Drosophila frizzled (Dfz1) was originally identified as a causative gene for a mutant with disoriented cuticular hairs and was then cloned and characterized as a seven-transmembrane-type protein similar to G protein-coupled receptors (GPCRs) (Vinson and Adler 1987; Lagerström and Schiöth 2008). The phenotype of the Dfz1 mutant was the aberrant polarization of epithelial cells within the epithelial plane or the dysregulation of planar cell polarity (PCP). Drosophila mutants of dishevelled, Van Gogh (Vang or strabismus), prickle, diego, and starry night (flamingo) show phenotypes similar to the Dfz1 mutant. Dfz1, Dishevelled, Vang, Prickle, Diego and Starry night are characterized as core PCP components (Katoh 2005; Wu and Mlodzik 2009).
Drosophila frizzled-2 (Dfz2) is a Dfz1-related seven-transmembrane protein that was cloned and characterized as a receptor for the secreted glycoprotein Wingless that is involved in the patterning of embryonic segments and their adult derivatives (Bhanot et al. 1996; Zhang and Carthew 1998). Genetic interaction experiments revealed that Dfz2, Wingless, Dishevelled, and Armadillo belong to the WNT group of segment polarity components.
Structural Features of Frizzled Receptors
Frizzled family members share a conserved domain architecture that consist of an N-terminal extracellular cysteine-rich domain (Frizzled-like CRD), seven-transmembrane domains, and the cytoplasmic K-T-X-X-X-W motif (Fig. 1b). Frizzled-like CRD is a WNT-binding domain (Fig. 1c) that is conserved among Frizzled family members, secreted-type WNT antagonist SFRPs, ROR family of WNT coreceptors, and a single transmembrane-type Frizzled-related protein (MFRP). By contrast, the K-T-X-X-X-W motif in the C-terminal cytoplasmic region is a binding motif for the PDZ domain of the Dishevelled family of scaffold proteins (Katoh and Katoh 2007; Dijksterhuis et al. 2014; Wang et al. 2016).
Context-Dependent WNT Signaling Through Frizzled Receptors
Canonical WNT signals induce the assembly of the canonical WNT signalosome, which consist of the WNT ligand, the Frizzled-Dishevelled complex, and the LRP5/6-AXIN complex. Due to its release from the AXIN-APC degradation complex, β-catenin is stabilized for nuclear accumulation and subsequent association with the TCF/LEF family of HMG-box transcription factors (Fig. 2). Because nuclear β-catenin is also associated with the BCL9-PYGO coactivator complex, the SWI/SNF-related chromatin regulator BRG1, the histone acetyl transferase CBP/p300, and the histone methyltransferase MLL, stabilized β-catenin induces the transcriptional activation of TCF/LEF target genes. FZD7, LEF1, DKK1, AXIN2, βTRCP1, MYC, and CCND1 are representative target genes of the canonical WNT signaling cascade (Katoh and Katoh 2007; Klaus and Birchmeier 2008). FZD7 and LEF1 are involved in positive-feedback regulation, whereas DKK1, AXIN2, and βTRCP1 are involved in negative-feedback regulation. Because MYC and CCND1 induce cell-cycle progression, canonical WNT signals promote the self-renewal of stem cells and the proliferation of progenitor or transit-amplifying cells in physiological and pathological situations.
Noncanonical WNT signals are transduced to a variety of signaling cascades, including RhoA-ROCK, RhoB-Rab4, Rac-JNK, aPKC, MAP3K7-NLK, Calcineurin-NFAT, and DAG-PKC branches (Fig. 2).
Noncanonical WNT signals are transduced to RhoA-ROCK, RhoB-Rab4, Rac-JNK, and aPKC signaling cascades in a Dishevelled-dependent and β-catenin-independent manner. Dishevelled-dependent RhoA-ROCK and Rac-JNK signaling branches are involved in PCP or polarized cell movements. DAAM1 and DAAM2 are Formin family proteins that regulate the dynamics of actin filaments, such as nucleation, elongation, and barbed-end capping. DAAM proteins are tethered in the cytoplasm based on intramolecular interactions in the absence of noncanonical WNT signals, whereas DAAM proteins are recruited to the membranous Frizzled-Dishevelled complex based on intermolecular interactions between DAAM and Dishevelled in the presence of noncanonical WNT signals. Because DAAM in the Frizzled-Dishevelled complex preferentially interacts with GTP-bound RhoA, noncanonical WNT signals activate the RhoA-ROCK signaling cascade via Dishevelled and DAAM to regulate actin remodeling during fetal morphogenesis and tumor invasion (Katoh and Katoh 2007; Bengoa-Vergniory and Kypta 2015).
Noncanonical WNT signals activate PLC for the catalysis of phosphatidylinositol diphosphate (PIP2) to inositol triphosphate (IP3) and DAG because Frizzled receptors are GPCR superfamily members (Lagerström and Schiöth 2008; Dijksterhuis et al. 2014). IP3 triggers the release of Ca2+ from the endoplasmic reticulum for the activation of the MAP3K7-NLK and Calcineurin-NFAT signaling cascades, whereas DAG induces PKC activation. MAP3K7 is a mitogen-activated protein kinase kinase kinase (MAP3K), which phosphorylates NLK, IKK, MAP2K3 (MEK3), and MAP2K6 (MEK6). Noncanonical WNT signals induce PLC-mediated Ca2+ release for the activation of MAP3K7, which leads to the phosphorylation and subsequent activation of NLK. NLK then phosphorylates TCF/LEF to inhibit the transcriptional activation of the canonical WNT signaling cascade. Therefore, noncanonical WNT signaling to the MAP3K7-NLK branch switches off the canonical WNT signaling cascade.
