Journal: Nature Communications

Article Title: An artificial cell capable of signal transduction mediated by ADRB2 for the regulation of glycogenolysis

doi: 10.1038/s41467-026-68503-3

Figure Lengend Snippet: a The signal transduction from extracellular ISO into intracellular cAMP and subsequent regulation of glycogenolytic metabolic pathway inside an artificial cell; ISO: isoproterenol; ADRB2: β2-adrenergic receptor; Gsα: Gs subunit α; ADCY5: adenylate cyclase V; PKA: Protein kinase A; PhK: Phosphorylase kinase; PYGM: Glycogen phosphorylase; PGM: Phosphoglucomutase; G6PDH: Glucose-6-phosphate dehydrogenase; GDP: Guanosine diphosphate; GTP: Guanosine triphosphate; ATP: Adenosine triphosphate; cAMP: Cyclic adenosine monophosphate; G-1-P: Glucose 1-phosphate; G-6-P: Glucose-6-phosphate; 6-PGL: 6-Phosphoglucono-lactone; NADP⁺(H): Nicotinamide adenine dinucleotide phosphate. Created in BioRender. Liu, Y. (2025) https://BioRender.com/689u8j4 . b Chemical reaction equations involved in the signal transduction and glycogenolytic metabolism; ADCY5: Adenylate cyclase V; p-PYGM: Phosphorylated glycogen phosphorylase.

Article Snippet: Rabbit anti-PhKA2 polyclonal antibody (No. 24658-1-AP), rabbit anti-PYGM-Specific polyclonal antibody (No. 19716-1-AP) and ADRB2 polyclonal antibody (No. 29864-1-AP) were purchased from Proteintech (China).

Techniques: Transduction

Journal: Nature Communications

Article Title: An artificial cell capable of signal transduction mediated by ADRB2 for the regulation of glycogenolysis

doi: 10.1038/s41467-026-68503-3

Figure Lengend Snippet: a Scheme of the signal transduction pathway from ISO to cAMP mediated by ADRB2. SDS-PAGE images of purified ADRB2 ( b ), Gsα ( c ) and ADCY5 ( d ) from Homo Sapiens . n = 3 independent replicates. e The size exclusion chromatography of ADRB2, Gsα, and ADRB2-Gsα complex. f The effect of pH on the ADCY5 activity by measuring the production of cAMP at 30 °C, 10.0 μM ISO and 2.0 mM Mg 2+ . g The effect of temperature on the ADCY5 activity by measuring the production of cAMP at pH 8, 10.0 μM ISO and 2.0 mM Mg 2+ . The data were expressed as mean ± SD, n = 3 independent replicates. h The Mg 2+ concentration effect on the ADCY5 activity by measuring the production of cAMP at 37 °C, pH 8 and 10.0 μM ISO. The data were expressed as mean ± SD, n = 3 independent replicates. i Initial reaction velocities of cAMP production catalyzed by ADCY5 as a function of ATP concentration from 0 to 14.0 mM in the solution (10.0 μM ISO, 2.0 mM Mg 2+ , 6.0 μg/mL ADRB2-Gsα complex, 6.0 μg/mL ADCY5, pH 8) at 37 °C. The data were expressed as mean ± SD, n = 3 independent replicates. j The cAMP production as a function of ADRB2-Gsα/ADCY5 molar ratios with ADCY5 fixed at 10 nM and ADRB2-Gsα varied from 1.1 to 21.7 nM in a solution (10.0 μM ISO, 2.0 mM Mg 2+ , pH 8) at 37 °C. The data were expressed as mean ± SD, n = 3 independent replicates. **** P < 0.0001, ns P > 0.05, two-sided, multiple comparisons by One-way ANOVA test. k ISO dose-dependent cAMP production as a function of time in a solution (17.4 nM ADRB2-Gsα, 10.0 nM ADCY5, 10 −5 −10 3 μM ISO, 2.0 mM Mg 2+ , pH 8) at 37 °C. l The corresponding cAMP production at 25 min of ( k ). The data were expressed as mean ± SD, n = 3 independent replicates. **** P < 0.0001, ns P > 0.05, two-sided, multiple comparisons by One-way ANOVA test. P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: Rabbit anti-PhKA2 polyclonal antibody (No. 24658-1-AP), rabbit anti-PYGM-Specific polyclonal antibody (No. 19716-1-AP) and ADRB2 polyclonal antibody (No. 29864-1-AP) were purchased from Proteintech (China).

Techniques: Transduction, SDS Page, Purification, Size-exclusion Chromatography, Activity Assay, Concentration Assay

Journal: Nature Communications

Article Title: An artificial cell capable of signal transduction mediated by ADRB2 for the regulation of glycogenolysis

