International Union of Basic and Clinical Pharmacology Review: WNT/Frizzled signalling: receptor–ligand selectivity with focus on FZD-G protein signalling and its physiological relevance: IUPHAR Review 3

Links to online information in IUPHAR-DB and the BPS Concise Guide to PHARMACOLOGY

Introduction

Frizzleds (FZDs) are seven-transmembrane (7TM) domain-spanning cell surface receptors, very much like the classical GPCRs. They are activated by the wingless/int1 (WNT) family of lipoglycoproteins (Schulte, 2010; Willert and Nusse, 2012). Cellular communication mediated by WNTs and FZDs is crucial for proper embryonic development, stem cell differentiation, organogenesis and patterning. Also in the adult organism, stem cells are regulated by WNTs and it is also emerging that WNTs have an important role in the regulation of basic physiology. Furthermore, WNT/FZD signalling plays a crucial role in the adult in the maintenance of tissue homeostasis, regeneration, plasticity and repair (Logan and Nusse, 2004; van Amerongen and Nusse, 2009; Chien et al., 2009). Even though many cellular processes are regulated by WNTs, surprisingly little is known about the underlying mechanisms involving the 10 different mammalian FZDs, their 19 WNT ligands and signal initiation at the level of the cell membrane.

From a pharmacological and mechanistic point of view, class FZD receptors as 7TM receptors are poorly understood. Hardly any quantitative information is available about ligand selectivity or efficacy and the most proximal signalling events induced by activated receptors have not been fully elucidated. The field is hampered by a complete lack of pharmacological compounds that can directly target FZDs, which originates in the long-standing difficulties to obtain recombinant pure and biologically active WNTs. Even though WNT/β-catenin pathway activation can be studied through the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assay called TOPflash assay (Molenaar et al., 1996; Van de Wetering et al., 1996), there is a lack of suitable tools for studying the β-catenin-independent pathways. This methodological imbalance has resulted in a strong bias towards WNT/β-catenin signalling.

The possibility of targeting WNT signalling therapeutically is widely regarded as attractive and molecules such as glycogen synthase kinase 3 (GSK3) inhibitors, tankyrase inhibitors, secreted FZD-related proteins, R-spondin, Dickkopf1 (DKK1), Norrin and molecules targeting the co-receptors of the WNT/FZD pathway are being considered as putative drugs for diseases such as different types of cancer, neurodegenerative diseases and osteoporosis (Logan and Nusse, 2004; Moon et al., 2004; Chien and Moon, 2007). Drug screening employing, for example, TOPflash reporter assays has identified novel and apparently promising compounds but has never identified FZD-targeting molecules. A recent screen with FZD internalization as readout has identified the antihelminthic drug niclosamide, even though the molecular mechanism and direct action through FZD remains elusive (Chen et al., 2009). Thus, more appropriate screening technologies could aid the development of FZD targeting molecules, so providing useful research tools and therapeutics (Koval and Katanaev, 2012).

In this review we aim to summarize the status quo of our knowledge about WNT/FZD selectivity, signalling trafficking and the functionality of class FZDs as GPCRs in vitro and in vivo. The nomenclature used in this review conforms to the BJP's Concise Guide to PHARMACOLOGY (Alexander et al., 2013).

WNT/FZD signalling pathways

So far, the underlying mechanisms and associated components that determine the outcome of WNT-induced FZD activation are poorly understood. First of all, the degree of WNT/FZD binding specificity in mammals is generally unknown and the question of whether certain WNT/FZD combinations can selectively activate certain signalling routes over others has not been systematically addressed. Secondly, coupling selectivity of activated FZDs to downstream signalling pathways remains obscure. There is emerging evidence that FZDs serve to induce complex signalling networks integrating multiple pathways rather than activating a single, linear signalling pathway. Moreover, different WNT signalling branches crosstalk as exemplified by the inhibition of WNT-3A-induced signalling by WNT-5A (Topol et al., 2003; Mikels and Nusse, 2006). Further, the selective recruitment and participation of co-receptors, such as single transmembrane-spanning receptors of the low-density lipoprotein receptor-related protein family (LRP5/6) and receptor TK-like orphan receptor (ROR1/2) and receptor-like TK, can influence the signalling outcome (van Amerongen et al., 2008; Hendrickx and Leyns, 2008). WNTs are also known to bind to transmembrane heparin sulphate proteoglycans such as glypican and syndecan, resulting in the accumulation of WNT molecules at the cell surface and the prevention of aggregate formation in the extracellular environment (Fuerer et al., 2010). The importance of this phenomenon for the WNT/FZD interaction is unclear, and the role of these cofactors and other extracellular matrix components create an additional level of complexity for ligand/receptor binding studies.

