting in a BRET decrease (low BRET state). BRET ratios are plotted as reciprocals of the I/I_o values. d_i–iii Agonist-induced IP₁ accumulation in HEK293 cells with and without ATP (100 µM) or CCh (100 µM) priming using Iso (d_i), PGE₁ (d_ii), and NECA (d_iii) to stimulate β₂AR, EP₂/EP₄, and A_2A/A_2B, respectively. e Iso-induced PIP₂ depletion after Gq priming. Schematic of the PIP₂ hydrolysis NanoBiT-based biosensor. PIP₂ hydrolysis is reflected by rapid translocation of the Small BiT (SmBiT)-tagged PH ___domain of PLCδ1 from plasma membrane-localized Large BiT (LgBiT)-CAAX to the cytosol resulting in decreased luminescence. Real-time recordings in (a, b) are mean values + SEM. IP₃ (c) and PIP₂ (e) recordings, concentration-effect curves (a, b), and bar charts (c–e) are mean values ± SEM for n independent biological experiments (a: n = 4; b: n = 3; c: n = 5; d: solvent and ATP n = 4, CCh n = 3; e: n = 4). Ca²⁺ measurements are duplicates; DMR, IP₁ accumulation, and PIP₂ depletion are triplicate, and IP₃-BRET time-courses are quadruplicate determinations. Statistical significance was calculated with a two-way ANOVA with Dunnett’s (c) and Šídák’s (d, e) post-hoc analysis. Source data are provided as a Source Data file. c and e was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en”.