ZNRF3/RNF43 E a Direct Linkage of Extracellular Recognition and E3 Ligase Activity to Modulate Cell Surface Signalling

Progress in Biophysics and Molecular Biology 118 (2015) 112e118

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Progress in Biophysics and Molecular Biology

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Review ZNRF3/RNF43 e A direct linkage of extracellular recognition and E3 ligase activity to modulate cell surface signalling

* Matthias Zebisch a, E. Yvonne Jones b, a Evotec (UK) Ltd, 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, United Kingdom b Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom article info abstract

Article history: The interactions of extracellular ligands with single membrane spanning receptors, such as kinases, Available online 1 May 2015 typically serve to agonise or antagonise the intracellular activation of signalling pathways. Within the cell, E3 ligases can act to alter the localisation and activity of proteins involved in signalling systems. Keywords: Structural and functional characterisation of two closely related single membrane spanning molecules, Transmembrane E3 RING ligase RNF43 and ZNRF3, has recently revealed the receptor-like functionalities of a ligand-binding ectodomain Wnt signalling combined with the intracellular architecture and activity of an E3 ligase. This direct link provides a Rspondin hereto novel mechanism for extracellular control of ubiquitin ligase activity that is used for the modu- LGR4/5/6 lation of Wnt signalling, a pathway of major importance in embryogenesis, stem cell biology and cancer. In this review we discuss recent ﬁndings for the structure and interactions of the extracellular region of RNF43/ZNRF3 and draw parallels with the properties and function of signalling receptor ectodomains. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction ...... 112 2. The extracellular architecture of ZNRF3/RNF43 ...... 113 3. The oligomeric state of the ZNRF3/RNF43 ectodomain ...... 114 4. Ligand recognition by the ZNRF3/RNF43 ectodomain ...... 114 5. ZNRF3/RNF43 ectodomain interactions in ternary complexes ...... 116 6. Conclusions and questions ...... 117 Acknowledgements ...... 117 References ...... 117

1. Introduction whilst others comprise a single protein (Metzger et al., 2012). ZNRF3 and RNF43 resemble a type I transmembrane receptor with ZNRF3 and RNF43 belong to a small set of proteins (members of an N-terminal region followed by a single membrane spanning the Goliath and Godzilla families) which bear the hallmark se- region, but their C-terminal region comprises the RING-type E3 quences of an ubiquitin ligase and a transmembrane region (de Lau ligase domain rather than a typical ‘signalling’-type domain. These et al., 2014). This is an unusual combination. E3 ubiquitin ligases distinctive proteins were ‘orphan’ ubiquitin ligases of unknown function to target the substrate protein for ubiquitin modiﬁcation. function until 2012 when they became a focus of particular interest RING-type E3 ligases constitute a particularly large and varied to researchers working on the control of the Wnt signalling system family, some members are huge multi-component complexes (Koo et al., 2012; Hao et al., 2012). The Wnt family of extracellular ligands trigger the canonical Wnt/b-catenin signalling pathway through interaction with mem-

* Corresponding author. bers of the Frizzled family of seven transmembrane domain (7TM) E-mail address: [email protected] (E.Y. Jones). receptors and a co-receptor, low-density-lipoprotein-receptor- http://dx.doi.org/10.1016/j.pbiomolbio.2015.04.006 0079-6107/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118 113

Fig. 1. Function of PA-TM-RING E3 ligases ZNRF3 and RNF43 in Wnt signalling. (a) To transmit canonical signals, Wnt proteins need to bind to their two cognate receptors Frizzled (denoted as Fz) and LRP5/6. ZNRF3 and RNF43 are Wnt-feedback induced cell surface E3 ligases, which appear to speciﬁcally target Frizzled receptors for degradation. This limits the cellular Wnt response. (b) In the presence of Rspondin ligands and LGR receptors a ternary complex is formed and rapidly endocytosed. This relieves the E3 feedback inhibition of Wnt allowing a much higher Wnt response.