To date, several distinct FZD signalling pathways have been identified and can be divided into β-catenin-dependent and β-catenin-independent branches (Macdonald et al., 2007; Semenov et al., 2007; Schulte, 2010). In the β-catenin-dependent pathway, WNT binding to FZD induces FZD to interact with the co-receptor LRP6, or the similar LRP5 (van Amerongen et al., 2008; MacDonald et al., 2009; MacDonald and He, 2012). The formation of a WNT-FZD-LRP5/6 complex and the recruitment of the scaffolding protein Dishevelled (DVL) leads to the co-clustering of these proteins in signalosomes, which in turn triggers LRP6 phosphorylation (Bilic et al., 2007). Subsequently, the activated co-receptor causes a redistribution of axin to the cell membrane and thus an incomplete and dysfunctional destruction complex – composed of GSK3β, axin, adenomatous polyposis coli (APC) and other proteins – in the cytosol. This, in turn, causes β-catenin to accumulate within the cytosol and enter the nucleus, where it regulates TCF/LEF-dependent transcription. Classical WNT/β-catenin target genes include cyclin D1, c-myc, COX-2 and inducible NOS (Ramsay et al., 2003; Barker, 2008; Du et al., 2009; MacDonald et al., 2009). β-Catenin regulation of TCF/LEF transcription factors can be assessed with the TCF luciferase reporter/TOPflash assay (Molenaar et al., 1996; Van de Wetering et al., 1996), a method that is extensively used. The components of the WNT/β-catenin pathway have been and are being mapped in detail (MacDonald et al., 2009). However, the signal initiation events involving FZD-DVL-LRP5/6 complex formation, kinase activation and recruitment, phosphorylation of DVL and inhibition of the destruction complex are not understood at a molecular level. Generally, this pathway is considered to be independent of heterotrimeric G proteins, although accumulating evidence suggests the opposite (Liu et al., 2001; Katanaev et al., 2005; Halleskog and Schulte, 2013a). For example, G_αq and G_αo have been shown to dissociate the axin-GSK3β and axin2-GSK3β complex, respectively (Liu et al., 2005), and G_α12 and G_αs act on axin, which is the key negative regulator of the β-catenin-dependent WNT/FZD signalling pathway (Castellone et al., 2005; Stemmle et al., 2006).

The β-catenin-independent pathways comprise a steadily increasing number of complex signalling routes: FZD/planar cell polarity (PCP), WNT/Ca²⁺, WNT/cAMP, WNT/ROR, WNT/RAP and WNT/RAC and WNT/RHO pathways (Semenov et al., 2007; Schulte, 2010). In fact, these pathways are so diverse that it is a strong oversimplification to name them collectively as ‘non-canonical’ or ‘β-catenin-independent’.

Because detailed information about the individual signalling pathways is available in recent, detailed review articles, we will focus here on what is relevant for the discussion about FZDs as unconventional GPCRs (Macdonald et al., 2007; Semenov et al., 2007; Chien et al., 2009; Schulte, 2010).

The first indications of a G protein-dependent WNT/FZD/Ca²⁺ pathway were obtained using injections of RNA coding for different WNTs and FZDs into Danio rerio and Xenopus laevis embryos. The induction of intracellular calcium release upon XWNT-5A and rat FZD₂ could be mimicked by 5-HT_2C receptor overexpression and inhibited by using agents that inhibit specific G protein subunits such as pertussis toxin (PTX) or overexpression of Gα sequestering βγ subunits (Slusarski et al., 1997a,b1997b). These studies were followed up by experiments addressing WNT-induced PKC membrane recruitment in X. leavis embryos, a pertussis-sensitive response to intracellular Ca²⁺ mobilization (Sheldahl et al., 1999). After having named this WNT-induced and G protein-dependent pathway WNT/Ca²⁺ pathway (Kuhl et al., 2000), PTX-sensitive and rapid Ca²⁺ release upon WNT stimulation has been shown to be functional in several cellular systems (Sheldahl et al., 2003; Kremenevskaja et al., 2005; Dejmek et al., 2006; Ma and Wang, 2006; Halleskog et al., 2012; Halleskog and Schulte, 2013a). In addition to the WNT/Ca²⁺ pathway, other G protein-dependent signalling routes involving classical GPCR-associated second messengers and signalling pathways emerge, such as the WNT/cAMP, WNT/ERK1/2 and inositol phosphate signalling (Saneyoshi et al., 2002; Semenov et al., 2007; Bikkavilli and Malbon, 2009; Hansen et al., 2009; Cervenka et al., 2011; Halleskog et al., 2012; Halleskog and Schulte, 2013a).

