ARGENT Regulated Heterodimerization

2006

• Bayle, J.H., Grimley, J.S., Stankunas, K., Gestwicki, J.E., Wandless, T.J., and Crabtree, G.R. (2006) Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem Biol 13:99-107.
  Characterizes the ability of several rapamycin analogs, including AP21967, to act as heterodimerizers in transcription and protein relocalization assays. Pairings of rapalogs and engineered binding proteins are identified that are able to operate independently.
• Dirnberger, D., Unsin, G., Schlenker, S., and Reichel, C. (2006) A small-molecule-protein interaction system with split-ubiquitin as sensor. Chembiochem 7:936-942.
  Describes a yeast three-hybrid system based on use of heterodimerizers of dexamethasone and methotrexate, in which the readout is reconstitution of a split ubiquitin protein leading to degradation of a reporter protein.
• Dudas, M., Kim, J., Li, W.Y., Nagy, A., Larsson, J., Karlsson, S., Chai, Y., and Kaartinen, V. (2006) Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion. Dev Biol 296:298-314.
  Uses AP21967-mediated heterodimerization of ALK5 to show that its activity can be induced by interaction with multiple TGF-beta type II receptor kinases.
• Silvius, J.R., Bhagatji, P., Leventis, R., and Terrone, D. (2006) K-ras4B and prenylated proteins lacking "second signals" associate dynamically with cellular membranes. Mol Biol Cell 17:192-202.
  Uses AP21967-mediated heterodimerization to study the dynamics of membrane localization of prenylated or myristoylated proteins.
• Zhan, L., Xiang, B., and Muthuswamy, S.K. (2006) Controlled activation of ErbB1/ErbB2 heterodimers promote invasion of three-dimensional organized epithelia in an ErbB1-dependent manner: implications for progression of ErbB2-overexpressing tumors. Cancer Res 66:5201-5208.
  Uses AP1510-mediated homodimerization and AP21967-mediated heterodimerization to compare the tumor-promoting activities of ErbB2 homodimers and ErbB1-ErbB2 heterodimers.
• Zhu, S., Zhang, H., and Matunis, M.J. (2006) SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes. Exp Cell Res 312:1042-1049.
  Uses AP21967-mediated heterodimerizatin to study the function of small ubiquitin-related modifiers (SUMOs) by inducing their association with candidate substrates.

2005

• Ameres, S.L., Drueppel, L., Pfleiderer, K., Schmidt, A., Hillen, W., and Berens, C. (2005) Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer. Embo J 24:358-367.
  AP21967-mediated heterodimerization of DNA binding domains is used to reversibly alter DNA structure to study its role in controlling gene expression.
• Binkowski, B.F., Miller, R.A., and Belshaw, P.J. (2005) Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules. Chem Biol 12:847-855.
  Uses the rapamycin-based heterodimerization in yeast to generate peptides that interact with protein targets in a ligand-reversible manner. The system is validated using rapamycin and the non-immunosuppressive analog AP23102.
• Graveley, B.R. (2005) Small molecule control of pre-mRNA splicing. RNA 11:355-358.
  An inducible system to control pre-mRNA splicing is described in which a nontoxic rapamycin derivative is used to heterodimerize an RNA binding domain and a splicing activation domain.
• Inoue, T., Heo, W.D., Grimley, J.S., Wandless, T.J., and Meyer, T. (2005) An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat Methods 2:415-418.
  A system for inducible plasma membrane targeting using a rapamycin analog is optimized to allow the rapid activation and inactivation of Rho small GTPases on a timescale of seconds.>
• Karpova, A.Y., Tervo, D.G., Gray, N.W., and Svoboda, K. (2005) Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons. Neuron 48:727-735.
  Describes the development of dimerization-based switches, called MISTs, for reversible inactivation of neurotransmitter release. The switches are based on sequestration of proteins essential to synaptic vesicle fusion, using AP20187-mediated homodimerization or AP21967-mediated heterodimerization. Switches functioned in cultured neurons, brain slices and (for AP20187 injected into the brain) in transgenic mice expressing MISTs selectively in Purkinje neurons.

