The WNT signaling pathway regulates patterning and morphogenesis during development (Hoppler and Moon, 2014). In adults, WNT ligands promote the renewal of stem cells, maintaining tissue homeostasis during routine turnover or following injury (Clevers et al., 2014). Some WNT responses in vertebrates depend on a second signal provided by the R-spondin (RSPO) family of four secreted proteins (de Lau et al., 2012). RSPOs have emerged as important stroma-derived growth factors that drive the renewal of epithelial stem cells in many adult vertebrate tissues (de Lau et al., 2014). RSPOs markedly amplify target cell sensitivity to WNT ligands by neutralizing two transmembrane E3 ubiquitin ligases, ZNRF3 and RNF43, which reduce cell-surface levels of WNT receptors (Hao et al., 2012; Koo et al., 2012). Indeed, chromosomal translocations that increase RSPO expression or mutations that inactivate ZNRF3/RNF43 can drive cancer (Hao et al., 2016).

RSPOs contain tandem furin-like repeats (FU1 and FU2), a thrombospondin type 1 (TSP) domain and a basic region (BR). RSPOs simultaneously engage ZNRF3/RNF43 through their FU1 domain and leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4-6 through their FU2 domain (Chen et al., 2013; Peng et al., 2013; Wang et al., 2013; Xu et al., 2013; Zebisch et al., 2013), triggering the clearance of ZNRF3/RNF43 and the consequent rise in WNT receptor levels on the cell surface. LGR4-6 are selectively expressed in various tissue stem cells (Clevers et al., 2014) and are considered the primary high-affinity receptors for RSPOs (Carmon et al., 2011; de Lau et al., 2011; Glinka et al., 2011).

We now show that RSPO2 and RSPO3, but not RSPO1 or RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. Using purified mutant and chimeric RSPOs and cell lines lacking various receptors, we elucidated the requirements for this mode of signaling. The ZNRF3/RNF43-interacting FU1 domain is necessary for LGR-independent signaling, while the LGR-interacting FU2 domain is dispensable. The FU1 domain of RSPO3 is also sufficient to confer the capacity to signal without LGRs when transplanted to RSPO1. The TSP/BR domains of RSPOs and their interaction with heparan sulfate proteoglycans (HSPGs), previously considered dispensable for potentiation of WNT/β-catenin signaling (Kazanskaya et al., 2004; Ohkawara et al., 2011), are essential in the absence of LGRs. These results define two alternative modes of RSPO-mediated signaling that share a common dependence on ZNRF3/RNF43, but differ in their use of either LGRs or HSPGs.

In previous work (Lebensohn et al., 2016), we generated and thoroughly characterized a haploid human cell line (HAP1-7TGP) that harbors a fluorescent transcriptional reporter for WNT/β-catenin signaling. Both the fluorescence of this synthetic reporter and the transcription of endogenous WNT target genes in HAP1-7TGP cells can be activated by WNT ligands and these WNT responses can be strongly potentiated by RSPOs (Lebensohn et al., 2016). HAP1-7TGP cells do not secrete WNT ligands and thus their response to RSPOs requires the co-administration of a low concentration of WNT. A comprehensive set of unbiased genetic screens in HAP1-7TGP cells identified many of the known components required for a signaling response to WNT and RSPO ligands, establishing this cell line as a valid and genetically tractable system to study this pathway (Lebensohn et al., 2016).

We made the serendipitous and unexpected observation that RSPO3 could potently enhance WNT reporter fluorescence driven by a low concentration of WNT3A in two independently derived HAP1-7TGP clonal cell lines carrying loss-of-function mutations in LGR4 (LGR4^KO cells; see Materials and methods and Supplementary file 1) (Figure 1A). In contrast, RSPO1 did not enhance the response to WNT3A in LGR4^KO cells. RSPO1 and RSPO3 had equivalent activity in wild-type (WT) HAP1-7TGP cells, demonstrating that both ligands were functional, and the response to RSPO3 in both WT and LGR4^KO cells depended on the presence of WNT3A (Figure 1A).

