The ability to rapidly recruit signaling proteins to the plasma membrane (PM) is a powerful tool for the control of signal transduction in living cells. To date, several methods based on chemically induced dimerization have been developed and used for various cell biological studies. However, they often suffer from the need to tune the relative expression level of two protein components, and reversible dimerization systems are still limited. As a tool for dimerization-free, small-molecule-mediated protein localization control, we previously devised a chemogenetic approach termed SLIPT (self-localizing ligand-induced protein translocation). In SLIPT, a small molecule hybrid ligand with organelle/membrane localization ability is used to recruit its binding protein from the cytoplasm to the target site. In this presentation, we report a versatile chemogenetic SLIPT system for PM-specific protein recruitment. In this system, an engineered Escherichia coli dihydrofolate reductase (eDHFR) termed iK6DHFR, which contains a hexalysine (K6) sequence inserted into the loop region of eDHFR, is fused to target proteins as a SLIPT tag. The iK6DHFR-fused proteins can be recruited specifically from the cytoplasm to the PM within a few minutes upon addition of the myristoyl-D-Cys-tethered trimethoprim ligand (mDcTMP). This SLIPT-PM system was applicable to control various signal transductions such as Raf/ERK activation, Ca2+ spike, and cAMP generation. We further established a method to manipulate PM recruitment and release of signaling proteins in a reversible and repeatable manner, which allowed us to artificially induce oscillatory dynamics of cell signaling. In our presentation, we will present the outline and applications of the SLIPT-PM platform.

SLIPT-PM: A new versatile chemogenetic platform for controlling plasma membrane signaling

ACS Spring 2022

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