Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals

Luker, Gary D.; Smith, Madeline; Luker, Gary D.; Gammon, Seth T.; Piwnica‐Worms, Helen; Piwnica‐Worms, David

doi:10.1073/pnas.0404041101

Cited by 398 publications

(413 citation statements)

References 39 publications

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“…The split protein complementation assay technique has been previously described to detect a variety of different protein-protein interactions by using split proteins derived from dihydrofolate reductase, β-lactamase, Renilla and firefly luciferases, and the GFP and YFP reporters [27][28][29][30][31][32][33][34]. However, this technique has never been applied to proteins in the Wnt signaling pathway.…”

Section: An Egfp Complementation Assay For Detection Of Wnt Signalingmentioning

confidence: 99%

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

Carmon

Loose

2008

Biochemical and Biophysical Research Communications

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Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, diiferentiation, and cell fate. In the β-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of β-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the β-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain of Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling. KeywordsWnt signaling; Wnt7a; Fzd5; split eGFP; protein complementationThe β-catenin/canonical Wnt pathway defines a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled (Fzd) family. Currently, 19 Wnt proteins and 10 different Fzd members have been identified in the human genome[1;2]. The Wnt-Fzd interaction promotes complex formation with a membrane-associated Lrp 5/6 co-receptor and activates signaling through the direct recruitment of a Dishevelled (Dsh) protein to the intracellular portion of Fzd[3;4]. Dsh inhibits GSK-3 association with the Axin/APC complex of proteins that normally mark β-catenin for proteolytic degradation [5]. Intracellular pools of cytoplasmic β-catenin become stabilized, enter into the nucleus, and interact with the TCF/ LEF family of transcription factors to promote specific gene expression [6;7]. The Wnt-Fzd interaction has also been shown to activate two distinct non-canonical pathways; the planar cell polarity/JNK pathway and a Ca 2+ -mediated pathway[2;8;9].Wnt was initially discovered as a proto-oncogene in mammary tumors activated by integration of the mouse mammary tumor virus [10]. More recently, studies have linked the β-catenin/ canonical Wnt signaling pathway to disease onset and tumorogenesis [11][12][13]. The development of cancer has been attributed to mutations resulting in constitutive activation of *Corresponding Author: Phone: 713-500-7440, Fax: 713-500-7455, David.S.Loose@uth.tmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Me...

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Section: An Egfp Complementation Assay For Detection Of Wnt Signalingmentioning

confidence: 99%

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

Carmon

Loose

2008

Biochemical and Biophysical Research Communications

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“…Therefore, we first set out to discover appropriate split‐luciferase fragments that can be bacterially expressed and purified without large stabilizing fusion proteins. Reported examples of split‐firefly luciferase pairs (Fluc) were used as a starting point to explore three N ‐ and C ‐terminal fragments Fluc(1–416)/(398–550),29 Fluc(1–437)/(438–550),30 and Fluc(1–475)/(265–550) (Table S1) 31. First, these fragment pairs were linked via a flexible (GGS) 12 amino acid linker.…”

mentioning

confidence: 99%

Supramolecular Control over Split‐Luciferase Complementation

et al. 2016

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Supramolecular split‐enzyme complementation restores enzymatic activity and allows for on–off switching. Split‐luciferase fragment pairs were provided with an N‐terminal FGG sequence and screened for complementation through host‐guest binding to cucurbit[8]uril (Q8). Split‐luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20‐fold upregulation of luciferase activity. Supramolecular split‐luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak‐binding FGG peptide revealed a 300‐fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks.

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“…47 Although the FDG-PET methodology and criteria for tumor responses varied in different studies, changes in FDG uptake by tumors after the first chemotherapy cycle correlate significantly with patient survival in these various studies. 46 These data suggest that FDG-PET might become an early indicator for treatment efficacy in clinical studies. 47 The National Cancer Institute has recently published guidelines for conducting clinical trials with use of FDG-PET to monitor therapies.…”

Section: Assessment Of Tumor Response To Therapymentioning

confidence: 94%

“…Rapamycininduced formation of the FRB and FKB12A complex restores the enzymatic function of luciferase; following injection of luciferin, light emission can be monitored noninvasively by optical imaging. 45,46 Although studies involving genetically encoded reporters cannot be used in the clinical setting, they can provide fundamental information on the ability of new drugs to target protein-protein interactions in animal models. These reporters might be used for high-throughput screening of drugs in cell culture assays.…”

Section: Measuring Target Inhibitionmentioning

confidence: 99%

Technology Insight: novel imaging of molecular targets is an emerging area crucial to the development of targeted drugs

et al. 2008

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SUMMARYTargeted drugs hold great promise for the treatment of malignant tumors; however, there are several challenges for efficient evaluation of these drugs in preclinical and clinical studies. These challenges include identifying the 'correct', biologically active concentration and dose schedule, selecting the patients likely to benefit from treatment, monitoring inhibition of the target protein or pathway, and assessing the response of the tumor to therapy. Although anatomic imaging will remain important, molecular imaging provides several new opportunities to make the process of drug development more efficient. Various techniques for molecular imaging that enable noninvasive and quantitative imaging are now available in the preclinical and clinical settings, to aid development and evaluation of new drugs for the treatment of cancer. In this Review, we discuss the integration of molecular imaging into the process of drug development and how molecular imaging can address key questions in the preclinical and clinical evaluation of new targeted drugs. Examples include imaging of the expression and inhibition of drug targets, noninvasive tissue pharmacokinetics, and early assessment of the tumor response.

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Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals

Cited by 398 publications

References 39 publications

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

Supramolecular Control over Split‐Luciferase Complementation

Technology Insight: novel imaging of molecular targets is an emerging area crucial to the development of targeted drugs

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