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While LGR4 is the only RSPO receptor expressed in HAP1 cells (Table 1) (Dubey et al., 2016), we excluded the possibility of compensatory up-regulation of LGR5 or LGR6 by simultaneously disrupting both genes in LGR4^KO cells, generating multiple independent clonal cell lines lacking all three RSPO receptors (hereafter called LGR4/5/6^KO cells; Supplementary file 1). LGR4/5/6^KO cells retained an intact WNT signaling cascade, responding normally to a saturating dose of WNT3A (Figure 1B). All four RSPOs (1-4) strongly potentiated WNT signaling in WT cells, establishing ligand integrity. However, RSPO1 and RSPO4 were completely inactive in LGR4/5/6^KO cells, even at concentrations that induced maximum WNT reporter induction in WT cells, whereas RSPO2 and RSPO3 strongly potentiated signaling driven by low concentrations of WNT3A in the absence of all three LGRs (Figure 1B). Therefore, RSPO2 and RSPO3 possess a unique quality absent in RSPO1 and RSPO4 that enables them to potentiate WNT responses without LGRs.

Dose-response analysis revealed that RSPO1 and RSPO3 enhanced WNT signaling in WT cells with nearly identical pharmacodynamics—both the efficacy (maximum reporter activity) and the potency (measured by the EC50, defined as the RSPO concentration that induced half-maximum reporter activity) were similar for both ligands (Figure 1C). In LGR4/5/6^KO cells, RSPO1 had no detectable activity at concentrations up to 160 ng/ml, 400-fold higher than its EC50 in WT cells. While RSPO3 potentiated WNT signaling in LGR4/5/6^KO cells, its efficacy was reduced by 33% and its EC50 was increased by 16-fold compared to WT cells (Figure 1C). The distinct pharmacodynamics of RSPO3 in the two cell types suggested that its reception was mediated by different mechanisms in the presence and absence of LGRs.

We sought to determine which domains of RSPO3 were required for LGR-independent signaling using a ligand mutagenesis strategy (Figure 2A). Our experimental strategy leveraged a comparison between RSPO1 and RSPO3, since the former depended strictly on LGRs while the latter could signal in their absence. Unless otherwise noted, each WT and mutant RSPO ligand described hereafter was produced as a fusion protein carrying an N-terminal hemagglutinin (HA) tag and a tandem C-terminal tag composed of an immunoglobulin fragment crystallizable (Fc) domain followed by a 1D4 epitope tag (Molday and Molday, 2014) used for immuno-affinity purification (see Materials and methods and Figure 1—figure supplement 1A and B for a description of ligand purification and characterization). Importantly, the pharmacodynamics of the tagged RSPO proteins were similar to those of their untagged counterparts in both WT and LGR4/5/6^KO cells (Figure 1—figure supplement 1C and D).

Figure 2

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Previous studies have shown that the FU1 and FU2 domains in all RSPOs, which bind to ZNRF3/RNF43 and LGRs, respectively, are both necessary and sufficient to potentiate WNT responses, while the TSP and BR domains are dispensable (Kazanskaya et al., 2004). Simultaneous deletion of the FU1 and FU2 domains of RSPO3 abolished signaling in both WT and LGR4/5/6^KO cells (Figure 2B). Point mutations in the FU1 domain of RSPO3 (R67A/Q72A; Figure 2A) known to weaken the interaction between RSPOs and ZNRF3/RNF43 (Xie et al., 2013) entirely abolished RSPO3 signaling in LGR4/5/6^KO cells (Figure 2B and D) and substantially reduced (but did not abolish) RSPO3 signaling in WT cells (EC50 increased by 21-fold; Figure 2B and C). Thus, the reduction in the affinity between RSPO3 and ZNRF3/RNF43 caused by the FU1 R67A/Q72A mutation impaired LGR-independent signaling to a much greater extent than LGR-dependent signaling. Indeed, the equivalent R66A/Q71A mutation in RSPO1 (Figure 2A), which only signals in an LGR-dependent manner, also impaired but did not completely abolish signaling in WT cells (Figure 2B).

