TY - JOUR
T1 - A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells
AU - Varnum, Megan M.
AU - Clayton, Kevin A.
AU - Yoshii-Kitahara, Asuka
AU - Yonemoto, Grant
AU - Koro, Lacin
AU - Ikezu, Seiko
AU - Ikezu, Tsuneya
N1 - Funding Information:
This work was supported by National Institutes of Health Predoctoral Training Grant 5T32 GM008541 (to M. M. V. and K. A. C.) and Grant RF1 AG054199-01 (to T. I.), a Henry I. Russek Award (to M. M. V.), Boston University Undergraduate Research Opportunities Program Awards (to G. Y. and L. K.), the Alzheimer’s Association (to T. I.), the BrightFocus Foundation (to T. I.), and the Massachusetts Neuroscience Consortium (to T. I.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Acknowledgments—We thank Dr. Naohiro Kato at the Louisiana State University for pDuex-Bait and pDuex-Prey vectors, the Flow Cytometry Core Facility at Boston University School of Medicine for expertise and guidance in experiments, and Dr. Hirohide Asai for assisting in gene constructions.
Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/6/23
Y1 - 2017/6/23
N2 - Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer’s disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.
AB - Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer’s disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.
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U2 - 10.1074/jbc.M116.759159
DO - 10.1074/jbc.M116.759159
M3 - Article
C2 - 28490631
AN - SCOPUS:85021352419
SN - 0021-9258
VL - 292
SP - 10651
EP - 10663
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 25
ER -