related protein 5 or 6 (LRP5/6) (Macdonald et al., 2009; Clevers and the characteristics of the ZNRF3/RNF43 ectodomain as revealed by Nusse, 2012). Wnt signalling plays a central role in tissue homeo- multiple crystal structures in unliganded and liganded states (Chen stasis and stem cell maintenance in the adult as well as in em- et al., 2013; Zebisch et al., 2013; Peng et al., 2013b). We discuss bryonic development (Clevers and Nusse, 2012). It is subject to properties common to other examples of this domain architecture exquisite control by a particularly varied set of modulation mech- and highlight distinctive features. In particular, we focus on the anisms (Niehrs, 2012; Malinauskas and Jones, 2014). In addition to atomic level determinants of ZNRF3/RNF43 binding to the Rspon- extracellular inhibitor binding to either the Wnt ligand or its re- din family of ligands (Chen et al., 2013; Zebisch et al., 2013; Peng ceptors, these mechanisms include the actions of a membrane- et al., 2013b). We also review current structural and biophysical bound protease (Tiki) that specifically cleaves the amino-terminal evidence that points to a propensity of the ZNRF3, but not the region of Wnts (Zhang et al., 2012), and of Notum, a novel extra- RNF43, ectodomain to oligomerise (Zebisch et al., 2013; Peng et al., cellular protein deacylase that removes a distinctive palmitoleoyl 2013b), an intriguing difference of unknown physiological moiety from Wnts that is essential for their ligand function relevance. (Kakugawa et al., 2015; Zhang et al., 2015). ZNRF3/RNF43 adds an additional mode of action to this broad range of mechanisms; 2. The extracellular architecture of ZNRF3/RNF43 furthermore this mode of action is itself subject to modulation. ZNRF3/RNF43 acts on members of the Frizzled family of Wnt Several contemporaneous crystal structure determinations receptors, mediating the ubiquitination of specific lysines in the revealed the three dimensional details of the ZNRF3/RNF43 extra- cytoplasmic loops of these 7TM integral membrane proteins (Fig. 1) cellular region (Chen et al., 2013; Zebisch et al., 2013; Peng et al., (Koo et al., 2012; Hao et al., 2012). The ubiquitination results in 2013b). The ectodomain comprises a single globular domain of removal of Wnt receptors from the cell surface and consequent ~150 amino acids, which has at its core a relatively compact, albeit down regulation of Wnt signalling. This functionality is conserved distorted, b-sandwich type structure (Fig. 2a). The two b-sheets are from Caenorhabditis elegans (ZNRF3/RNF43 homolog PLR-1) to formed by four strands (b2, b1, b7, b3) and three strands (b4, b5, b6) human (Moffat et al., 2014). Unsurprisingly, the type I trans- respectively. The b-sandwich is splayed apart to incorporate an a- membrane architecture, with cell surface exposed ectodomain, helix and has two additional a-helices packed against the outer face appears central to the substrate specificity of these E3 ligases. Co- of the b4, b5, b6 sheet. A single disulphide bridge links the b3eb4 immunoprecipitation data point to ZNRF3 forming a specific and b4eaA loops. Comparisons of ZNRF3 structures determined in complex with Frizzled and LRP5/6 receptors (Hao et al., 2012) and different crystal lattices indicate that a part of the b3eb4 loop the extracellular cysteine-rich domain of Frizzled and the PLR-1 harbouring predominantly acidic residues, the b4eaA and aCeb7 ectodomain have both been shown to be necessary for the ubiq- loops, and the long b1eb2 hairpin are the most flexible compo- uitin ligase function in the C. elegans system (Moffat et al., 2014). In nents of an otherwise rather rigid domain (Fig. 2b) (Zebisch et al., vertebrates, the receptor-like structure of ZNRF3 and RNF43 also 2013). The single most striking feature of the ZNRF3/RNF43 fold provides a mechanism for the modulation of their activity. ZNRF3/ is the b1eb2 hairpin, which extends beyond the main globular core. RNF43 can itself be targeted for removal from the cell surface by the The topology of the ZNRF3/RNF43 domain is a variant of the binding of a ligand, Rspondin, to its ectodomain. Rspondin binding protease-associated (PA) domain fold. This fold is so named because results in formation of a ternary complex involving leucine-rich sequence analysis (Mahon and Bateman, 2000) showed that it oc- repeat containing G protein-coupled receptor 4, 5 or 6 (LGR4/5/ curs, as an insert, in various protease families. In proteases the PA 6), Rspondin and ZNRF3/RNF43 that triggers membrane clearance fold contributes to specificity rather than catalytic activity of the E3 ligase (Fig. 1)(Hao et al., 2012). The removal of ZNRF3/ (Bruinenberg et al., 1994). The signature sequence of the PA fold RNF43 allows Wnt receptors to re-accumulate at the cell surface could also be detected in a variety of receptors involving in protein resulting in enhancement of Wnt signalling. trafficking in plants and animals, for example the plant specific We review here recent structural and biophysical studies of the vacuolar receptor BP-80 and the mammalian transferrin receptor, ZNRF3/RNF43 ectodomain that have provided atomic level insight as well as, intriguingly, in C-RZF, a RING zinc finger containing into the mechanisms controlling this novel E3 ligase. We describe protein (Tranque et al., 1996). Mahon and Bateman predicted that 114 M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118