Point mutations in the FU2 domain of RSPO3 (F106E/F110E; Figure 2A) that weaken interactions with LGRs (Xie et al., 2013) had little impact on RSPO3 signaling in LGR4/5/6^KO cells, consistent with the lack of LGRs in these cells (Figure 2B and D). In WT cells, the F106E/F110E mutation in RSPO3 did not prevent signaling, but reduced the efficacy by 48% and increased the EC50 by 2.9-fold (Figure 2C). Thus, RSPO3 signaling in WT cells includes contributions from both LGR-dependent and independent pathways. In contrast, the F106E/F110E mutation in RSPO1 abolished signaling in WT cells, demonstrating that signaling by RSPO1 depends entirely on its interaction with LGRs (Figure 2B).

The C-terminal TSP and BR domains of RSPOs (denoted TSP/BR when discussed together) are considered dispensable for LGR-mediated signaling (Glinka et al., 2011). When we deleted these domains individually in RSPO3, there were only minor effects on signaling in WT cells (Figure 2E and F). Deletion of both domains in RSPO3 increased the EC50 in WT cells by 333-fold, but did not change the efficacy (Figure 2F). Therefore, while the TSP/BR domains are not essential for signaling in WT cells, consistent with prior work, their loss substantially reduces the potency of RSPO3. In contrast, RSPO3 lacking the TSP/BR domains failed to potentiate WNT responses in LGR4/5/6^KO cells at the concentrations tested (Figure 2E and G). The signaling properties of RSPO3 lacking the TSP/BR domains were unchanged when the dimerizing Fc tag was removed (Figure 2F and G).

These mutagenesis experiments demonstrated that the FU1 and TSP/BR domains of RSPO3 are required for its ability to potentiate WNT responses in the absence of LGRs, while the FU2 domain is dispensable. These domain requirements are distinct from those for LGR-mediated signaling by RSPO1, which depends on the FU1 and FU2, but not on the TSP/BR domains. In WT cells, RSPO3 signaling proceeded through both LGR-dependent and independent mechanisms because disruption of the FU2 or the TSP/BR domains partially impaired but did not abolish signaling (Figure 2C and F). The ZNRF3/RNF43-interacting FU1 domain is essential for signaling both in the presence and absence of LGRs.

To identify the region of RSPO3 that confers the capacity to signal without LGRs, we constructed a series of chimeric ligands by combining regions of RSPO1 and RSPO3 (Figure 3A). Remarkably, replacing the FU1 domain of RSPO1 with the FU1 domain of RSPO3 enabled RSPO1 to potentiate WNT signaling in LGR4/5/6^KO cells (Figure 3B and D). Conversely, replacing the FU1 domain of RSPO3 with that of RSPO1 drastically reduced the signaling capacity of RSPO3 in LGR4/5/6^KO cells (Figure 3B and D). In important control experiments, all chimeric ligands showed equivalent activity in WT cells, establishing ligand integrity (Figure 3B and C). Thus, a difference in the interaction between ZNRF3/RNF43 and the FU1 domains of RSPO1 and RSPO3 is the crucial determinant of the requirement for LGRs. Of note, the affinities of the FU1-FU2 fragment of RSPO2 (25 nM) and RSPO3 (60 nM) for ZNRF3 have been reported to be much higher than those of RSPO1 (6.8 μM) and RSPO4 (300 μM) (Zebisch et al., 2013). Indeed, these affinities correlate with the capacity of RSPO2 and RSPO3, but not RSPO1 or RSPO4, to promote LGR-independent signaling (Figure 1B). While the TSP/BR domains of RSPO3 were required for LGR-independent signaling, they were not sufficient because replacing the TSP/BR domains of RSPO1 with those of RSPO3 did not confer on RSPO1 the capacity to signal in LGR4/5/6^KO cells (Figure 3E). In fact, the TSP/BR domains of RSPO1 and RSPO3 seemed interchangeable for signaling activity in both WT and LGR4/5/6^KO cells (Figure 3E).

