The iDimerize Inducible Homodimer System brings protein-protein interactions under real-time, small molecule control. A protein of interest is fused to the DmrB binding domain, and dimerization is induced by adding the B/B Homodimerizer (AP20187). This cell-permeable ligand induces two or more copies of the DmrB fusion protein to interact. iDimerize Inducible Homodimer technology has been used to create conditional alleles of cell surface receptors, signaling molecules, and other proteins which are regulated by protein-protein interactions. Induced protein homodimerization is already widely used in vitro and in vivo to control signaling pathways, and it can be used to induce any type of event that is activated or controlled by protein-protein interactions, including proliferation, differentiation, adhesion, transformation, and apoptosis. Plasmid and lentiviral (Lenti-X) vector formats are available.
Note: While it is possible to use the iDimerize Inducible Homodimer System to induce heterodimerization, you will obtain a mixture of homodimers and heterodimers. To induce heterodimers specifically and exclusively, use the iDimerize Inducible Heterodimer System.
B/B Homodimerizer Ligand (AP20187)
The B/B Homodimerizer is a synthetic, cell-permeable ligand that can be used to induce homodimerization of fusion proteins containing the DmrB domain. It is nontoxic in vitro and in mice (recommended dosage range, 0.01–100 nM for in vitro use or 0.005–10 mg/kg in mice). The B/B Homodimerizer is identical to the AP20187 ligand, which was previously supplied by ARIAD Pharmaceuticals Inc.
B/B Washout Ligand
B/B Washout Ligand dissociates protein interactions that were induced by the B/B Homodimerizer (T1/2 ~10 minutes). The B/B Washout Ligand has a much greater effect on target cells that have been treated with B/B Homodimerizer than simply replacing medium containing B/B Homodimerizer with medium without B/B Homodimerizer.
iDimerize Inducible Homodimer Systems (with Tet-On 3G)
One challenge of ligand-dependent dimerization experiments is that non ligand-induced dimerization events may occur if the protein of interest is expressed at high levels. This is especially problematic if the target protein is a membrane protein, because the local concentration can increase quickly due to the limited space on the membrane. We’ve combined iDimerize and Tet-On 3G technologies to eliminate these unwanted events. First, use doxycycline to optimize the protein's expression to physiologically-relevant levels. Then induce dimerization by adding the dimerizer ligand to your culture medium.
Application: Signal Transduction
Many signaling cascades are activated almost exclusively by the interactions of signaling proteins. Cell surface receptor proteins cluster in response to extracellular factors, which leads to the recruitment and activation of intracellular signaling proteins, transcription activation, effector protein production, and finally to protein activation or secretion. Any step of this signaling pathway that relies on a dimerization event can be brought under dimerizer control by fusing the proteins involved to the DmrB domain.
Example 1: Inducing a Programmed Cell Death Pathway (Inducible Apoptosis)
The Fas receptor (FasR) is a transmembrane protein located on the surface of cells that activates programmed cell death (apoptosis) when induced to trimerize by fas ligand (FasL) molecules located on the surface of adjacent cells (e.g., cytotoxic T cells). FasR-FasL binding plays an important role in the regulation of the immune system and cancer progression. This trimerization (and the resulting apoptotic signaling cascade) can be mimicked using the iDimerize Inducible Homodimer System: cells expressing a FasR-DmrB fusion protein can induce cell death on demand, simply by adding the B/B Homodimerizer. An in vivo model (the MaFIA mouse; 1) utilizes the Fas receptor to systematically and reversibly eliminate macrophages from transgenic mice.
Example 2: Inducible Mouse Model for Prostate Cancer
iDimerize Inducible Homodimer technology was used to characterize the role of different FGF receptor subtypes (FGFR1 and FGFR2) in prostate cancer. Transgenic mice (which express conditionally active alleles of FGFR1 or FGFR2 in prostate tissue) were treated with B/B Homodimerizer and monitored for prostate cancer. FGFR1, but not FGFR2, was found to play a role in prostate cancer development (2).