Fig. 2. Structure and flexibility of the PA domain of PA-TM-RING E3 ligases. (a) Cartoon fold representation of the three available crystal structures coloured from blue (N-terminus) to red (C-terminus). For ZNRF3 b strands are numbered and a helices are labelled from A to C. (b) Tube presentation of the ZNRF3 (apo) PA domain. The tube diameter reflects the average displacement of backbone atoms and hence indicates flexible regions (arrows).

C-RZF is an integral membrane protein comprising N-terminal PA ZNRF3 ectodomain crystallised as a dimer. This finding is paral- domain, transmembrane region (TM) and C-terminal RING domain lelled by results from the Gros laboratory (Peng et al., 2013b). The (Mahon and Bateman, 2000). Subsequently, a number of such PA- ZNRF3 dimer is two-fold symmetric, with a substantial interface TM-RING proteins have been identified. Of the 11 human PA-TM- area of 992 ± 109 Å2 (Zebisch et al., 2013). The two b-sandwich RING family members, several have been implicated in the regu- subunits are aligned in parallel with the core of the dimer interface lation of cellular endosomal trafficking and the PA domain has been formed by a face-to-face interaction between strands b3 and b7 shown to determine their endosomal localisation (van Dijk et al., (Fig. 3a). A second interface is contributed by the extended b1eb2 2014). The most extensively characterised endosomal PA-TM- hairpins which each wrap around the helix aA and b3eb4 loop of RING ligase is GRAIL (gene related to anergy in lymphocytes, also the opposite subunit. In this binding mode the C termini of the two known as RNF128), which has been studied for its role in the subunits are appropriately positioned to attach to the same cell cellular immune system, specifically the control of helper T cell surface, suggestive of ZNRF3 being able to associate physiologically responsiveness (Anandasabapathy et al., 2003; Whiting et al., as a dimer. In contrast, we and others have found that in all crystal 2011). Within the endocytic pathway the luminal PA domain of structures to date RNF43 is monomeric (Chen et al., 2013; Zebisch GRAIL makes specific interactions with a number of trans- et al., 2013). membrane receptors including CD40L and CD83 (co-stimulatory We had not expected to find a dimeric ZNRF3 ectodomain in our molecules required for T-cell activation) to capture them for ubiq- crystal structures, because the protein had not shown any indica- uitination (Lineberry et al., 2008). The crystal structure of the GRAIL tion of dimerisation during gel filtration prior to crystallisation. PA domain has been determined (J.R. Walker and colleagues, However, analysis by analytical ultracentrifugation does provide Structural Genomics Consortium; Protein Data Bank ID code 3ICU). evidence of this molecule's propensity to dimerise in solution, The ectodomains of ZNRF3/RNF43 and GRAIL show only very albeit weakly, and mutagenesis studies have confirmed that this low level similarities in sequence analysis (for example a sequence mode of dimerisation is consistent with the dimer form seen in the identity of 13.4% for 127 residues between ZNRF3 and GRAIL crystal structures (Zebisch et al., 2013). Conversely, we could find (Zebisch et al., 2013), however, a structure based search of the no evidence for even transient RNF43 dimer formation in solution. Protein Data Bank reveals the closeness of the match in terms of Interestingly, the dimerization interface residues are conserved three-dimensional fold (Fig. 2a). Structural superposition of ZNRF3 across species in ZNRF3, but this conservation does not extend to and GRAIL gives a r.m.s.d. of 2.5 Å for 131 equivalent Ca pairs RNF43 (Fig. 3b). Similarly, the crystal structure of the GRAIL PA (Zebisch et al., 2013). Thus the PA fold has been identified as a domain does not show the dimeric arrangement seen for ZNRF3. putative protein recognition domain in a distinctive set of trans- Thus the oligomeric properties of the ZNRF3 PA domain currently membrane ubiquitin ligases which function in the endocytic appear to be rather distinctive. There are numerous examples of pathway and at the cell surface. A flurry of recent studies on ZNRF3/ proteins sharing closely related b-sandwich type folds, but different RNF43 has provided detailed insights into the structural de- oligomeric architectures. For instance, the immunoglobulin (Ig) terminants governing PA domain interactions. fold frequently serves as a protein recognition domain, and has evolved to function as a monomeric domain in some cell surface 3. The oligomeric state of the ZNRF3/RNF43 ectodomain receptors and as a dimeric domain in others (for examples see (Chothia and Jones, 1997). It remains to be seen what the functional Structural studies on ZNRF3 and RNF43 ectodomains revealed consequences of ZNRF3 dimerization might be. very similar three dimensional structures consistent with these two proteins sharing some 39% sequence identity. However, the 4. Ligand recognition by the ZNRF3/RNF43 ectodomain crystal structures of ZNRF3 and RNF43 ectodomains highlight one major difference in their properties; ZNRF3 shows a strong pro- The ZNRF3/RNF43 ectodomain currently appears to be a prime pensity to dimerize whilst RNF43 does not (Zebisch et al., 2013; candidate for a role in the targeting of the Wnt receptor, Frizzled, Peng et al., 2013b). for ubiquitination. The simplest hypothesis is that the PA domain of In our own studies we determined the structure of the ZNRF3 the ZNRF3/RNF43 ectodomain contributes to this targeting by ectodomain in multiple different crystal forms, either as the iso- interaction with the N-terminal cysteine-rich domain of the Friz- lated protein or in complex with Rspondin ligand (Zebisch et al., zled, either directly, or in combination with other complex com- 2013). For most of the unliganded structures, and in all ponents. However, the structural determinants for this putative ZNRF3eRspondin complexes (discussed in section 4), we found the ZNRF3/RNF43 ectodomain function have not been characterised, M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118 115