2004

• de Graffenried, C.L., Laughlin, S.T., Kohler, J.J., and Bertozzi, C.R. (2004) A small-molecule switch for Golgi sulfotransferases. Proc Natl Acad Sci U S A 101:16715-16720.
  Demonstrates that the activity and localization of Golgi-resident sulfotransferases can be put under control of the heterodimerizer rapamycin. Furthermore, ascomycin, which binds FKBP but not FRB, was shown to inhibit rapamycin-induced activities.
• Luker, K.E., Smith, M.C., Luker, G.D., Gammon, S.T., Piwnica-Worms, H., and Piwnica-Worms, D. (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci U S A 101:12288-12293.
  A firefly luciferase protein fragment complementation system is developed and used to visualize rapamycin-induced protein-protein interactions in vivo.
• Paulmurugan, R., Massoud, T.F., Huang, J., and Gambhir, S.S. (2004) Molecular imaging of drug-modulated protein-protein interactions in living subjects. Cancer Res 64:2113-2119.
  A luciferase protein fragment complementation system is developed and used to visualize rapamycin-induced protein-protein interactions in vivo.
• Pecot, M.Y., and Malhotra, V. (2004) Golgi membranes remain segregated from the endoplasmic reticulum during mitosis in mammalian cells. Cell 116:99-107.
  Uses rapamycin, and cells expressing an FKBP-tagged Golgi enzyme and a FRAP-tagged protein localized to the endoplasmic reticulum to demonstrate that Golgi membranes remain separate from the endoplasmic reticulum during mitosis.
• Terrillon, S., and Bouvier, M. (2004) Receptor activity-independent recruitment of betaarrestin2 reveals specific signalling modes. Embo J 23:3950-3961.
  Uses AP21967 to study the intrinsic signaling properties of beta-arrestin2 via the forced recruitment to vasopressin V1a or V2 receptors.

2003

• Hoogenraad, C.C., Wulf, P., Schiefermeier, N., Stepanova, T., Galjart, N., Small, J.V., Grosveld, F., De Zeeuw, C.I., and Akhmanova, A. (2003) Bicaudal D induces selective dynein-mediated microtubule minus end-directed transport. EMBO J 22:6004-6015.
  Uses the heterodimerizer AP21967 to inducibly tether the N-terminal domain of Bicaudal D to various membrane organelles to study the dramatic effect that it has on their subcellular distribution. The supplementary material includes videos showing AP21967-induced movement of organelles.
• Jullien, N., Sampieri, F., Enjalbert, A., and Herman, J.P. (2003) Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res 31:e131.
  Describes the development of an inducible Cre recombinase which is activated when rapamycin, or the nonimmunosuppressive analog AP23102, induces the association of 2 complementing fragments of Cre.
• Kohler, J.J., and Bertozzi, C.R. (2003) Regulating cell surface glycosylation by small molecule control of enzyme localization. Chem Biol 10:1303-11.
  Uses the heterodimerizer rapamycin to control the Golgi localization and hence catalytic activity of alpha1,3 fucosyltransferase in living cells, providing a method for bringing the production of sialyl Lewis x under small molecule control.
• Mootz, H.D., Blum, E.S., Tyszkiewicz, A.B., and Muir, T.W. (2003) Conditional protein splicing: a new tool to control protein structure and function in vitro and in vivo. J Am Chem Soc 125:10561-9.
  Demonstrates that a conditional protein splicing system, in which rapamycin or AP21967 heterodimerizes two halves of an artificially split intein, functions in mammalian cells.
• Schlatter, S., Senn, C., and Fussenegger, M. (2003) Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins. Biotechnol Bioeng 83:210-25.
  Describes development of a novel translation control system whereby an inactive translational initiation factor, eIF4G, is reconstituted via rapamycin-induced heterodimerization.
• So, C.W., Lin, M., Ayton, P.M., Chen, E.H., and Cleary, M.L. (2003) Dimerization contributes to oncogenic activation of MLL chimeras in acute leukemias. Cancer Cell 4:99-110.
  Demonstrates that AP21967-mediated dimerization of MLL is sufficient to enhance the self-renewal of hematopoietic progenitors and provides support for an oligomerization-dependent mechanism for oncogenic conversion of MLL.
• Stankunas, K., Bayle, J.H., Gestwicki, J.E., Lin, Y.M., Wandless, T.J., and Crabtree, G.R. (2003) Conditional protein alleles using Knockin mice and a chemical inducer of dimerization. Mol Cell 12:1615-24.
  Describes a novel regulatory system based on the observation that GSK-3beta is destabilized when fused to a mutant version of FRB, resulting in a loss-of-function, and that stability and function can be restored in the presence of the FRB ligand rapamycin or a rapamycin analog. This system may provide a general method of making conditional alleles to facilitate the study of protein function in vitro and in vivo.

