Elsevier

European Journal of Pharmacology

Volume 763, Part B, 15 September 2015, Pages 191-195
European Journal of Pharmacology

Frizzleds and WNT/β-catenin signaling – The black box of ligand–receptor selectivity, complex stoichiometry and activation kinetics

https://doi.org/10.1016/j.ejphar.2015.05.031Get rights and content

Abstract

The lipoglycoproteins of the mammalian WNT family induce β-catenin-dependent signaling through interaction with members of the Class Frizzled receptors and LDL receptor-related protein 5/6 (LRP5/6) albeit with unknown selectivity. The 10 mammalian Frizzleds (FZDs) are seven transmembrane (7TM) spanning receptors and have recently been classified as G protein-coupled receptors (GPCRs). This review summarizes the current knowledge about WNT/FZD selectivity and functional selectivity, the role of co-receptors for signal specification, the formation of receptor complexes as well as the kinetics and mechanisms of signal initiation with focus on WNT/β-catenin signaling. In order to exploit the true therapeutic potential of WNT/FZD signaling to treat human disease, it is clear that substantial progress in the understanding of receptor complex formation and signal specification has to precede a mechanism-based drug design targeting WNT receptors.

Introduction

The Class Frizzled receptors consisting of Frizzled 1–10 (FZD1–10) and SMO belong to the superfamily of seven transmembrane (7TM) spanning or G protein-coupled receptors (GPCRs) (Dijksterhuis et al., 2013, Foord et al., 2005, Schulte, 2010). FZDs are bound and activated by several different ligands, among which the WNT lipoglycoproteins (Willert and Nusse, 2012) and Norrin are the most important (Ye et al., 2010). In mammals, there are 19 different WNTs and due to difficulties with WNT purification, maintenance of their biological activity and suitable assay systems, the WNT–FZD selectivity remains obscure and is an intense matter of investigation (Dijksterhuis et al., 2013, Willert and Nusse, 2012, Willert, 2008). Another FZD ligand that is unrelated to WNTs, is Norrin, which selectively acts through FZD4 (Ye et al., 2010). Historically, research into WNT/FZD signaling was largely centered on the transcriptional regulator β-catenin. This review summarizes the current knowledge about the factors that specify ligand-induced and FZD-mediated initiation of the WNT/β-catenin pathway without much emphasis on β-catenin-independent signaling routes. WNT/β-catenin signaling (the outdated nomenclature of “canonical” signaling will herein be referred to as the WNT/β-catenin pathway (MacDonald and He, 2012; Macdonald et al., 2007; Schulte, 2010)) is initiated by ligand binding to the FZD and recruitment of the co-receptor LRP5/6 in an oligomeric complex in so-called signalosomes (Bilic et al., 2007). Inside the cell, a complex cascade involving key players such as casein kinase 1, axin, the phosphoprotein Disheveled (DVL) and glycogen synthase kinase 3, leads to the cytosolic stabilization of the transcriptional regulator β-catenin, its nuclear translocation and the activation of TCF/LEF-mediated gene transcription (Clevers and Nusse, 2012, MacDonald and He, 2012). Despite extensive efforts during the last 30 years, it is surprising that our understanding of this physiologically and pathophysiologically central pathway still contains so many gaps. These gaps include WNT–FZD selectivity, receptor complex stoichiometry, the kinetics of receptor complex formation, signal initiation and transduction and the molecular details of signal initiation. One example is the activation mechanisms of casein kinases responsible e.g. for WNT-induced phosphorylation of LRP5/6 and the phosphoprotein DVL.

Section snippets

Monomeric, dimeric, heterodimeric ternary complexes in signalosomes

The simplistic view of the receptor complexes initiating the WNT/β-catenin pathway involves a WNT-bound FZD that mediates recruitment of LRP5/6 to an oligomeric complex (Cong et al., 2004) through WNT-selective binding sites on the extracellular epidermal growth factor (EGF) repeats of LRP5/6 (Bourhis et al., 2010). From early data, we know that certain WNTs show a tendency to activate β-catenin-dependent over β-catenin-independent signaling pathways (Shimizu et al., 1997) and deeper structural

Required transmembrane components as cofactors (TSPAN12, GPR124)