Fig. 3. Dimer structure of ZNRF3 ectodomain. (a) Cartoon/surface representation for the two chains. Secondary structure elements involved in formation of the dimer interface are indicated. (b) Sequence alignment of selected ZNRF3 and RNF43 family members (PA domain only): human (h), mouse (m), Xenopus (x) and zebraﬁsh (z). Residues involved in ZNRF3 dimerization are highlighted with a triangle and those involved in ligand binding with a full circle. N-glycosylation sites of RNF43 are indicated with a pink hexagon. A single cysteine bridge is conserved between RNF43 and ZNRF3.

and direct binding between ZNRF3 and Frizzled 8 cysteine-rich The Rspondins (a family of four secreted proteins) had for some domain was not detectable in a surface plasmon resonance based time been known to act as secreted agonists of canonical Wnt binding assay (Peng et al., 2013b). In contrast, we and others have signalling in vertebrates, but until recently their mode of action succeeded in mapping the atomic details of the ligandeectodomain remained controversial (de Lau et al., 2014). The identification of interaction that triggers removal of ZNRF3/RNF43 from the cell LGR4, 5 and 6 as high affinity cell surface receptors for Rspondin surface and consequent increase in Wnt signalling. proteins provided the first robust molecular level insight into interactions which mediate Rspondin function (Carmon et al., 2011; 116 M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118

