Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION domain (CRD) necessary and sufficient for activities The structural basis of R-spondin (Kazanskaya et al. 2004; Glinka et al. 2011), a thrombospondin recognition by LGR5 and RNF43 (TSP) type I repeat domain for heparan sulfate proteoglycan binding (Ohkawara et al. 2011), and a C-terminal basic region Po-Han Chen,1 Xiaoyan Chen,1 Zhenghong Lin,2 of variable lengths. Underlying the critical role of the CRD Deyu Fang,2 and Xiaolin He1,3 in signaling, RSPO1 mutations in this region lead to XX sex reversal and skin abnormalities (palmoplantar keratosis) 1Department of Molecular Pharmacology and Biological (Parma et al. 2006), and RSPO4 mutations result in de- Chemistry, Northwestern University Feinberg School velopmental defect in fingernails and toenails (anonychia) of Medicine, Chicago, Illinois 60611, USA; 2Department (Blaydon et al. 2006). The identities of RSPO receptors had of Pathology, Northwestern University Feinberg been controversial. It was originally suggested that RSPOs School of Medicine, Chicago, Illinois 60611, USA bind to Fz or LRP6 (Wei et al. 2007; Ohkawara et al. 2011); in another study, RSPOs were reported to antagonize the R-spondins (RSPOs) enhance Wnt signaling, affect stem cell Wnt inhibitor Kremen. None of the results from these behavior, bind to leucine-rich repeat-containing G-protein- studies were confirmed, however (Binnerts et al. 2007). coupled receptors 4–6, (LGR4–6) and the transmembrane Most recently, several independent studies identified E3 ubiquitin ligases RING finger 43/zinc and RING finger 3 LGR4–6 (leucine-rich repeat [LRR]-containing G-protein- (RNF43/ZNRF3). The structure of RSPO1 bound to both coupled receptors [GPCRs] 4–6) as receptors for RSPO1–4 (Carmon et al. 2011; de Lau et al. 2011; Glinka et al. 2011; LGR5 and RNF43 ectodomains confirms their physical link- Ruffner et al. 2012). LGR4–6 mark adult stem cell age. RSPO1 is sandwiched by LGR5 and RNF43, with its rod compartments in several organ systems and are necessary module of the cysteine-rich domain (CRD) contacting LGR5 for proper kidney, mammary, and intestinal development andahairpininsertedintoRNF43.LGR5doesnotcontact (Barker and Clevers 2010; Snippert et al. 2010; Barker RNF43 but increases the affinity of RSPO1 to RNF43, sup- et al. 2012; Huch et al. 2013). LGR4–6 are closely related porting LGR5 as an engagement receptor and RNF43 as an to GPCRs for glycoprotein hormones FSH (LGR1 as the effector receptor. Disease mutations map to the RSPO1– receptor), LH (LGR2), TSH (LGR3), and relaxins (LGR7 RNF43 interface, which promises therapeutic targeting. and LGR8) (Hsu et al. 2000; Kong et al. 2010), all of which share a similar architecture: a large N-terminal extracellular Supplemental material is available for this article. LRR domain for ligand binding, a seven-helix transmem- Received April 14, 2013; revised version accepted May 17, brane domain, and a short cytosolic region. Intriguingly, 2013. unlike the hormone receptors, LGR4–6 are not known to use G proteins for signaling, provoking an intriguing ques- tion about their role as RSPO receptors. LGR5–RSPO1 has been reported to form a ternary Wnt signaling plays crucial roles in development across complex with the transmembrane RING-type E3 ubiquitin species. Underlying its evolutionary significance is the ligase ZNRF3 (zinc and RING finger 3) or RNF43 (RING large repertoire and dynamic assembly of receptors, core- finger 43) (Hao et al. 2012; Koo et al. 2012). This finding ceptors, agonists, and antagonists involved in Wnt signal- suggested a mechanism by which RSPO1 potentiates Wnt ing regulation (Clevers and Nusse 2012; Niehrs 2012). The signaling. Being a Wnt target gene, ZNRF3/RNF43 bal- core apparatus comprises the serpentine transmembrane ances Wnt signaling by providing a steady-state membrane receptor Frizzled (Fz), the coreceptor LRP6 in the case of clearance of the Fz–LRP complex (Hao et al. 2012; Koo canonical Wnt/b-catenin pathways, and other receptors et al. 2012). Through interacting with and inducing endo- such as Syndecan and ROR for noncanonical pathways cytosis of ZNRF3/RNF43, RSPO1 inhibits ZNRF3/RNF43 (Wnt/PCP, for instance). Recent revelation of the core Wnt– activities and henceforth stabilizes the surface level of Fz– Fz complex structure resolved long-standing questions on LRP for canonical and noncanonical Wnt signaling (Hao the molecular basis of Wnt–Fz recognition (Janda et al. et al. 2012). Importantly, the RSPO1-mediated ZNRF3 2012). Beyond Wnt–Fz, much remains unknown about membrane clearance is LGR-dependent, which is sup- how other regulators assemble to modulate Wnt pathways. ported by biochemical data that the three proteins could The secreted glycoproteins R-spondins 1–4 (RSPO1–4) form a physical complex (Hao et al. 2012). were discovered as potent activators of Wnt signaling To clarify the structural roles of RSPOs, LGRs, and (Kamata et al. 2004; Kazanskaya et al. 2004; de Lau