An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening
Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Pharmacology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphar.2022.902290/full |
| _version_ | 1811256724692140032 |
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| author | Wenfeng Liu Chang Liu Chang Liu Pei-Gen Ren Pei-Gen Ren Jun Chu Jun Chu Liang Wang |
| author_facet | Wenfeng Liu Chang Liu Chang Liu Pei-Gen Ren Pei-Gen Ren Jun Chu Jun Chu Liang Wang |
| author_sort | Wenfeng Liu |
| collection | DOAJ |
| description | Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but also high-content screening of compounds against GPCRs. We previously reported the high-performance circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1. Here, we developed improved G-Flamp1 variants G-Flamp2 and G-Flamp2b. Compared to G-Flamp1, G-Flamp2 exhibited increased baseline fluorescence (1.6-fold) and larger fluorescence change (ΔF/F0) (1,300% vs. 1,100%) in HEK293T cells, while G-Flamp2b showed increased baseline fluorescence (3.1-fold) and smaller ΔF/F0 (400% vs. 1,100%). Furthermore, live cell imaging of mitochondrial matrix–targeted G-Flamp2 confirmed cytosolic cAMP was able to enter the mitochondrial matrix. G-Flamp2 imaging also showed that adipose tissue extract activated the Gi protein-coupled orphan GPCR GPR50 in HEK293T cells. Taken together, our results showed that the high-performance of G-Flamp2 would facilitate sensitive intracellular cAMP imaging and activity measurement of compounds targeting GPCR-cAMP signaling pathway during early drug development. |
| first_indexed | 2024-04-12T17:45:34Z |
| format | Article |
| id | doaj.art-c2aea713826f4192b69a13c45844ca90 |
| institution | Directory Open Access Journal |
| issn | 1663-9812 |
| language | English |
| last_indexed | 2024-04-12T17:45:34Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Pharmacology |
| spelling | doaj.art-c2aea713826f4192b69a13c45844ca902022-12-22T03:22:41ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122022-05-011310.3389/fphar.2022.902290902290An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug ScreeningWenfeng Liu0Chang Liu1Chang Liu2Pei-Gen Ren3Pei-Gen Ren4Jun Chu5Jun Chu6Liang Wang7Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging & CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaCenter for Energy Metabolism and Reproduction, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaShenzhen College of Advanced Technology, University of Chinese Academy of Sciences, Shenzhen, ChinaCenter for Energy Metabolism and Reproduction, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaShenzhen College of Advanced Technology, University of Chinese Academy of Sciences, Shenzhen, ChinaGuangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging & CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaShenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science, Shenzhen, ChinaGuangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging & CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, ChinaCyclic adenosine 3′,5′-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but also high-content screening of compounds against GPCRs. We previously reported the high-performance circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1. Here, we developed improved G-Flamp1 variants G-Flamp2 and G-Flamp2b. Compared to G-Flamp1, G-Flamp2 exhibited increased baseline fluorescence (1.6-fold) and larger fluorescence change (ΔF/F0) (1,300% vs. 1,100%) in HEK293T cells, while G-Flamp2b showed increased baseline fluorescence (3.1-fold) and smaller ΔF/F0 (400% vs. 1,100%). Furthermore, live cell imaging of mitochondrial matrix–targeted G-Flamp2 confirmed cytosolic cAMP was able to enter the mitochondrial matrix. G-Flamp2 imaging also showed that adipose tissue extract activated the Gi protein-coupled orphan GPCR GPR50 in HEK293T cells. Taken together, our results showed that the high-performance of G-Flamp2 would facilitate sensitive intracellular cAMP imaging and activity measurement of compounds targeting GPCR-cAMP signaling pathway during early drug development.https://www.frontiersin.org/articles/10.3389/fphar.2022.902290/fullsingle-FP indicatorfluorescence imagingcircular permutationcAMPGPCRdrug discovery |
| spellingShingle | Wenfeng Liu Chang Liu Chang Liu Pei-Gen Ren Pei-Gen Ren Jun Chu Jun Chu Liang Wang An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening Frontiers in Pharmacology single-FP indicator fluorescence imaging circular permutation cAMP GPCR drug discovery |
| title | An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening |
| title_full | An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening |
| title_fullStr | An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening |
| title_full_unstemmed | An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening |
| title_short | An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening |
| title_sort | improved genetically encoded fluorescent camp indicator for sensitive camp imaging and fast drug screening |
| topic | single-FP indicator fluorescence imaging circular permutation cAMP GPCR drug discovery |
| url | https://www.frontiersin.org/articles/10.3389/fphar.2022.902290/full |
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