Figure 3

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These results suggested that the WNT-potentiating activity of RSPO3 in the absence of LGRs depends on its interaction with ZNRF3/RNF43 through the FU1 domain and additional interactions with an alternative co-receptor through the TSP/BR domains. We considered the previous observation that the TSP/BR domains of RSPOs can bind to heparin (Nam et al., 2006). Addition of heparin to the culture medium completely blocked potentiation of WNT signaling by RSPO3 in LGR4/5/6^KO cells, but had only a partial inhibitory effect in WT cells, in which RSPO3 can also signal through LGRs (Figure 4A).

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The TSP/BR domains of RSPOs can mediate interactions with the two major families of cell-surface HSPGs, the glycophosphatidylinositol (GPI)-linked glypicans and the transmembrane syndecans (Ohkawara et al., 2011). In humans, both protein families are encoded by multiple, partially redundant genes: six glypican genes (GPC1-6) and four syndecan genes (SDC1-4) (Park et al., 2000), all of which are expressed in HAP1 cells except for GPC5 (Table 1). Since all glypicans and syndecans must be post-translationally modified with heparan sulfate chains for receptor function, we disrupted EXTL3, a gene encoding a glycosyltransferase that is specifically required for HSPG biosynthesis, but dispensable for the synthesis of other glycosaminoglycans and proteoglycans (Takahashi et al., 2009). The loss of EXTL3 in LGR4/5/6^KO cells led to an 81% reduction in RSPO3-mediated potentiation of WNT signaling (Figure 4B). In contrast, the loss of EXTL3 in WT cells only reduced signaling by 34%, likely because RSPO3 can also signal through LGRs in WT cells. In an important control, the loss of EXTL3 did not affect signaling induced by addition of a sub-saturating concentration of WNT3A alone or by inhibition of the β-catenin destruction complex kinase GSK3 in either WT or LGR4/5/6^KO cells (Figure 4—figure supplement 1A).

To distinguish between glypicans and syndecans, we took advantage of the fact that only glypicans are anchored to the cell surface by a GPI linkage. Disrupting PIGL, a gene required for GPI-anchor biosynthesis, or disrupting both GPC4 and GPC6, the two glypican genes identified in our previous haploid genetic screens (Lebensohn et al., 2016), in LGR4/5/6^KO cells did not impair LGR-independent potentiation of WNT signaling by RSPO3 (Figure 4C). As we had found previously in WT HAP1-7TGP cells (Lebensohn et al., 2016), disrupting PIGL or GPC4 and GPC6 in LGR4/5/6^KO cells reduced signaling by a sub-saturating dose of WNT3A (Figure 4—figure supplement 1B), indicating that glypicans are required to mediate cellular responses to low doses of WNT3A. Disrupting all four syndecans (SDC1-4) simultaneously in LGR4/5/6^KO cells did not impair LGR-independent potentiation of WNT signaling by RSPO3 (Figure 4D), nor signaling induced by sub-saturating WNT3A or by inhibition of GSK3 (Figure 4—figure supplement 1C).

We conclude from these results that either glypicans or syndecans (or another HSPG) can mediate LGR-independent signaling, probably through interactions between their heparan sulfate chains and the TSP/BR domains of RSPO3. Therefore, disrupting any single family of HSPGs does not impair signaling (Figure 4C and D), while disrupting EXTL3, an enzyme required for the biosynthesis of heparan sulfate chains present in all HSPGs, substantially reduces LGR-independent signaling (Figure 4B).