doi: 10.1038/s41467-026-68503-3

Figure Lengend Snippet: a The representative fluorescence images of Cy5-ADRB2-GUV (with NBD-PE in the bilayer) taken with red channel (left image), green channel (middle image), and their merged image (right image) at ADRB2 and lipids molar ratio of 10 3 :10 6 . The scale bar is 10.0 μm. b The percentage of ADRB2-GUVs with varied ADRB2 to lipids molar ratios ranging from 1:10 6 to 10 4 :10 6 . The data were expressed as mean ± SD, n = 3 independent replicates. **** P < 0.0001 1:10 6 vs. 10:10 6 , **** P < 0.0001 10:10 6 vs. 10 2 :10 6 , *** P = 0.0007 10 2 :10 6 vs. 10 3 :10 6 , ns P > 0.05 10 3 :10 6 vs. 10 4 :10 6 , two-sided, multiple comparisons by One-way ANOVA test. c The percentage of ADRB2 with correct orientation on GUVs determined by WB (top image) and band intensities (bottom graph). The data were expressed as mean ± SD, n = 3 independent replicates. d Schematic diagram of the Gsα detection mechanism using BODIPY TR GTP γ S. Created in BioRender. Liu, Y. (2025) https://BioRender.com/g8zgjzy . e The representative time-series images of GUVs stimulated by 1.0 μM ISO for the binding events of BODIPY TR GTP γ S to Gsα from 30 s to 15 min. The scale bar is 10.0 μm. n = 3 independent replicates. f The corresponding fluorescence intensity of GUV in ( e ) (red curve) and the fluorescence intensity of GUV with no ISO stimulation (blue curve). n = 3 independent replicates. g The representative fluorescence images of FITC labeled ADCY5-GUV (with TR-DHPE in the bilayer) taken with green channel (left image), red channel (middle image), and their merged image (right image) at ADCY5 and lipids molar ratio of 10 3 :10 6 . The scale bar is 10.0 μm. n = 3 independent replicates. h The flow cytometry histogram of FITC-ADCY5-GUVs with varied ADCY5 to lipids molar ratios ranging from 10 −1 :10 6 to 10 3 :10 6 . n = 3 independent replicates. i The percentage of ADCY5-GUVs with varied ADCY5 to lipids molar ratios ranging from 10 −1 :10 6 to 10 3 :10 6 . The data were expressed as mean ± SD, n = 3 independent replicates. **** P < 0.0001 10 −1 :10 6 vs. 1:10 6 , **** P < 0.0001 1:10 6 vs. 10:10 6 , **** P < 0.0001 10:10 6 vs. 10 2 :10 6 , **** P < 0.0001 10 2 :10 6 vs. 10 3 :10 6 , two-sided, multiple comparisons by One-way ANOVA test. P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.

Article Snippet: Rabbit anti-PhKA2 polyclonal antibody (No. 24658-1-AP), rabbit anti-PYGM-Specific polyclonal antibody (No. 19716-1-AP) and ADRB2 polyclonal antibody (No. 29864-1-AP) were purchased from Proteintech (China).

Techniques: Fluorescence, Binding Assay, Labeling, Flow Cytometry

Journal: Nature Communications

Article Title: An artificial cell capable of signal transduction mediated by ADRB2 for the regulation of glycogenolysis

doi: 10.1038/s41467-026-68503-3

Figure Lengend Snippet: a Schematic illustration of the signal transduction of an artificial cell from extracellular ISO to intracellular cAMP. Created in BioRender. Liu, Y. (2025) https://BioRender.com/ik5i982 . b Fluorescence images of GUVs containing Cy5-ADRB2-Gsα complexes and FITC-ADCY5 taken by red channel (left image), green channel (middle image), and their merged image (right image). The scale bar is 10.0 μm. n = 3 independent replicates. c The representative time-series images of artificial cells stimulated by 1.0 μM ISO for the intracellular cAMP production from 0 to 20 min, with top row images taken by the blue channel and bottom row images taken by the green channel. The scale bar is 10.0 μm. n = 3 independent replicates. d The corresponding FRET ratios of Epac1-cAMP in the artificial cell in ( c ). The data were expressed as mean ± SD, n = 3 independent replicates. e The cAMP production in artificial cells with the addition of 1.0 μM ISO as a function of time from 0 to 30 min using fluorescence spectroscopy. n = 3 independent replicates. f The cAMP production in artificial cells with the addition of varied ISO concentration (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 μM) at 30 min. n = 3 independent replicates. g The signal amplification folds of artificial cells from extracellular ISO to intracellular cAMP as a function of ISO concentration (0.4 to 10.0 μM) at 30 min. n = 3 independent replicates. h The representative time-series images of Epac1-cAMP in artificial cells with sequential antagonist alprenolol treatment for 10 min from 10 to 20 min, and competitive 10.0 μM ISO activation for 10 min from 20 to 30 min, with top row images taken by blue channel and bottom row images taken by green channel. The scale bar is 10.0 μm. n = 3 independent replicates. i The corresponding Fret ratio of Epac1-cAMP in the artificial cell in ( h ). The data were expressed as mean ± SD, n = 3 independent replicates. j The representative time-series images of Epac1-cAMP in artificial cells with sequential inverse agonist carazolol treatment for 10 min from 10 to 20 min, and 10.0 μM ISO activation for 10 min from 20 to 30 min, with top row images taken by blue channel and bottom row images taken by green channel. The scale bar is 10.0 μm. n = 3 independent replicates. k The corresponding FRET ratio Epac1-cAMP in the artificial cell in ( j ). The data were expressed as mean ± SD, n = 3 independent replicates. l FRET ratio of Epac1-cAMP in the artificial cell measured by fluorescence spectroscopy as a function of time with the addition of 1.0 μM alprenolol antagonism at 10 min and the addition of 10.0 μM ISO at 20 min (blue curve), with the addition of 1.0 μM carazolol inverse agonism at 10 min and the addition of 10.0 μM ISO at 20 min (green curve). The data were expressed as mean ± SD, n = 3 independent replicates. Source data are provided as a Source Data file.

Article Snippet: Rabbit anti-PhKA2 polyclonal antibody (No. 24658-1-AP), rabbit anti-PYGM-Specific polyclonal antibody (No. 19716-1-AP) and ADRB2 polyclonal antibody (No. 29864-1-AP) were purchased from Proteintech (China).

Techniques: Transduction, Fluorescence, Spectroscopy, Concentration Assay, Amplification, Activation Assay