Fig. 4. Complex formation between PA-TM-RING E3 ligases and Rspondins. (a) The structure of a signalling competent fragment of Rspondin 2 revealed a ladder like structure of 6 b-hairpins belonging to the 2 Furin-like repeats. The regions most prominently involved in receptor binding are indicated by arrows. Rspondin 2 contains an exposed Methionine residue in b-hairpin 2 which plugs into a hydrophobic pocket in the surface of ZNRF3 and RNF43 (b, c). (b) 2:2 complex of ZNRF3eRspo2 viewed along the dimer axis (towards the membrane). (c) A similar view for the 1:1 complex of RNF43eRspo2. de Lau et al., 2011; Glinka et al., 2011; Ruffner et al., 2012). Subse- with ligand (Zebisch et al., 2013). Mutagenesis of interface resi- quent studies revealed that Rspondin proteins can also interact dues, in combination with biophysical and cellular assays, has directly with the ZNRF3/RNF43 ectodomain, recruiting the E3 ligase been carried out to confirm the functional relevance of the into a ternary complex with LGR4/5/6 (Hao et al., 2012). The ligandereceptor interaction detailed in the crystal structures available data point to formation of this LGR4/5/ (Zebisch et al., 2013; Peng et al., 2013a, 2013b). Indeed, mutations 6eRspondineZNRF3/RNF43 complex serving as the trigger for implicated in genetic diseases and cancer map to the observed membrane clearance of ZNRF3/RNF43. RspondineZNRF3/RNF43 interface (Chen et al., 2013; Zebisch In 2013 multiple laboratories, including our own, reported et al., 2013; Peng et al., 2013b). structure/function studies of the Rspondin ligands in isolation and The ligand binding surface on ZNRF3 does not impinge on the in complex with their LGR4/5/6 and ZNRF3/RNF43 receptors (Xu dimer interface. Thus, while the overall stoichiometry for all the et al., 2013; Wang et al., 2013; Chen et al., 2013; Zebisch et al., RspondineRNF43 complexes reported to date is 1:1, it is 2:2 for all 2013; Peng et al., 2013a, 2013b). Sequence analyses of the Rspon- the RspondineZNRF3 complexes (Fig. 4b,c). Indeed, analytical ul- dins indicate a three domain architecture comprising two furin-like tracentrifugation measurements provide some indication that cysteine rich regions (Fu domains) plus a thrombospondin type 1 increased ZNRF3 dimerisation occurs in solution on formation of repeat domain. Of these only the two Fu domains are required to the RspondineZNRF3 complex (Zebisch et al., 2013). Structurally enhance canonical Wnt signalling and molecular level studies have there is also some evidence to support the notion that Rspondin focused on this portion of the protein. Crystal structures of binding contributes indirectly to stabilization of the ZNRF3 dimer, Rspondin Fu1eFu2 proteins revealed a ladder-like arrangement of however, the functional relevance of this phenomena remains un- b hairpins, three b hairpins from each Fu domain (Fig. 4). Of these b resolved (Zebisch et al., 2013). hairpins the second in Fu1 is particularly long, a distinctive feature we termed the ‘Met-finger’ since in our Fu1eFu2 crystal structures 5. ZNRF3/RNF43 ectodomain interactions in ternary for Rspondin 2 this b hairpin presents a methionine residue at its complexes tip (Zebisch et al., 2013). Exposed hydrophobic residues are frequently major players in proteineprotein interactions. Indeed, The interaction of Rspondin ligands with the ectodomain of the the Met-finger proved to be a key determinant in the interaction of LGR4/5/6 receptor has been detailed in a number of structural and Rspondin 2 ligand and ZNRF3/RNF43 ectodomain. biophysical studies (Xu et al., 2013; Wang et al., 2013; Chen et al., Multiple structures of Rspondin Fu1eFu2 in complex with 2013; Peng et al., 2013a; Zebisch and Jones, in press). All of the ZNRF3 or RNF43 ectodomain reveal a one-to-one interaction analyses, regardless of the particular Rspondin or LGR involved, between the ZNRF3/RNF43 PA domain and the Rspondin Fu1 report the same 1:1 ligandereceptor interface. This conserved domain (Fig. 4)(Chen et al., 2013; Zebisch et al., 2013; Peng et al., interaction is mediated by the concave surface of the LGR4/5/6 2013b). The interface is extensive, for example for Rspondin 2 ectodomain (a horseshoe-like structure comprising 17 leucine rich interacting with ZNRF3 it involves a discontinuous area of repeats) and in large part the Fu2 domain of the Rspondin. 2 990 ± 105 Å and, as in many ligandereceptor interactions, Although the Rspondin Fu1 domain also contributes to the inter- comprises a mixture of hydrophobic and complementary charged action with LGR4/5/6 the ZNRF3 binding surface is not involved. interactions (Zebisch et al., 2013). A number of the residues Thus, Rspondin appears competent to act as a classical ‘cross- involved in these interactions are conserved across species for linking’ type secreted ligand, able to recruit two different cell sur- ZNRF3 and RNF43 (Fig. 3b). Notably, the second b hairpin of Fu1 face receptors, ZNRF3/RNF43 and LGR4/5/6, into a ternary complex. (the Met-finger) inserts into a hydrophobic pocket situated be- Such ternary complex formation is consistent with the hypothesis tween the b3 strand and the aCeb7 loop of the ZNRF3/RNF43 that the association of LGR4/5/6, Rspondin and ZNRF3/RNF43 ectodomain. A comparison of high resolution ZNRF3 ectodomain provides the trigger for the membrane clearance of ZNRF3/RNF43 structures in isolation and in complex with ligand reveals no and its Frizzled specific E3 ligase activity (Fig. 1)(Hao et al., 2012). major conformational difference, however, the flexibility of the Importantly, the crystal structure of a 1:1:1 complex of b3eb4andaCeb7 loops appears to be reduced on interaction LGR5eRspondineRNF43 has been determined, providing a clear M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118 117

