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

Abstract

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...

WNT7A Regulates Tumor Growth and Progression in Ovarian Cancer through the WNT/β-Catenin Pathway

“…Because WNT7A stimulates cell growth outside of the ovary (18–20), we first assessed cell proliferation either by counting total cell numbers at 24, 48 and 72 h, or by BrdU incorporation at 48 h (Fig. 2b).…”

“…Further, stimulated Top-Flash reporter activity by WNT7A and FZD5 was inhibited by co-transfection with SFRP2, a WNT receptor antagonist, or dnTCF4 in SKOV3 and HEY cells (P<0.01). We also co-transfected with WNT7A and FZD10, because Carmon et al showed that WNT7A signaling was mediated by FZD5 and FZD10 in endometrial cancer cells (20, 21). However, we did not see any significant Top-Flash activity when cells were co-transfected with FZD10 (data not shown).…”

“…Further, WNT7A increases cell proliferation via activation of the canonical WNT/β-catenin pathway (18, 19). In endometrial carcinomas, WNT7A acting via β-catenin signaling induces cell proliferation (20), where SFRP4 (a WNT receptor antagonist) suppresses the activity of WNT7A leading to inhibition of cell division (21). While WNT7A dependent regulation of cell proliferation and adhesion in other tissues and tumors has been reported, the role of WNT7A in ovarian cancer has not been examined due to the absence of WNT7A in normal ovary.…”

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

“…Differential expression of WNT7A can be explained as due the fact that WNT7A can induce both the canonical and the noncanonical WNT pathway, depending on the receptor to which it binds: the former mediated by FZD5 [40], and the latter, by binding FZD9 [41] or FZD10 [42].…”

Wnt pathway genes in osteoporosis and osteoarthritis: differential expression and genetic association study

“…In the present study, we found upregulation of the FZD5 gene in OA. FZD5 binds WNT7A and WNT5B and may have a more important role in the non-canonical than in the canonical pathway [39,40]. The secreted frizzled-related proteins (SFRPs) bind Wnt ligands.…”