2002

• Abida, W.M., Carter, B.T., Althoff, E.A., Lin, H., and Cornish, V.W. (2002) Receptor-dependence of the transcription read-out in a small-molecule three-hybrid system. Chembiochem 3:887-95.
  Further characterizes the use of a dexamethasone-methotrexate (Dex-Mtx) heterodimerizer in yeast.
• Althoff, E.A., and Cornish, V.W. (2002) A bacterial small-molecule three-hybrid system. Angew. Chemie 41:2327-30.
  Describes the development of a novel alternative heterodimerizer, Mtx-SLF, which can heterodimerize dihydrofolate reductase- and FKBP- containing fusion proteins. SLF is a monomeric FKBP ligand based on the early-generation homodimerizer AP1510. These reagents are used to construct a three-hybrid system that functions in bacteria.
• Baker, K., Bleczinski, C., Lin, H., Salazar-Jimenez, G., Sengupta, D., Krane, S., and Cornish, V.W. (2002) Chemical complementation: a reaction-independent genetic assay for enzyme catalysis. Proc Natl Acad Sci USA 99:16537-42.
  Uses a dexamethasone-methotrexate (Dex-Mtx) heterodimerizer system to develop a general yeast-based screen for enzymatic activity.
• Chang, D.W., Xing, Z., Pan, Y., Algeciras-Schimnich, A., Barnhart, B.C., Yaish-Ohad, S., Peter, M.E., and Yang, X. (2002) c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. Embo J 21:3704-3714.
  Uses rapamcyin-mediated heterodimerization and AP20187-mediated homodimerization to explore the role of c-FLIP-L in the CD95-mediated apoptotic signaling pathway.
• Chen, M., Orozco, A., Spencer, D.M., and Wang, J. (2002) Activation of initiator caspases through a stable dimeric intermediate. J Biol Chem 277:50761-7.
  Uses the rapamycin analog AP20840 to heterodimerize different caspases in order to study the mechanism by which they are activated.
• Clemons, P.A., Gladstone, B.G., Seth, A., Chao, E.D., Foley, M.A., and Schreiber, S.L. (2002) Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors. Chem Biol 9:49-61.
  Describes the development of an FK506-based heterodimerizeration system. A varient of FK506 was developed that does not bind appreciably to wild type calcineurin but instead interacts interacts simultaneously with FKBP12 and a mutant, chimeric domain derived from the calcineurin A and B chains. The system was used to control the activity of a transcription factor in yeast and mammalian cells.
• de Wildt, R.M., Tomlinson, I.M., Ong, J.L., and Holliger, P. (2002) Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes. Proc Natl Acad Sci USA 99:8530-5.
  Uses the rapamycin-dependent interaction of FKBP and FRAP to demonstrate the ability of the SAC (selection by avidity capture) approach to detect receptor-ligand interactions in vitro. FKBP is fused to the g3p coat protein of filamentous phage, and FRAP is fused to GST.
• Galarneau, A., Primeau, M., Trudeau, L.E., and Michnick, S.W. (2002) beta-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 20:619-22.
  Uses rapamycin to heterodimerize two complementing fragments of beta-lactamase to demonstrate the ability of the system to detect inducible protein interactions.
• Li, B., Desai, S.A., MacCorkle-Chosnek, R.A., Fan, L., and Spencer, D.M. (2002) A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis. Gene Ther 9:233-44.
  Uses a rapamycin analog to conditionally activate Akt (via membrane recruitment), thereby protecting cells from apoptosis.
• Mootz, H.D., and Muir, T.W. (2002) Protein splicing triggered by a small molecule. J. Am. Chem. Soc. 124:9044-9045.
  Describes the development of a system for conditional protein splicing by using rapamycin to heterodimerize two halves of an artificially split intein.
• Scheid, M.P., Marignani, P.A., and Woodgett, J.R. (2002) Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. Mol Cell Biol 22:6247-60.
  Uses AP21967-mediated membrane recruitment of protein kinase B/Akt to study the mechanism by which the kinase becomes activated.
• Wehrman, T., Kleaveland, B., Her, J.H., Balint, R.F., and Blau, H.M. (2002) Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments. Proc Natl Acad Sci USA 99:3469-74.
  Uses rapamycin to heterodimerize two complementing fragments of beta-lactamase to demonstrate the ability of the system to detect inducible protein interactions in mammalian cells.

