Investigating the Applicability of Immune tSH2-ITAM Binding as a Molecular Biosensor for FGFR1 RTK Activation

Abstract

Receptor tyrosine kinases are important for studying cell signaling pathways. Their binding activity to extracellular signaling proteins at the plasma membrane activates phosphorylation cascades that induce the downstream expression of target genes involved in various cellular processes including cellular proliferation and differentiation. Unfortunately, we lack biosensors that successfully report specific RTK activation patterns in single living cells. Therefore, in this paper we sought to better understand the dynamics of RTK activation through the development of an efficient RTK-specific biosensor capable of reporting real-time activation of these receptors at the plasma membrane. Based on our understanding of orthogonal binding of the ZAP70 protein tSH2 domain to ITAM regions of transmembrane T cell receptors, we inserted a tSH2-ITAM reporter system into single living cells to act as a biomarker for ZAP70 protein binding at the pTyr residues of FGFR1 upon being activated by FGF ligand stimulation. We saw RTK activation efficiently reported as visualized by swift localization of ztSH2 to the FGFR1-ITAM construct localized at the plasma membrane. However, the absence of a negative control with cells lacking the tSH2-ITAM system necessitates further validation of the biosensor. The results implicate a potential generalizable, live-imaging biosensor capable of reporting multi-RTK activation patterns to highlight new understanding of cell signaling dynamics and their connections to cell proliferation and many other important cellular processes.