Frizzleds and WNT/β-catenin signaling – The black box of ligand–receptor selectivity, complex stoichiometry and activation kinetics
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 WNT/β-catenin signaling (the outdated nomenclature of “canonical” signaling will herein be referred to as the WNT/β-catenin pathway (MacDonald and He, 2012; Macdonald et al., 2007; Schulte, 2010))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
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