2001

• Biggar, S.R., and Crabtree, G.R. (2001) Cell signaling can direct either binary or graded transcriptional responses. Embo J 20:3167-76.
  Uses the heterodimerizer FK506 to explore the mechanism by which transcriptional activators effect changes in gene expression.
• Koide, K., Finkelstein, J.M., Ball, Z., and Verdine, G.L. (2001) A synthetic library of cell-permeable molecules. J. Am. Chem. Soc. 123:398-408.
  Generates a library of synthetic heterodimerizers and demonstrates their ability to penetrate cells as a first step toward creating novel heterodimerizers that target known proteins.
• Otto, K.G., Jin, L., Spencer, D.M., and Blau, C.A. (2001) Cell proliferation through forced engagement of c-Kit and Flt-3. Blood 97:3662-4.
  Rapamycin-mediated heterodimerization of the signaling domains of c-kit and Flt-3 stimulates proliferation of cells normally dependent on IL-3 for growth.

2000

• Biggar, S.R., and Crabtree, G.R. (2000) Chemically regulated transcription factors reveal the persistence of repressor-resistant transcription after disrupting activator function. J Biol Chem 275:25381-90.
  Uses rapamycin and FK506 as heterodimerizers to study the function of transcriptional repressors.
• Castellano, F., and Chavrier, P. (2000) Inducible membrane recruitment of small GTP-binding proteins by rapamycin-based system in living cells. Methods Enzymol 325:285-95.
  Reviews recent work using the rapamycin heterodimerizing system to study the function of signaling proteins by recruiting them to the membrane.
• Castellano, F., Montcourrier, P., and Chavrier, P. (2000) Membrane recruitment of rac1 triggers phagocytosis. J Cell Sci 113:2955-61.
  Rapamycin-mediated recruitment of Rac1 to the cell surface triggers phagocytosis.
• Griffith, E.C., Licitra, E.J., and Liu, J.O. (2000) Yeast three-hybrid system for detecting ligand-receptor interactions. Methods Enzymol 328:89-103.
  Provides detailed protocols for the use of the three hybrid system for identifying or characterizing novel small molecule protein ligands.
• Lin, H., Abida, W.M., Sauer, R.T., and Cornish, V.W. (2000) Dexamethasone-Methotrexate: An efficient chemical inducer of protein dimerization in vivo. J. Am. Chem. Soc. 122:4247-4248.
  Describes the development of a novel alternative heterodimerizer, Dex-Mtx, that can dimerize DHFR- and glucocorticoid receptor- containing fusion proteins in vitro.