In accordance with the ternary complex model known from classical GPCRs (De Lean et al., 1980), which describes the allosteric interaction between heterotrimeric G proteins and agonists mediated by the GPCR, it is natural to assume that FZDs directly interact with heterotrimeric G protein to regulate distinct signalling routes, such as WNT/Ca²⁺ and WNT/cAMP signalling. As a side note, it should be mentioned that the same could be assumed for FZD-β-arrestin interaction, which, however, appeared to be indirectly bridged by the phosphoprotein DVL to mediate agonist-induced internalization (Chen et al., 2003). So far, a growing body of evidence indicates that WNT binding to FZDs induces G protein activation, clearly suggesting that FZDs can interact with heterotrimeric G-proteins. For example, WNTs induce G protein activation by inducing the guanine-nucleotide exchange on Gα_i/o proteins (Katanaev and Buestorf, 2009; Kilander et al., 2011a,b2011b; Koval and Katanaev, 2011; Halleskog et al., 2012). Although these studies provide a direct measure of WNT-induced activation of heterotrimeric G proteins in reconstituted systems, membrane preparations from primary cells and brain tissue, the underlying mechanisms of FZD-G protein coupling remain obscure.

Historically, the WNT/β-catenin, PCP, WNT/RHO and WNT/RAC signalling are seen as heterotrimeric G protein-independent, whereas the WNT/Ca²⁺ branch is recognized as the G protein-dependent WNT pathway. However, over the years several important studies have shown that heterotrimeric G proteins play a more global role in the WNT signalling network including the WNT/β-catenin pathway (Liu et al., 2001; Katanaev et al., 2005; Egger-Adam and Katanaev, 2010). In a recent study from our laboratory using primary microglia cells, we have, for example, shown that PTX does not only block the WNT/Ca²⁺-mediated ERK1/2 phosphorylation but also the parallel WNT/β-catenin signalling, including the WNT-3A-induced phosphorylation of LRP6, the formation of PS-DVL3 and the stabilization of β-catenin (Halleskog and Schulte, 2013a). In addition, when comparing the signalling capacities of several recombinant WNTs in the N13 microglia-like cell line, we observed that only WNT-3A induced WNT/β-catenin signalling, whereas all the WNTs (WNT-3A,-4,-5A,-5B,-7A,-9B) led to the activation of PTX-sensitive heterotrimeric G proteins (Kilander et al., 2011b). These findings show that heterotrimeric G proteins probably have a more central role in the field of WNT signalling than previously appreciated.

Intracellular binding partners for FZDs: heterotrimeric G proteins and DVL

The two most prominent and widely discussed intracellular binding partners for class FZD receptors are the scaffold protein DVL and heterotrimeric G proteins.

In humans, there are three variants of DVL, which consist of 670 (DVL1), 736 (DVL2) or 716 (DVL3) amino acids. The protein has three main functional domains: the N-terminal end of the protein consists of a so-called DIX (Dishevelled and Axin) polymerization domain, at the C-terminal region, there is a DEP (Dishevelled, Egl-10 and Pleckstrin) domain, functionally involved in the regulation of small GTPases. In addition, the protein has a centrally located PDZ (Psd-95/Disc large/ZO-1 homologous) binding domain, which enables interactions with other proteins. DVL is a central scaffold protein localized at the crossroads of several FZD-induced pathways and plays not only an important role in the β-catenin-dependent but also in the independent signalling pathway, such as the WNT/β-catenin, the PCP pathway, the WNT/RAC, the WNT/RHO and possibly also the WNT/Ca²⁺ pathway (Gao and Chen, 2010). DVL exists in multiprotein complexes with punctuate appearance in the cytosol (Schwarz-Romond et al., 2005). These multiprotein complexes can be dissolved, for example, by casein kinase overexpression, by WNT-5A stimulation and by expression of FZD (Cong et al., 2004a; Strutt et al., 2006; Bryja et al., 2007a,b2007b). Co-expression with FZDs recruits DVL to the membrane and so far, it is generally unknown if this interaction is static or dynamic, for example, upon agonist treatment (Cong et al., 2004b).

