Construction of Light-Activated Neurotrophin Receptors Using the Improved Light-Induced Dimerizer (iLID). Issue 13 (12th June 2020)

• Record Type: Journal Article
• Authors: Hope, Jen M.; Liu, Aofei; Calvin, Ghawayne J.; Cui, Bianxiao
• Is Part Of: Journal of molecular biology. Volume 432:Issue 13(2020)
• Page Start: 3739
• Page End: 3748
• Publication Date: 2020-06-12
• Subjects: optogenetics -- RTK -- cell signaling -- receptor tyrosine kinases; RTK receptor tyrosine kinase -- ICD intracellular kinase domain -- iLID improved light-induced dimer -- KTR kinase translocation reporter -- PHAKT1 pleckstrin homology domain of Akt1; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.04.018
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Receptor tyrosine kinases (RTKs) play crucial roles in human health, and their misregulation is implicated in disorders ranging from neurodegenerative diseases to cancers. The highly conserved mechanism of activation of RTKs makes them especially appealing candidates for control via optogenetic dimerization methods. This work offers a strategy for using the improved light-induced dimer (iLID) system with a constructed tandem dimer of its binding partner nano (tdnano) to build light-activatable versions of RTKs. In the absence of light, the iLID-RTK is cytosolic, monomeric, and inactive. Under blue light, the iLID + tdnano system recruits two copies of iLID-RTK to tdnano, dimerizing, and activating the RTK. We demonstrate that iLID opto-iTrkA and opto-iTrkB are capable of reproducing downstream ERK and Akt signaling only in the presence of tdnano. We further show with our opto-iTrkA that the system is compatible with multi-day and population-level activation of TrkA in PC12 cells. By leveraging genetic targeting of tdnano, we achieve RTK activation at a specific subcellular location even with whole-cell illumination, allowing us to confidently probe the impact of context on signaling outcome. Graphical abstract: Unlabelled Image Highlights: The authors construct a tandem dimer of SspB/nano with a long, flexible linker. tdnano permits simultaneous recruitment and dimerization of iLID opto-RTKs under blue light. iLID opto-iTrkA and opto-iTrkB do not activate downstream signaling in the absence of tdnano. The fusion of genetically encoded localization tags to tdnano allows location-specific activation of opto-RTKs. The design of iLID + tdnano is generalizable to other RTKs.