crystallographic analysis of a LGR5eRspondineZNRF3 ternary complex in our laboratory has conﬁrmed the prediction of a ZNRF3 dimer-mediated 2:2:2 assembly (Zebisch and Jones, in press). This striking difference in ternary complex architecture implies that there are functional differences between RNF43 and ZNRF3 awaiting discovery. More generally, the mechanism of action by which ternary complex formation triggers clearance from the cell surface is still obscure. It has been reported to require an active ZNRF3/RNF43 RING domain (Hao et al., 2012), but the consequences of extracellular ternary complex formation for interactions in the cytoplasmic region are not known. Thus, whilst progress in understanding the structure and function of the ZNRF3 and RNF43 E3 ligases has been rapid since 2012 much remains to be learnt.

Acknowledgements

Our studies on the ZNRF3/RNF43 system are funded by Cancer Research UK (to E.Y.J., A10976). M.Z. held an IEF Marie Curie fellowship. The Wellcome Trust Centre for Human Genetics is supported by Wellcome Trust Centre grant 090532/Z/09/Z.

References

Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., Soares, L., 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4þ T cells. Immunity 18, 535e547. Fig. 5. Architecture of the ternary LGR5eRSPO1eRNF43 complex. Rspondin cross- Bruinenberg, P.G., Doesburg, P., Alting, A.C., Exterkate, F.A., de Vos, W.M., Siezen, R.J., links LGR to the PA domain of the E3 ligases. LGR5 does not contact RNF43 directly. 1994. Evidence for a large dispensable segment in the subtilisin-like catalytic domain of the Lactococcus lactis cell-envelope proteinase. Protein Eng. 7, 991e996. Carmon, K.S., Gong, X., Lin, Q., Thomas, A., Liu, Q., 2011. R-spondins function as li- demonstration of the cross-bridge function of Rspondin in action gands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc. Natl. Acad. Sci. U. S. A. 108, 11452e11457. (Fig. 5)(Chen et al., 2013). Chen, P.H., Chen, X., Lin, Z., Fang, D., He, X., 2013. The structural basis of R-spondin The four Rspondins appear to be promiscuous cross-linkers, able recognition by LGR5 and RNF43. Genes Dev. 27, 1345e1350. to bind LGR4, 5 or 6, and to bind both ZNRF3 and RNF43. However, Chothia, C., Jones, E.Y., 1997. The molecular structure of cell adhesion molecules. Annu. Rev. Biochem. 66, 823e862. these ligands do show some differences in their binding properties, Clevers, H., Nusse, R., 2012. Wnt/beta-catenin signaling and disease. Cell 149, resulting in a range of potencies and efficacies (Kim et al., 2008; 1192e1205. Chen et al., 2013; Zebisch et al., 2013; Moad and Pioszak, 2013; de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., e Haegebarth, A., Peters, P.J., van de, W.M., et al., 2011. Lgr5 homologues associate Warner et al., 2015). The Rspondin LGR4/5/6 interaction is uni- with Wnt receptors and mediate R-spondin signalling. Nature 476, 293e297. formly high (nanomolar) affinity, whilst RspondineZNRF3/RNF43 de Lau, W.B.M., Peng, W.C., Gros, P., Clevers, H., 2014. The R-spondin/Lgr5/Rnf43 binding shows a greater range in affinities (for example 25 nM module: regulator of Wnt signal strength. Genes Dev. 28, 305e316. m Glinka, A., Dolde, C., Kirsch, N., Huang, Y.L., Kazanskaya, O., Ingelfinger, D., versus 300 M for mouse Rspondin 2 and 4 binding to ZNRF3, Boutros, M., Cruciat, C.M., Niehrs, C., 