et al. ZNRF3/RNF43 in Wnt signaling, we determined the 2012; Cruciat and Niehrs 2013). Their enigmatic nature is crystal structure of the human ternary LGR5–RSPO1– highlighted by their Wnt-dependent synergistic effects, RNF43 complex at 2.5 A˚ resolution. Our results reveal their presence only in vertebrates plus selected inverte- the RSPO CRD fold, provide direct evidence that RSPOs brates, and their low sequence homology with other Wnt physically tether LGR4–6 and ZNRF3/RNF43 corecep- signaling molecules (de Lau et al. 2012; Cruciat and Niehrs tors, and provide insights into the roles of LGR4–6 and 2013). RSPOs consist of an N-terminal Cys-rich furin-like ZNRF3/RNF43 in the signaling complex assembly. Results and Discussion [Keywords: Wnt signaling; LGR5; R-spondin; RNF43; furin-like repeat; E3 ubiquitin ligase] 3Corresponding author Assembly of the ternary complex E-mail [email protected] Article published online ahead of print. Article and publication date are online The LGR5 ectodomain (ECD), RNF43 protease-associated at http://www.genesdev.org/cgi/doi/10.1101/gad.219915.113. (PA) domain, and RSPO1 CRD were used for the structural GENES & DEVELOPMENT 27:000–000 Ó 2013 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/13; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Chen et al. studies (Fig. 1A). Because LGR5 aggregates easily by itself, Divergence of the LGR5 scaffold from glycoprotein it was coexpressed with RSPO1 in HEK293S cells (Materials hormone receptors and Methods; Supplemental Material), and affinity-purified RSPO1–LGR5 was mixed with RNF43 to reconstitute the Conforming to the general architecture of LRR domains ternary complex. The structure was determined by single (Bella et al. 2008), LGR5 contains an LRR N-terminal isomorphous replacement with anomalous scattering domain that caps the most N-terminal LRR, followed by (SIRAS) (Supplemental Table 1). Consistent with previous 17 LRRs that form a sleigh-like solenoid. Due to the large findings, the structure confirms the physical linkage size of the LGR5 ECD, the entire solenoid adopts a between the three proteins (Hao et al. 2012), with RSPO1 slightly right-handed twist around LRR11 and LRR12. The large number of LRRs in LGR5 ECD encircles almost CRD sandwiched between two coreceptors (Fig. 1B). RNF43 ˚ and LGR5 C termini point in the same direction, leading half a circle with a diameter of ;80 A, forming an arch that engulfs the entire vertical length of RSPO1 and to the cell membrane, suggesting that the observed struc- ˚ tural assembly mimics the biologically relevant complex. RNF43 (;50 A) under its concave face. Structure-based Overall, the position of RSPO1 recognition by LGR5 is sequence alignment suggests that the 17 LRRs should be reminiscent of the position of FSH recognition by FSHR at conserved from LGR4 to LGR6 and thus establishes the the N-terminal LRR region (Supplemental Fig. 1), despite structural basis of functional equivalence between LGR4, the fact that RSPO1 and FSH are of different folds. FSHR LGR5, and LGR6. uses its additional C-terminal hinge domain (discussed The most C-terminal region of the LGR4–6 ECD below) to clamp FSH in place (Jiang et al. 2012). In LGR5, contains a conserved hinge motif that differs from the however, the C-terminal region does not form direct one for glycoprotein hormone receptors (Hsu et al. 2000). contact with either RSPO1 or RNF43; instead, the RNF43 For LGR4–6, this region consists of a helix with se- PA domain binds to the other side of RSPO1, clamping the quence PYAYQCC (residue 475–481) that caps the most ligand in an analogous fashion. C-terminal LRRs and is connected to another conserved Interestingly, each asymmetric unit reveals two copies sequence, GXFKPCE (residue 552–558), by a large flexible of LGR5–RSPO1–RNF43 heterotrimers arranged around a hinge loop whose electron density is lacking in our twofold axis (Supplemental Fig. 2). The 2:2:2 complex is structure (residue 483–537). In FSHR, this so-called hinge mediated between LGR5–LGR5 and LGR5–RNF43 in- loop is similarly present. Notably, a sulfur-modified Tyr teractions that involve the sides of the LRR solenoids, (sTyr) on this loop serves as the secondary binding site for forming a back-to-back dimer resembling the TLR4-MD2 FSH (Supplemental Fig. 1). For LGR4–6, the absence of structure (Kim et al. 2007). Our gel filtration data, however, this critical sTyr, the lack of sequence conservation in show primarily a 1:1:1 assembly ratio of LGR5, RSPO1, and the hinge loop residues, and the flexibility of the LRR RNF43, at least within the range of protein concentrations C-terminal domain/hinge domain in the crystal structure in our experiments. Due to uncertainties regarding the altogether suggest that the membrane-proximal region is physiological relevance of the 2:2:2 complex, the following unlikely to interact with RNF43 or RSPO1 and diverges discussion focuses on the single LGR5–RSPO1–RNF43 from the LGR1–3 hormone recognition mode.