Adding to the complexity of our current view, additional transmembrane proteins have recently been implicated in the WNT/LRP5/6 receptor complex by aiding or fine-tuning the initiation of WNT/β-catenin signals. Interestingly, the tetraspanin 12 protein (TSPAN12), expressed in the retinal vasculature, phenocopies the depletion of FZD4, LRP5 and Norrin (Junge et al., 2009). As with FZD4 and LRP5, several TSPAN12 mutations are associated with familial exudative vitreoretinopathy (Gal et al., 2014,

Evidence for the involvement of heterotrimeric G proteins in WNT/β-catenin signaling

Historically, the WNT/β-catenin pathway – in contrast to the β-catenin-independent WNT/Ca2+ pathway – is seen to be independent of heterotrimeric G proteins (Clevers and Nusse, 2012, Kuhl et al., 2000, Slusarski et al., 1997), even though substantial evidence for the involvement of heterotrimeric G proteins in WNT/β-catenin signaling has been presented over the years both in cells and living organisms (Egger-Adam and Katanaev, 2010, Halleskog and Schulte, 2013, Katanaev and Buestorf, 2009,

Combined or in parallel? – a compound response defined by different receptor complexes

The general perception of WNT-induced β-catenin signaling is that of a rather linear pathway from the cell membrane to transcriptional regulation (Clevers and Nusse, 2012, Macdonald et al., 2007). However, strong evidence is accumulating indicating that the WNTs that are seen as strong activators of the WNT/β-catenin pathway, such as WNT-3A, also induce β-catenin-independent signaling pathways either solely or in parallel to a WNT/β-catenin input (Bikkavilli et al., 2008a, Bikkavilli et al.,

Signaling kinetics – β-catenin signaling and other pathways

Equaling in importance to the mechanisms of signal initiation by WNTs through various cell surface receptors, the kinetics of WNT signaling are very poorly understood. When it comes to the transcriptional regulation by β-catenin, the time frame of endpoint readouts (phosphorylation of LRP6, PS-DVL formation, β-catenin stabilization, TOPflash, morphological changes, proliferation etc) range commonly from 30 min to 24 h or longer (Bryja et al., 2007a, Bryja et al., 2007b, Liu et al., 2005, Willert

Promiscuity vs specificity – WNT/FZD (functional) selectivity

As of late, the field of pharmacology has seen the development of novel concepts such as functional selectivity of different ligands through the same receptor, also referred to as signaling bias, pluridimensional efficacy or functional selectivity (Kenakin, 2011, Stallaert et al., 2011). This particular concept has been developed while studying classical GPCRs, but recent data suggest that also the WNT/FZD system could employ the concept of functional selectivity to mediate intracellular

Conclusions

In summary, I have aimed to provide a glimpse of the current knowledge and pinpoint gaps in our understanding of WNT signal initiation that – from the view point of a receptor pharmacologist – require intense research efforts in order to shed some light on the nature of WNT receptor complexes and proximal steps of signal initiation. Given the high therapeutic potential of WNT receptors in disease as diverse as cancer, neurological disorders, bone disease, degenerative disease and immunological

Acknowledgments

Members of my research team are acknowledged for constant inspiration and discussions. I especially thank Shane C. Wright for constructive comments on the manuscript. Funding in my research group comes from Karolinska Institutet, the Swedish Research Council (K2008-68P-20810-01-4, K2008-333 68X-20805-01-4, K2012-67X-20805-05-3), the Swedish Cancer Society (CAN 2008/539, 2011/690, 2014/659), the Knut and Alice Wallenberg Foundation (KAW2008.0149), Engkvist Foundations, the Czech Science

References (69)

  • M. Kuhl et al.

    The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape

    Trends Genet.

    (2000)
  • X. Liu et al.

    Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins

    Curr. Biol.

    (2005)
  • I. Samarzija et al.

    Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

    Biochem. Biophys. Res. Commun.

    (2009)
  • G. Schulte et al.

    The Frizzled family of unconventional G-protein-coupled receptors

    Trends Pharmacol. Sci.

    (2007)
  • X. Ye et al.

    The Norrin/Frizzled 4 signaling pathway in retinal vascular development and disease

    Trends Mol. Med.