2011. LGR4 and LGR5 are R-spondin re- respectively (Zebisch et al., 2013)). This, and other differences in the ceptors mediating Wnt/beta-catenin and Wnt/PCP signalling. EMBO Rep. 12, characteristics of the ligand interactions for this two receptor based 1055e1061. system, has led to the view that LGR4/5/6 acts as the engagement Hao, H.X., Xie, Y., Zhang, Y., Charlat, O., Oster, E., Avello, M., Lei, H., Mickanin, C., Liu, D., Ruffner, H., et al., 2012. ZNRF3 promotes Wnt receptor turnover in an R- receptor, LGR4/5/6 bound Rspondin recruiting ZNRF3/RNF43 into spondin-sensitive manner. Nature 485, 195e200. the ternary complex where it acts as the effector receptor (Chen Kakugawa, S., Langton, P.F., Zebisch, M., Howell, S.A., Chang, T.H., Liu, Y., Feizi, T., et al., 2013; Xie et al., 2013). Bineva, G., O'Reilly, N., Snijders, A.P., et al., 2015. Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187e192. Kim, K.A., Wagle, M., Tran, K., Zhan, X., Dixon, M.A., Liu, S., Gros, D., Korver, W., 6. Conclusions and questions Yonkovich, S., Tomasevic, N., et al., 2008. R-spondin family members regulate the Wnt pathway by a common mechanism. Mol. Biol. Cell 19, 2588e2596. Koo, B.K., Spit, M., Jordens, I., Low, T.Y., Stange, D.E., van de, W.M., Mohammed, S., The distinctive, ligand-binding, ectodomains of the ZNRF3 and Heck, A.J., Maurice, M.M., Clevers, H., 2012. Tumour suppressor RNF43 is a RNF43 E3 ligases prompt interesting comparisons with the prop- stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665e669. erties and function of classical signalling receptor ectodomains. Lineberry, N., Su, L., Soares, L., Fathman, C.G., 2008. The single subunit trans- One recurrent question arising from studies of cell surface signal- membrane E3 ligase gene related to anergy in lymphocytes (GRAIL) captures ling assemblies concerns the role of high order oligomerisation and and then ubiquitinates transmembrane proteins across the cell membrane. e clustering in function. This is a very pertinent question to ask in the J. Biol. Chem. 283, 28497 28505. Macdonald, B.T., Tamai, K., He, X., 2009. Wnt/beta-catenin signaling: components, light of our current knowledge of ZNRF3/RNF43-based complexes. mechanisms, and diseases. Dev. Cell 17, 9e26. Can the 2:2 stoichiometry found for RspondineZNRF3 complexes Mahon, P., Bateman, A., 2000. The PA domain: a protease-associated domain. Pro- e (i.e. ZNRF3 homodimerization) co-exist with the mode of receptor tein Sci. 9, 1930 1934. e e Malinauskas, T., Jones, E.Y., 2014. Extracellular modulators of Wnt signalling. Curr. heterodimerization seen in the LGR5 Rspondin RNF43 ternary Opin. Struct. Biol. 29C, 77e84. complex? We hypothesised, based on plausible modelling of our Metzger, M.B., Hristova, V.A., Weissman, A.M., 2012. HECT and RING finger families ZNRF3 dimer structures into the LGR5eRspondineRNF43 crystal of E3 ubiquitin ligases at a glance. J. Cell Sci. 125, 531e537. Moad, H.E., Pioszak, A.A., 2013. Reconstitution of R-spondin:LGR4:ZNRF3 adult stem structure, that the ZNRF3-based ternary complex would exhibit a cell growth factor signaling complexes with recombinant proteins produced in 2:2:2 stoichiometry (Zebisch et al., 2013). Subsequent Escherichia coli. Biochemistry 52, 7295e7304. 118 M. Zebisch, E.Y. Jones / Progress in Biophysics and Molecular Biology 118 (2015) 112e118