In addition to DVL, the solid evidence for G protein involvement in WNT/FZD signalling shows that heterotrimeric G proteins – according to the ternary complex model valid for GPCRs – interact directly with FZDs. Agonist-bound GPCRs act as a guanine nucleotide exchange factor for the G proteins inducing the GTP-bound active state (Pfeuffer and Helmreich, 1988; Chung et al., 2011). The G protein heterotrimer consists of three subunits, Gα, Gβ, Gγ. Currently, it is known that there are 16 Gα, 5 Gβ genes and 12 genes encoding Gγ (Downes and Gautam, 1999). Commonly, the G proteins can be divided according to their Gα subunit into four groups: Gα_i, Gα_s, Gα_q and Gα_12/13 (Neubig and Siderovski, 2002). See also Figure 1.

Despite the fact that there is experimental evidence indicating that both DVL and heterotrimeric G proteins bind and signal through class FZD receptors, the exact binding sites have not yet been clearly defined. Thus, it is still unclear whether these two proteins compete for FZD binding or if they actually cooperate. While it is known that DVL-FZD interaction is mainly based on three mechanisms involving (i) the KTxxxW–DVL interaction; (ii) DEP–domain interaction with a bipartite motif in the i3 of FZD; and (iii) DEP-domain-mediated electrostatic interactions with the negatively charged head groups of membrane lipids, we can only extrapolate from what is known for other GPCRs and speculate about FZD-G protein contact surfaces. Because classical GPCRs engage mainly the i3 and the C terminus for G protein interaction (Wess, 1998), it appears that the G protein and DVL contact surfaces on FZDs show substantial overlap.

Mutagenesis has revealed several residues in the intracellular loops and in the C-terminal domain of FZDs that are crucial for FZD signalling. Specifically, the mutation of the highly conserved internal KTxxxW motif after the seventh hydrophobic domain, or single amino acid exchanges in the first (R340A) or the third (L524A) intracellular loops of human FZD₅, completely abolished FZD signalling (Cong et al., 2004b; Schulte and Bryja, 2007). The KTxxxW motif, which is supposedly located two amino acids after the seventh transmembrane domain on the intracellular HELIX 8 (Wang et al., 2013), is essential for the activation of the WNT/β-catenin signalling pathway (Umbhauer et al., 2000). Flanking sequences of the KTxxxW motif significantly influence the affinity of peptides derived from FZD₇ for the PDZ domain of DVL (Punchihewa et al., 2009) suggesting a role in FZD–DVL interaction. Mutation in the KTxxxW motif of Drosophila melanogaster FZD, which blocks DVL binding, results in a protein that is deficient in cell-autonomous planar polarity activity (Wu and Mlodzik, 2008; Wu et al., 2008). Apart from the KTxxxW motif, the C-terminal tail is not well conserved among FZDs, suggesting differences in the pattern of downstream signalling pathways.

Structural features of FZDs and WNTs

Class FZD receptors were separated from the classical class A, B, C family GPCRs due to particular structural and functional features (Foord et al., 2005). Class FZD receptors contain conserved cysteines in the extracellular loops 1 and 2, as is common for GPCRs. These cysteines could engage in stabilizing disulfide bonds. Also, a series of charged residues at the C-and N-terminal ends of intracellular loop 3, which are known to be essential for receptor–G protein coupling, are present in FZD. But class FZD receptors also lack other domains, which are important in many GPCRs. For example, they lack the DRY motif at the C-terminal end of the third transmembrane domain known to be important for G protein coupling and specificity (Schulte, 2010). The recent X-ray structure of the related Smoothened (SMO) indicates clearly that domains common to class A GPCRs are not present in class FZD receptors (Wang et al., 2013). Even though the SMO structure provides an interesting insight into possible mechanisms, more detailed structural and functional analysis is required to understand the functional features of this receptor family. Nevertheless, SMO does signal through G proteins and can couple to them, as demonstrated in biochemical and genetic models (Riobo and Manning, 2007; Philipp and Caron, 2009; Ayers and Therond, 2010). There is strong experimental evidence that the constitutive active state of SMO results in GDP/GTP exchange in heterotrimeric G proteins (Riobo et al., 2006), and genetic experiments indicate that SMO recruits G_αi/o proteins to modulate cAMP levels (Ogden et al., 2008). Furthermore, in a study using micelles, an eighth helix – similar to other GPCRs including SMO – was found in the c-terminal region of FZD₁ (Gayen et al., 2013).

