Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: a Representative Ca 2+ traces and their quantification evoked in HEK-∆ f PLCβ1–4 cells transiently transfected to re-express PLCβ3 in the absence and presence of the Gβγ-scavenger masGRK3ct. Cells were primed with CCh at t = 20 s followed by addition of Iso at t = 140 s. b Same experimental setup as in ( a ) using HEK293-wt cells and 1 µM CCh as the first stimulus. The concentration-response curves derived from the mean net peak responses are divided into a high-potency and a low-potency component, reflecting Gα s -cAMP “α” and Gs-βγ “βγ” contribution, respectively. Bar graphs represent the fractional distribution of high- and low-potency Iso-Ca 2+ and its alteration with co-expressed masGRK3ct. Representative traces are mean + SEM, and data points in concentration-response curves are mean ± SEM of n biologically independent experiments ( a : vector n = 6, masGRK3ct n = 5; b : n = 5), each performed in duplicate. Source data are provided as a Source Data file.

Article Snippet: Membranes were stripped and reprobed in ROTI®Block with an antibody against the anti-PLCβ3 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-133231, 1:500) and incubated overnight at 4 °C.

Techniques: Transfection, Concentration Assay, Derivative Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: A molecular mechanism to diversify Ca 2+ signaling downstream of Gs protein-coupled receptors

doi: 10.1038/s41467-024-51991-6

Figure Lengend Snippet: a , b Representative Ca 2+ traces and corresponding quantification of maximum Ca 2+ amplitudes in HEK293 ( a ) and HEK-ΔGs ( b ) cells transfected with either empty vector DNA or cDNA plasmids coding for PLCβ3-wt, PLCβ3 ΔXY or PLCβ3 F715A upon Iso stimulation. Rightmost panels: Western blot quantification of each PLCβ variant. The statistical significance of expression level differences was determined using a one-way ANOVA with Tukey´s post-hoc analysis. c , d Naive HEK293 cells were transfected to express either PLCβ3 ΔXY ( c ) or PLCβ3 F715A ( d ) in the absence (vector) or presence of the Gβγ scavenger masGRK3ct. e Iso-induced PIP 2 depletion in HEK-ΔGq/11/12/13 cells transfected to express the PIP 2 hydrolysis NanoBiT-based biosensor along with PLCβ F715A , β 2 AR, and masGRK3ct or empty vector DNA as control. f IP 3 BRET recordings and corresponding quantification in HEK-ΔGq/11/12/13 cells, transfected to express the IP 3 -BRET sensor along with PLCβ F715A in the absence (empty vector) or presence of masGRK3ct upon addition of Iso or buffer. g Cartoon representation depicting the cellular consequences of cAMP production as well as IP 3 formation on mobilization of cytosolic Ca 2+ from ER sources. cAMP and IP 3 synergize to sensitize ER-localized IP 3 R channels for mobilization of cytosolic Ca 2+ . Mutant PLCβ3 variants with crippled autoinhibition produce IP 3 without Gq priming in response to Gβγ only. PLCβ mut = PLCβ 3 ΔXY , or PLCβ 3 F715A . Cells in a – d were FR-pretreated (1 µM) to exclude any potential Gq contribution. Representative Ca 2+ recordings in a – d are shown as mean + SEM, summarized data are mean ± SEM of n independent biological replicates ( a , b : n = 3; c , d : n = 4), each performed in duplicate. PIP 2 depletion data ( e ) are mean + SEM of n = 3 experiments, each performed in duplicate. BRET IP 3 real-time recordings ( f ) are depicted as mean + SEM of n = 3 experiments, one performed in triplicate and two in nonuplicate; their summarized data are shown as mean ± SEM; statistical significance was determined using a two-tailed student’s t test. Source data are provided as a Source Data file. g 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 ”.

Article Snippet: Membranes were stripped and reprobed in ROTI®Block with an antibody against the anti-PLCβ3 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-133231, 1:500) and incubated overnight at 4 °C.

Techniques: Transfection, Plasmid Preparation, Western Blot, Variant Assay, Expressing, Control, Mutagenesis, Two Tailed Test