A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

• Published in: The Journal of biological chemistry Vol. 292; no. 25; pp. 10651 - 10663
• Main Authors: Varnum, Megan M., Clayton, Kevin A., Yoshii-Kitahara, Asuka, Yonemoto, Grant, Koro, Lacin, Ikezu, Seiko, Ikezu, Tsuneya
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.06.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Alzheimer disease; Alzheimer Disease - genetics; Alzheimer Disease - metabolism; Alzheimer's disease; Animals; cell culture; Cell Line; Flow Cytometry - methods; Genetic Complementation Test - methods; genetic polymorphism; Humans; Lipodystrophy - genetics; Lipodystrophy - metabolism; Luciferases, Renilla - metabolism; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; microglia; Microglia - metabolism; Molecular Bases of Disease; Osteochondrodysplasias - genetics; Osteochondrodysplasias - metabolism; phagocytosis; plasmid; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Subacute Sclerosing Panencephalitis - genetics; Subacute Sclerosing Panencephalitis - metabolism; Syk Kinase - genetics; Syk Kinase - metabolism; phagocytosis; Alzheimer disease; cell culture; plasmid; genetic polymorphism; microglia; Alzheimer's disease
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.759159

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.