    (2010)
  • Y. Zhou et al.

    Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling

    Dev. Cell

    (2014)
  • R.K. Bikkavilli et al.

    G alpha(o) mediates WNT–JNK signaling through Dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

    J. Cell Sci.

    (2008)
  • R.K. Bikkavilli et al.

    p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta

    J. Cell Sci.

    (2008)
  • J. Bilic et al.

    Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation

    Science

    (2007)
  • V. Bryja et al.

    beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo

    Proc. Natl. Acad. Sci. USA

    (2007)
  • V. Bryja et al.

    Beta-arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways

    EMBO Rep.

    (2008)
  • C. Carron et al.

    Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway

    J. Cell Sci.

    (2003)
  • G. Civenni et al.

    Wnt1 and Wnt5a induce cyclin D1 expression through ErbB1 transactivation in HC11 mammary epithelial cells

    EMBO Rep.

    (2003)
  • F. Cong et al.

    Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors, frizzled and LRP

    Development

    (2004)
  • C.E. Dann et al.

    Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains

    Nature

    (2001)
  • J.P. Dijksterhuis et al.

    Systematic mapping of WNT-frizzled interactions reveals functional selectivity by distinct WNT–frizzled pairs

    J Biol Chem

    (2015)
  • J.P. Dijksterhuis et al.

    WNT/frizzled signaling: receptor–ligand selectivity with focus on FZD-G protein signaling and its physiological relevance

    Br. J. Pharmacol.

    (2013)
  • D. Egger-Adam et al.

    Trimeric G protein-dependent signaling by Frizzled receptors in animal development

    Front. Biosci.

    (2008)
  • D. Egger-Adam et al.

    The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

    Dev. Dyn.

    (2010)
  • S.M. Foord et al.

    International union of pharmacology. XLVI. G protein-coupled receptor list

    Pharmacol. Rev.

    (2005)
  • M. Gal et al.

    Novel mutation in TSPAN12 leads to autosomal recessive inheritance of congenital vitreoretinal disease with intra-familial phenotypic variability

    Am. J. Med. Genet. Part A

    (2014)
  • C. Halleskog et al.

    WNT signaling in activated microglia is pro-inflammatory

    Glia

    (2011)
  • N.C. Inestrosa et al.

    Emerging roles of Wnts in the adult nervous system

    Nat. Rev. Neurosci.

    (2010)
  • C.Y. Janda et al.

    Structural basis of Wnt recognition by frizzled

    Science

    (2012)
  • Cited by (37)

    • The Pharmacology of WNT Signaling

      2022, Comprehensive Pharmacology
    • Targeting leukemia stem cells in T-cell acute lymphoblastic leukemia (T-ALL)

      2020, Biological Mechanisms and the Advancing Approaches to Overcoming Cancer Drug Resistance
    • The Wnt/β-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation

      2019, Colloids and Surfaces B: Biointerfaces
      Citation Excerpt :

      WNT proteins act through Frizzled (FZD) receptors, which transduce the signal through either the canonical β-catenin pathway or the non-canonical pathway [4]. Canonical signaling is referred to as the Wnt/β-catenin pathway and consists of a heterotrimeric complex composed of WNT ligands, low-density lipoprotein receptor-related protein transmembrane coreceptors (LRP), and signaling transmembrane receptors FZD [5–7]. This pathway is initiated when a ligand binds to the FZD and recruits the coreceptor LRP5/6 in an oligomeric complex called signalosome [8].

    • Molecular signaling in bone cells: Regulation of cell differentiation and survival

      2019, Advances in Protein Chemistry and Structural Biology
      Citation Excerpt :

      Wnts are a family of secrete glycoproteins originally described in Drosophila, as the Wingless (Wg) gene that is involved in wing development and in mice, as the Int-1 gene that is involved in breast cancer development (Nusse et al., 1991; Nusse & Varmus, 1992). Wnts induce intracellular signaling by binding to members of the frizzled (FZD) family of receptors, which comprise 10 members (Schulte, 2015). FZDs are seven-transmembrane proteins, with a structure similar to G protein-coupled receptors.

    View all citing articles on Scopus
    View full text