Pathophysiology of Frizzled Family Members
The FZD3 and FZD6 genes are human orthologs of Dfz1, whereas the VANGL1 and VANGL2 genes are human homologs of Drosophila Vang. FZD3, FZD6, VANGL1, and VANGL2 are core components of human PCP, as Dfz1 and Vang are core components of Drosophila PCP (Katoh 2005). Because loss-of-function mutations in VANGL1 and VANGL2 occur in patients with neural tube defects (NTD) that are characterized by congenital defects of neural tube closure (De Marco et al. 2014), there has been a search for loss-of-function FZD3/6 mutations or variations in NTD patients. Although single nucleotide polymorphisms (SNPs) of human FZD3 or FZD6 were not associated with NTD in a case-control study, including 338 NTD cases and 338 controls (Wen et al. 2010), the rare FZD6 variations R405Q, R511C/H, and C615X were identified in NTD cases based on a resequencing analysis of 473 NTD cases and 639 controls (De Marco et al. 2014).
The FZD4 gene at human chromosome 11q14.2 (Kirikoshi et al. 1999) is mutated in autosomal-dominant familial exudative vitreoretinopathy (FEVR) and sporadic exudative vitreoretinopathy that display variable clinical manifestations, such as retinal exudates, retinal neovascularization, retinal detachment, vitreous hemorrhage, and total blindness (Musada et al. 2016). M105V, I114S, M157T, D428fs, and K429fs are representative FZD4 mutations in FEVR patients. M105V, I114S, and M157T FZD4 are missense mutations in the extracellular WNT-binding CRD, whereas D428fs and K429fs FZD4 are truncation mutations.
Mutations in the FZD5 gene occur rarely in patients with autosomal-dominant coloboma (Liu et al. 2016). A219fs FZD5 mutant encodes a secreted-type FZD5 protein that blocks both canonical and noncanonical WNT signaling. Because FZD5 is involved in the regulation of progenitor cell expansion during retinal development, the A219fs FZD5 mutation causes eye malformation owing to incomplete fusion of the optic fissure.
FZD7 is expressed in blastocyst, undifferentiated embryonic stem (ES) cells, ES-derived endodermal progenitors, ES-derived neural progenitors, fetal cochlea, adult gastrointestinal tract, and regenerating liver. The expression of FZD7 on mesenchymal stem cells is downregulated during osteogenesis. FZD7 is upregulated in gastric, esophageal, colorectal, and liver cancers. FZD7 is one of the target genes of the canonical WNT signaling cascade as mentioned above. FZD7 transduces WNT3, WNT6, or WNT8B signals to the canonical WNT signaling cascade and also WNT11 or WNT5A signals to the noncanonical WNT signaling cascades. FZD7 orchestrates cellular proliferation, epithelialization, migration, and tissue movement in a context-dependent manner during embryogenesis, adult-tissue homeostasis, and carcinogenesis (Sagara et al. 1998; Vincan and Barker 2008).
The FZD9 gene at human chromosome 7q11.23 was initially cloned and characterized as one of the genes within the region that is commonly deleted in patients with Williams syndrome, which is a neurodevelopmental disorder with dysmorphic facial features, cardiovascular disease, mild mental retardation, and a unique cognitive profile; however, because the FZD9 gene is not always deleted in patients with Williams syndrome, it is believed that FZD9 is not responsible for the major features that are observed in Williams syndrome (Botta et al. 1999).
The FZD10 gene at human chromosome 12q24.33 was cloned and characterized as the last member of the Frizzled family (Koike et al. 1999). FZD10 is upregulated in several types of human cancers, such as cervical cancer, glioblastoma, colorectal cancer, fibrolamellar hepatocellular carcinoma, and synovial sarcoma (Koike et al. 1999; Simon et al. 2015; Nagayama et al. 2005). Anti-FZD10 polyclonal antibody effectively mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against synovial sarcoma cells in a nude mouse xenograft model (Nagayama et al. 2005). Humanized anti-FZD10 monoclonal antibody radiolabelled with 90Y (OTSA101-DTPA-90Y) is in a Phase I clinical trial for the treatment of patients with synovial sarcoma (ClinicalTrials.gov database 2016).
Frizzled family members are atypical GPCRs that function as WNT receptors. Canonical WNT signals are transduced through Frizzled to the TCF/LEF transactivation cascade in a β-catenin-dependent manner to upregulate target genes, such as FZD7, LEF1, DKK1, AXIN2, βTRCP1, MYC, and CCND1. Noncanonical WNT signals are transduced through Frizzled to a variety of downstream cascades in a β-catenin-independent manner. Dishevelled-mediated noncanonical WNT signaling to RhoA-ROCK and Rac-JNK signaling cascades are involved in PCP and cellular migration during embryonic morphogenesis and tumor invasion, whereas PLC-mediated noncanonical WNT signaling to the MAP3K7-NLK signaling cascade is involved in the inhibition of the canonical WNT signaling cascade. Because WNT-Frizzled signaling cascades play key roles in a variety of cellular processes during embryogenesis, adult-tissue homeostasis, and carcinogenesis, Frizzled receptors are utilized as stem-cell markers in the field of regenerative medicine and also as therapeutic targets in the field of clinical oncology.
- ClinicalTrials.gov database. US National Institute of Health. https://clinicaltrials.gov. Accessed 28 Aug 2016.