Our study shows that RSPOs can potentiate WNT signals in the absence of LGRs, expression of which has been hitherto considered the hallmark of RSPO-responsive cells. This quality is unique to RSPO2 and RSPO3 and is endowed by their ZNRF3/RNF43-interacting FU1 domain, since transplanting the FU1 domain from RSPO3 renders RSPO1 capable of signaling in the absence of LGRs (Figure 3B, D). Furthermore, the TSP/BR domains of RSPO3, which had been deemed dispensible for LGR-dependent signaling (Glinka et al., 2011), are necessary for signaling in the absence of LGRs (Figure 2E, G). HSPGs, cell surface proteins capable of interacting with the TSP/BR domains (Ohkawara et al., 2011), are also selectively required for RSPO3-dependent potentiation of WNT signaling in the absence of LGRs (Figure 4B). Thus, our results suggest that the interaction of the TSP/BR domains of RSPO3 with HSPGs provides an alternative mechanism that neutralizes ZNRF3/RNF43 in the absence of LGRs (Figure 4E). HSPGs are known to mediate the efficient endocytosis of multiple cargoes (Christianson and Belting, 2014). Hence, we speculate that the simultaneous interaction of RSPO3 with ZNRF3/RNF43 through its FU1 domain and HSPGs through its TSP/BR domains provides an LGR-independent route for the endocytosis and clearance of ZNRF3/RNF43 from the cell surface (Figure 4E) and the consequent rise in WNT receptor levels.

Future work will be necessary to define the developmental, regenerative or oncogenic contexts in which this LGR-independent mode of signaling is used to amplify target cell responses to WNT ligands. The presence of three LGRs capable of mediating responses to four RSPO ligands (de Lau et al., 2011) poses significant experimental challenges to addressing this question genetically in mouse models, as we did in human haploid cells, since multiple genes would need to be simultaneously disrupted to test if any responses to RSPOs are retained in tissues lacking all three LGRs.

However, some information can be gleaned from the developmental phenotypes of mice carrying mutations in Rspo2 or Rspo3, the two members of the family capable of potentiating WNT signaling in cells lacking all three LGRs (Figure 1B). Rspo2 is expressed in the limb and lung buds of the early embryo and Rspo2^-/- mice have defects in both limb development and branching of bronchioles in the developing lung (Nam et al., 2007a; Nam et al., 2007b; Bell et al., 2008). Rspo3 has been shown to be important for angioblast and vascular development and Rspo3^-/- mouse embryos die around day E10 due to defects in placental vascularization (Aoki et al., 2007; Kazanskaya et al., 2008). While all three Lgrs have not been knocked out simultaneously in mice, Lgr4^-/-; Lgr5^-/- double knock-out embryos have impaired development of the intestine, kidney and skin (Kinzel et al., 2014). We speculate that the lung, limbs and vasculature, which depend on Rspo2 or Rspo3 but not Lgr4 and Lgr5 function, are tissues in which the LGR-independent mode of signaling may be important during development.

While LGRs are specifically expressed in some stem cell compartments (Clevers et al., 2014), HSPGs are expressed ubiquitously on the surface of most cells (Park et al., 2000). Thus, LGR-mediated RSPO signaling may be critical when high-level WNT signaling must be restricted to specific cells, as in the stem cell niches of various tissues, while HSPG-mediated RSPO signaling might confer amplification of WNT responses more broadly during the development of organs like the limbs, the lungs or the placenta.

In summary, our results define two alternative modes of RSPO-mediated amplification of WNT/β-catenin signaling that differ in their use of either LGRs or HSPGs (Figure 4E). The mutant and chimeric RSPO ligands we described should allow the selective modulation of these alternate modes of signaling.

The following Materials and methods relevant to this manuscript have been described previously (Lebensohn et al., 2016): cell lines and growth conditions, preparation of WNT3A conditioned medium (CM) and construction of the HAP1-7TGP WNT reporter haploid cell line.

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Author details

1. Andres M Lebensohn

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Department of Medicine, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   andreslebensohnphd@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4224-8819

2. Rajat Rohatgi

   1. Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
   2. Department of Medicine, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   rrohatgi@stanford.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

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