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Dynamic Measurements of Gi Signaling in Living Cells
Author(s) -
Tewson Paul,
Quinn Anne Marie,
Hughes Thomas
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb478
Subject(s) - ibmx , forskolin , phosphodiesterase , microbiology and biotechnology , chemistry , signal transduction , receptor , cyclase , g protein coupled receptor , cytosol , lysis , biology , biochemistry , enzyme
Receptors coupled to Gs and Gi activate and inhibit adenyl cyclase in a competitive manner. Each has it's own binding site on adenyl cyclase, and each can influence the enzyme activity positively or negatively. Gs signaling has been relatively easy to measure: activation of GPCRs coupled to Gs increases cytosolic levels of cAMP either globally or locally and transiently. Typically, Gs assays involve poisoning the degradative pathway with IBMX, and measuring cAMP accumulation in a cell lysate. Gi signaling has been much harder to measure, and involves a double negative: forskolin is added to artificially activate the adeyl cyclase, IBMX is added to poison the phosphodiesterase, and then activation of a Gi‐coupled receptor is measured as a lack of cAMP accumulation. To address the need for a better way to directly measure Gi activation, we developed a physiologically‐relevant assay to measure Gi signaling in living cells. The assay is based on cADDis, a genetically encoded fluorescent sensor that detects both increases and decreases in cAMP in real time. The cADDis sensor is packaged in BacMam, a viral vector, for efficient delivery to most cell types, including pancreatic islets, neurons and cardiomyocytes, as well as standard cell lines. The assay is robust enough for drug discovery, producing Z’ > 0.7. Here we show that activation of first the Gs pathway and then the Gi pathway produces a collision whereby the action of the first is clearly reversed by the action of the other. We demonstrate in real time, in living cells, the actual competition between Gs and Gi signaling at the adenyl cyclase. The interval between Gs and Gi activation can be varied, as well as the order of agonist application, and agonist concentrations, to clearly measure the delicate balance ‐ in time and efficacy ‐ of the stimulatory and inhibitory G protein pathways. Support or Funding Information National Science Foundation for SBIR Phase II 1430878 National Institute of Neurologic Disease and Stroke for SBIR Phase II R44 NS082222,Adenyl cyclase can be inhibited by different Gi‐coupled GPCRs. Panel A: cADDis fluorescence change on activation of Gi‐coupled Succinate Receptor with Succinic Acid (500um) Panel B: cADDis sensor fluorescence change on activation of Gi‐coupled M2 receptor with carbachol (30 uM) Panel C: cADDis fluorescence change on activation of Gi‐coupled A1 Adenosine Receptor, Cyclopentyladenosine addition (1 um)