Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology

Abstract

Chemically-inducible dimerization (CID) is a powerful tool that has proved useful in solving numerous problems in cell biology and related fields. In this review, we focus on case studies where CID was able to provide insight into otherwise refractory problems. Of particular interest are the cases of lipid second messengers and small GTPases, where the "signaling paradox" (how a small pool of signaling molecules can generate a large range of responses) can be at least partly explained through results gleaned from CID experiments. We also discuss several recent technical advances that provide improved specificity in CID action, novel CID substrates that allow simultaneous orthogonal manipulation of multiple systems in one cell, and several applications that move beyond the traditional CID technique of moving a protein of interest to a specific spatiotemporal location.

Fig. 1

Schematic illustration of a typical CID experiment. Initially, one protein component (in this case, FRB) is anchored at a target site (in this case plasma membrane), while the other protein component (FKBP) is fused to a protein of interest (POI). In the absence of dimerizer, the POI-FKBP fusion protein diffuses freely in the cytoplasm. Upon addition of the dimerizer rapamycin, a ternary complex is formed of FKBP–rapamycin–FRB, which brings the POI to the target site

Fig. 2

Variations on the CID theme: caged rapamycin and plant hormone CID systems. a Direct UV photocaging: the caged rapamycin derivative pRap is unable to induce FKBP-FRB dimerization. Activation by UV light releases rapamycin, which can then induce dimerization as normal. b Indirect UV photocaging: the cRb-A complex is unable to cross the cell membrane, and thus unable to interact with intracellular FRB or FKBP proteins. Activation by UV light releases HE-Rapa, which is able to cross the plasma membrane and induce FRB-FKBP dimerization. c Plant hormone CID: the plant hormone abscisic acid (ABA) is able to induce complex formation between the ABI and PYL protein moieties, in a manner analogous to rapamycin-induced dimerization of FRB and FKBP. d Plant hormone CID: The gibberellic acid derivative GA₃-AM, upon crossing the plasma membrane, is rapidly cleaved by cytosolic esterases to release GA₃, which can induce complex formation between GID1 and GAI proteins

Fig. 3

Further variations on the CID theme. a Kinase activation: Insertion of the iFKBP into the kinase domain of a protein can cause the protein to be catalytically inactive. Binding of the iFKBP to rapamycin and FRB induces a conformational change that restores kinase activity. b and c Membrane cross-linking: FRB and FKBP proteins are anchored to different cellular membranes. Addition of rapamycin leads to cross-linking of the membranes. d “Anchor-Away” technique: The POI is initially at its active location. Addition of rapamycin induces translocation to a different domain where is sequestered and inactive. e Inactive reservoir: POI is initially sequestered at a location where it is inactive (in this case the Golgi) rather than floating freely in the cytoplasm where it can have background activity. Addition of dimerizer induces translocation to the active site without membrane cross-linking. f Lipid synthesis versus lipid liberation: standard targeting of a PI(4) 5-kinase to plasma membrane induces formation of PI(4,5)P₂, but also depletes the plasma membrane supply of PI(4)P. If instead the PI(4,5)P₂ at the plasma membrane is initially “hidden” with a PH domain, which is then removed by sequestration to the mitochondrial membrane, the concentration of available PI(4,5)P₂ at the plasma membrane is suddenly increased without affecting concentrations of other membrane lipids