1999

• Briesewitz, R., Ray, G.T., Wandless, T.J., and Crabtree, G.R. (1999) Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces. Proc Natl Acad Sci USA 96:1953-8.
  Describes an innovative use of the heterodimerizer approach to enhance the affinity of small molecule inhibitors of target proteins. The inhibitor is coupled to a ligand for an endogenous cellular protein (in this case FKBP), which is thereby recruited to the target protein when the inhibitor binds, contributing additional binding energy. This paper illustrates the concept using heterodimerizers consisting of an SH2 domain-binding tetrapeptide linked to a synthetic FKBP ligand.
• Castellano, F., Montcourrier, P., Guillemot, J.C., Gouin, E., Machesky, L., Cossart, P., and Chavrier, P. (1999) Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation. Curr Biol 9:351-60.
  Rapamycin-mediated recruitment of Cdc42 or WASP to the cell surface triggers actin polymerization.
• Muthuswamy, S.K., Gilman, M., and Brugge, J.S. (1999) Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and heterodimers. Mol Cell Biol 19:6845-57.
  Uses the homodimerizer AP1510 and the heterodimerizer rapamycin to explore the differential signaling activities of ErbB1 and ErbB2 homodimers and heterodimers.

1998

• Klemm, J.D., Schreiber, S.L., and Crabtree, G.R. (1998) Dimerization as a regulatory mechanism in signal transduction. Annu Rev Immunol 16:569-92.
  Reviews the natural role of protein dimerization in cellular processes and describes how dimerizers provide a general method to regulate such processes.
• Stockwell, B.R., and Schreiber, S.L. (1998) Probing the role of homomeric and heteromeric receptor interactions in TGF-beta signaling using small molecule dimerizers. Curr Biol 8:761-70.
  Explores the role of receptor oligomerization in TGF-beta signaling using a variety of homo- and heterodimerizers including rapamycin and FK1012.

1997

• Graef, I.A., Holsinger, L.J., Diver, S., Schreiber, S.L., and Crabtree, G.R. (1997) Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70. Embo J 16:5618-28.
  Explores the effects of membrane recruitment, orientation and oligomerization of ZAP-70 using the homodimerizer FK1012 and the heterodimerizer rapamycin.
• Klemm, J.D., Beals, C.R., and Crabtree, G.R. (1997) Rapid targeting of nuclear proteins to the cytoplasm. Curr Biol 7:638-44.
  Describes a general method to inducibly relocalize a nuclear protein to the cytoplasm by using rapamycin to heterodimerize a nuclear protein of interest and a protein containing a nuclear export signal.
• Liberles, S.D., Diver, S.T., Austin, D.J., and Schreiber, S.L. (1997) Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proc Natl Acad Sci USA 94:7825-30.
  Describes the modification of the rapamycin heterodimerizer system to function with one class of non-immunosuppressive analogs. A mutant FRAP domain is identified that binds to an analog called Ma-rap that has greatly reduced affinity for wild type FRAP. The utility of the modified system for controlling the subcellular localization of a protein and the activity of a transcription factor is demonstrated.
• Rossi, F., Charlton, C.A., and Blau, H.M. (1997) Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation . Proc Natl Acad Sci USA 94:8405-10.
  Uses rapamycin to heterodimerize two complementing beta-galactosidase deletion mutants to test a novel system for detecting protein complexes within cells.

1996

• Belshaw, P.J., Ho, S.N., Crabtree, G.R., and Schreiber, S.L. (1996) Controlling protein association and subcellular localization with a synthetic ligand that induces heterodimerization of proteins. Proc Natl Acad Sci USA 93:4604-7.
  Uses an FKBP-cyclophilin heterodimerizer (FKCsA) to alter the subcellular localization of a fusion protein or control the activity of a transcription factor.
• Licitra, E.J., and Liu, J.O. (1996) A three-hybrid system for detecting small ligand-protein receptor interactions. Proc Natl Acad Sci USA 93:12817-21.
  Describes the use of the chemical dimerizer concept to develop a "three-hybrid" system for identifying or characterizing novel small molecule-protein interactions. In this proof-of-concept, a heterodimerizer comprising FK506 and dexamethasone is used to bring together transcription factor fragments fused to a glucocorticoid receptor and FKBP, activating reporter gene expression in yeast. By substituting dexamethasone (or FK506) for a novel ligand, and the glucocorticoid receptor for a cDNA library, this system could be used to discover receptors for novel ligands; or conversely to screen for new ligands to known receptors.