Journal: Nature chemical biology
Article Title: Endosome positioning coordinates spatially-selective GPCR signaling
doi: 10.1038/s41589-023-01390-7
Figure Lengend Snippet: A-B. Cells expressing a Nuclear- (A) or ER- (B) localized ExRai-AKAR2 sensor and the CID system were pre-treated with ethanol (“Vehicle”) or 1μM AP21967 (“Rapalog”) for 30 min before 10nM isoproterenol stimulation. The delta F/F (ΔF/F) ratio was quantified every 30 sec for 1000 sec (left) and the total nuclear (A) or ER (B) PKA activity was quantified by calculating the area under the curve (AUC, right). A. Data are mean from n = 2 biologically independent replicates; 11–13 cells total/condition. ** = p = 0.0019 by unpaired two-tailed Student’s t-test. B. Data are mean from n = 4 (“Vehicle”) and n = 5 (“Rapalog”) biologically independent replicates ± s.e.m.; 21–24 cells total/condition. * = p = 0.0193 by unpaired two-tailed Student’s t-test. C. CREB reporter expression in pCRE-NLS-DD-zsGreen1 cells stimulated with 1μM isoproterenol or 10nM isoproterenol/100μM IBMX in the presence of 1μM Shield for 4 h. Data are mean from n = 3 biologically independent replicates ± s.e.m.; 45 cells total/condition. **** = p < 0.0001, ** = p < 0.01 by two-way ANOVA test with Tukey. D-E. Cells expressing a Nuclear- (D) or ER- (E) localized ExRai-AKAR2 sensor and the CID system were pre-treated with ethanol (“Vehicle”) or 1μM AP21967 (“Rapalog”) for 30 min before isoproterenol stimulation. Vehicle- and Rapalog-treated cells were stimulated with 10nM isoproterenol and 5nM isoproterenol/100μM IBMX, respectively. The delta F/F (ΔF/F) ratio was quantified every 30 sec for 1000 sec (left) and the total nuclear (D) or ER (E) PKA activity was quantified by calculating the area under the curve (AUC, right). D. Data are mean from n = 3 (“Vehicle”) and n = 2 (“Rapalog+IBMX”) biologically independent replicates ± s.e.m.; 25 cells total/condition. E. Data are mean from n = 3 biologically independent replicates ± s.e.m.; 14–20 cells total/condition. ** = p = 0.0059 and * = p = 0.0141 by unpaired two-tailed Student’s t-test. F. Model: Endosomes enable site-selective outputs by functioning as vehicles to deliver the receptor in proximity to PKA and the nucleus and away from phosphodiesterases.
Article Snippet: ExRai-AKAR2-NLS was generated by subcloning ExRai AKAR2 (Addgene, Cat #161753) into a pcDNA3.1(+) backbone containing a nuclear localization signal (PKKKRKVEDA).
Techniques: Expressing, Activity Assay, Two Tailed Test
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