Taken together, these findings indicate that so far very little is known about the exact FZD structure. Nevertheless, it has been possible to crystallize and analyse the mouse FZD8-cysteine-rich domain (CRD) domain together with the secreted CRD antagonist FZD-related protein 3. Thereby, it was shown that the CRDs are ∼160 residue and primarily consist of α-helical proteins (Dann et al., 2001; Janda et al., 2012). In addition, lipid modification of WNTs complicates the expression, purification and crystallization of active WNTs (Hausmann and Basler, 2006). However, recently, Janda et al. were able to crystallize a Xenopus WNT-8 (XWNT8) in complex with mouse FZD8-CRD. This study revealed an unusual two-domain WNT structure, not obviously related to known protein folds, and suggests that binding to the FZD8-CRD shields the WNT lipid from aqueous solvent (Janda et al., 2012). These results highlight the presumed selectivity of WNT/FZD.

WNT binding to FZD and physiological relevance

Very few studies provide direct evidence and quantitative measurements of the WNT/FZD interaction. The conventional idea of a ligand-binding assay between hydrophilic small molecule ligands or peptides with their receptors cannot be applied to assess the WNT/FZD interaction quantitatively due to the lipophilic characteristics of the WNT proteins (Willert et al., 2003). Several studies have used isolated FZD-CRDs to measure WNT/FZD selectivity and affinity and have quantified WNT-CRD affinities in the lower nanomolar range (Kawasaki et al., 2007; Carmon and Loose, 2008; Sato et al., 2010). In the following paragraphs we attempt to provide a WNT/FZD binding profile based on what is known about the interactions between the ten mammalian FZD isoforms and the 19 WNTs in the context of their physiological relevance (mammalian vs. more primitive organisms). Figure 2 lists putative WNT/FZD combinations based either on direct binding experiments determined by immunoprecipitation or similar experiments that allow conclusions on protein–protein interaction, such as mass spectrometry, functional testing and signalling outcome. However, immunoprecipitation in this case is the most powerful and direct evidence for a receptor–ligand interaction, depending on the experimental conditions.

Evidence for the physiological relevance of FZD-G protein signalling

As pointed out in the previous sections, evidence exists on WNT/FZD selectivity but this has obviously not been systematically investigated, partly due to the difficulties in obtaining recombinant, pure and active WNTs (Willert et al., 2003; Schulte et al., 2005; Willert, 2008; Willert and Nusse, 2012). With increasing knowledge about the underlying signalling mechanisms and the refinement of methodologies to quantify pathway activation, the structure–function relationship between the 19 WNTs and 10 FZDs can be investigated in more detail and questions regarding β-catenin-dependent and-independent signalling and its association with FZD-induced activation of heterotrimeric G proteins can be studied in more depth.

As early as 1999, the first proof was obtained that WNT/β-catenin signalling is inhibited in F9 teratocarcinoma cells after inhibition of Gα₁ and Gα_o via PTX (Liu et al., 1999). In parallel, biochemical evidence emerged supporting the hypothesis that FZDs that mediate β-catenin stabilization are pre-coupled to Gα_o (Liu et al., 2005). As mentioned earlier, studies of β-catenin-independent WNT pathways have also provided evidence for G protein signalling linked with FZDs. Studies with zebrafish embryos showed that inhibition of G proteins blocked the changes in Ca²⁺ influx, which occur in response to WNT-5A and FZD₂ (Slusarski et al., 1997a,b1997b). Further, Katanaev et al. showed in vivo that FZDs act as guanine nucleotide exchange factors of PTX-sensitive G proteins mediating both WNT/β-catenin and PCP signals in Drosophila melanogaster (Katanaev et al., 2005).

For example, loss of WNT-7A resulted in a reduction in satellite cells in skeletal muscle 3 weeks after injury, demonstrating its physiological role in regulating the homeostatic level of satellite cells (Le Grand et al., 2009; von Maltzahn et al., 2012). In the same study, mass spectrometry and co-immunoprecipitation studies revealed an association between FZD₇ and Gα_s and possibly Gα_o.

