An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP
Xiaobo Li, … , Shengnan Luo, Tongsen Zheng
Xiaobo Li, … , Shengnan Luo, Tongsen Zheng
Published December 14, 2021
Citation Information: J Clin Invest. 2022;132(3):e144339. https://doi.org/10.1172/JCI144339.
View: Text | PDF
Research Article Immunology Oncology

An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP

Abstract

It has been revealed that 2′3′-cyclic-GMP-AMP (cGAMP), a second messenger that activates the antiviral stimulator of IFN genes (STING), elicits an antitumoral immune response. Since cGAMP cannot cross the cell membrane, it is not clear how intracellular STING has been activated by extracellular cGAMP until SLC19A1 was identified as an importer to transport extracellular cGAMP into the cytosol. However, SLC19A1-deficient cells also sense extracellular cGAMP, suggesting the presence of mechanisms other than the facilitating transporters for STING sensing extracellular cGAMP. Here, using immunoprecipitation, immunofluorescence, and flow cytometry, we identified an alternatively spliced STING isoform, plasmatic membrane STING (pmSTING), that localized in the plasma membrane with its C-terminus outside the cell, due to a lack of 1 transmembrane domain in its N-terminus compared with canonical STING. Further studies showed that extracellular cGAMP not only promoted the dimerization of pmSTING and interaction of pmSTING with TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3), but also enhanced the phosphorylation of TBK1 and IRF3 and the production of IFN in pmSTING-transfected cells. Additionally, we also identified similar pmSTING isoforms in other species including human. This study suggests a conserved role for pmSTING in sensing extracellular cGAMP and provides insight into the role of cGAMP as an immunotransmitter.

Authors

Xiaobo Li, Yuanyuan Zhu, Xiao Zhang, Xiang An, Mingjiao Weng, Jiaqi Shi, Song Wang, Caiqi Liu, Shengnan Luo, Tongsen Zheng

×

Figure 5

An alternatively spliced isoform of human STING localizes in the plasma membrane.

Options: View larger image (or click on image) Download as PowerPoint
An alternatively spliced isoform of human STING localizes in the plasma ...
(A) Flow cytometry was performed to detect cell surface STING with its C-terminus outside human PBMCs. (B) Human PBMCs were incubated with the indicated antibodies and then washed and lysed. Immunoblotting was performed to detect IgG in the cell lysate using a secondary antibody against rabbit IgG. (C) Colocalization of cell surface STING with surface protein of T cells (CD3) and myeloid cells (CD11b) from human PBMCs was detected by confocal microscopy. Scale bars: 10 μm. (D) Exon structure of the predicted human TMEM173 transcript variants based on the NCBI’s GENE database. (E) Predicted plasma membrane topology of STING isoforms in homo sapiens. (F) Two STING isoforms were detected by an antibody against the STING C-terminal epitope using immunoblotting of human PBMCs from 3 healthy donors. (G) Two STING isoforms with a different N-terminus were detected in human PBMCs (hPBMC) by RT-PCR. (H and I) 293T cells were transfected with h-erSTING-Flag, h-pmSTING-Flag, or a vector plasmid, respectively. An antibody against Flag was used to detect Flag projecting outside of cells by immunofluorescence (H) and flow cytometry (I), respectively. Scale bar: 20 μm.