Moffat, L.L., Robinson, R.E., Bakoulis, A., Clark, S.G., 2014. The conserved trans- Warner, M.L., Bell, T., Pioszak, A.A., 2015. Engineering high-potency R-spondin adult membrane RING finger protein PLR-1 downregulates Wnt signaling by reducing stem cell growth factors. Mol. Pharmacol. 87, 410e420. Frizzled, Ror and Ryk cell-surface levels in C. elegans. Development 141, Whiting, C.C., Su, L.L., Lin, J.T., Fathman, C.G., 2011. GRAIL: a unique mediator of CD4 617e628. T-lymphocyte unresponsiveness. FEBS J. 278, 47e58. Niehrs, C., 2012. The complex world of WNT receptor signalling. Nat. Rev. Mol. Cell Xie, Y., Zamponi, R., Charlat, O., Ramones, M., Swalley, S., Jiang, X., Rivera, D., Biol. 13, 767e779. Tschantz, W., Lu, B., Quinn, L., et al., 2013. Interaction with both ZNRF3 and LGR4 Peng, W.C., de, L.W., Forneris, F., Granneman, J.C., Huch, M., Clevers, H., Gros, P., is required for the signalling activity of R-spondin. EMBO Rep. 14, 1120e1126. 2013a. Structure of stem cell growth factor R-spondin 1 in complex with the Xu, K., Xu, Y., Rajashankar, K.R., Robev, D., Nikolov, D.B., 2013. Crystal structures of ectodomain of its receptor LGR5. Cell Rep. 3, 1885e1892. Lgr4 and its complex with R-spondin1. Structure 21, 1683e1689. Peng, W.C., de, L.W., Madoori, P.K., Forneris, F., Granneman, J.C., Clevers, H., Gros, P., Zebisch, M., Jones, E.Y., 2015. Crystal Structure of R-spondin 2 in Complex with the 2013b. Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 Ectodomains of its Receptors LGR5 and ZNRF3. J. Struct. Biol. (in press). and implications for signaling. PLoS One 8, e83110. Zebisch, M., Xu, Y., Krastev, C., Macdonald, B.T., Chen, M., Gilbert, R.J., He, X., Ruffner, H., Sprunger, J., Charlat, O., Leighton-Davies, J., Grosshans, B., Salathe, A., Jones, E.Y., 2013. Structural and molecular basis of ZNRF3/RNF43 trans- Zietzling, S., Beck, V., Therier, M., Isken, A., et al., 2012. R-spondin potentiates membrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat. Wnt/beta-catenin signaling through orphan receptors LGR4 and LGR5. PLoS Commun. 4, 2787. One 7, e40976. Zhang, X., Abreu, J.G., Yokota, C., Macdonald, B.T., Singh, S., Coburn, K.L., Tranque, P., Crossin, K.L., Cirelli, C., Edelman, G.M., Mauro, V.P., 1996. Identification Cheong, S.M., Zhang, M.M., Ye, Q.Z., Hang, H.C., et al., 2012. Tiki1 is required for and characterization of a RING zinc finger gene (C-RZF) expressed in chicken head formation via Wnt cleavage-oxidation and inactivation. Cell 149, embryo cells. Proc. Natl. Acad. Sci. U. S. A. 93, 3105e3109. 1565e1577. van Dijk, J.R., Yamazaki, Y., Palmer, R.H., 2014. Tumour-associated mutations of PA- Zhang, X., Cheong, S.M., Amado, N.G., Reis, A.H., Macdonald, B.T., Zebisch, M., TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a deter- Jones, E.Y., Abreu, J.G., He, X., 2015. Notum is required for neural and head in- minant for endosomal localization. Biochem. J. 459, 27e36. duction via Wnt deacylation, oxidation, and inactivation. Dev. Cell 32, 719e730. Wang, D., Huang, B., Zhang, S., Yu, X., Wu, W., Wang, X., 2013. Structural basis for R- spondin recognition by LGR4/5/6 receptors. Genes Dev. 27, 1339e1344.