Physiologically, the effect of XWNT-11/Xfz7 signalling was shown to regulate convergent extension movements. These movements affected the dorsal marginal zone of gastrulating Xenopus embryos. Interestingly, FZD₇ and the βγ-subunits of heterotrimeric G proteins were required for this process (Penzo-Mendez et al., 2003).

WNT-3A signals through the Gα_q/11 subunits of G proteins to activate phosphatidylinositol signalling and PKCδ in murine ST2 cells. However, direct evidence for any regulation of FZD was not obtained (Tu et al., 2007). Furthermore, PKA/CREB signalling may contribute to other WNT-regulated processes, such as stem cell renewal, cancer and embryonic patterning. The authors also suggested that Gα proteins modulate adenylyl cyclase activity, which may be a mechanism by which WNT proteins, mainly WNT-1 and WNT-7A, signal possibly through class FZD receptors (Chen et al., 2005). Lately, there has been an emerging interest in WNTs and their role in inflammation and immune response including neuroinflammatory diseases (Gattinoni et al., 2010; Miao et al., 2013). For example, it has been suggested that genetic variations within WNT signalling components contribute to the initiation and maturation of prevalent neurological disorders (De Ferrari and Moon, 2006). Studies from our lab characterized WNTs as modulators of the CNS immune response and showed that β-catenin levels are elevated in pro-inflammatory microglia of AD patients (Halleskog et al., 2011). WNT-3A and WNT-5A induce a pro-inflammatory response in primary mouse microglia through an ERK1/2-dependent pathway (Halleskog et al., 2012; Halleskog and Schulte, 2013a). In contrast, after LPS induced pro-inflammatory transformation of microglia, WNT-3A and WNT-5A were found to induce a dose-dependent decrease in the pro-inflammatory marker COX2.

Most interestingly, both the WNT-3A-induced ERK1/2 pathway and the β-catenin signalling require heterotrimeric G proteins (Halleskog and Schulte, 2013b).

When investigating physiological responses of WNT signalling, the possibility of heterotrimeric G protein binding to class FZD receptors is often being overlooked or simply not addressed, possibly due to lack of direct and straightforward methodological readouts.

Today, the TOPflash assay, which is a powerful tool for measuring transcriptional activity of β-catenin levels, has been preferentially used to study activation of the WNT signalling. This assay is also frequently used in the industry to find molecules that will interact with the WNT/FZD signalling pathway.

A direct and less pathway-biased readout system for class FZD receptors would be of great value. Recent data pinpoint the activation of heterotrimeric G proteins as a more general downstream event activated by WNTs (Katanaev and Buestorf, 2009; Kilander et al., 2011a). Using, for example, a [³⁵S]-GTPγS or the non-radioactive alternative europium-labelled GTP analogue (Eu-GTP) assay – frequently used for other GPCRs – as a readout system would potentially improve screening efforts for FZD-targeting drugs (Koval et al., 2010).

Conclusions

The signalling network induced by WNTs acting at class FZD receptors is complex. In order to understand signalling specification, it is crucial to systematically address receptor–ligand interactions and quantify which WNT/FZD combinations activate which downstream events.

The role of FZDs in many types of cancer and other diseases is diverse, rendering class FZD receptors an obvious target for therapeutic intervention (Logan and Nusse, 2004; Janssens et al., 2006; van Amerongen and Nusse, 2009; Chien et al., 2009; King et al., 2012). Small molecular libraries are waiting to be screened for specific interaction with FZDs. Agonists and antagonists targeting FZD as well as positive and negative allosteric modulators would be of great value and could serve as pharmacophores for the development of new drugs (Chien and Moon, 2007; Anastas and Moon, 2013). Nevertheless, suitable readout assays are the key to successful high throughput screening (Koval and Katanaev, 2012). TOPflash assays reporting the transcriptional activity of β-catenin have been of great importance in the understanding of the biological importance of the WNT/β-catenin pathway and have proven partially successful in drug screening as well (Huang et al., 2009). However, additional responses, such as activation of heterotrimeric G proteins or second messengers, which have been used for GPCR screening for decades, could complement the assay repertoire very well (Koval et al., 2011).

The discussion about signal specification also opens up possibilities to employ pathway-selective ligands for putative therapeutic intervention. It might be possible to develop biased ligands that, for example, can block the WNT-β-catenin pathway at the same time as they maintain β-catenin-independent pathways. So far, this is purely hypothetical, but the proof-of-concept for biased drugs with class A GPCRs might be transferrable to class FZD receptors (Whalen et al., 2011).

Conflict of interest

None.