The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

How animal body plans evolved and diversified is a major question in evolutionary developmental biology. To address this question, it is important to characterize the exact molecular mechanisms that establish the major embryonic axes which give rise to the adult animal body plan. The anterior-posterior (AP) axis is the first axis to be established in most animal embryos, and in echinoderm sea urchin embryos its formation is governed by an integrated network of three different Wnt signaling pathways: Wnt/β-catenin, Wnt/JNK, and Wnt/PKC pathway. The extent to which this embryonic patterning mechanism is conserved among deuterostomes, or more broadly in metazoans, is an important open question whose answers could lead to a deeper appreciation of the evolution of the AP axis. Because Ambulacrarians (echinoderms and hemichordates) reside in a key phylogenetic position as the sister group to chordates, studies in these animals can help inform on how chordate body plans may have evolved. Here, we assayed the spatiotemporal gene expression of a subset of sea urchin AP Wnt patterning gene orthologs in the hemichordate, Schizocardium californicum. Our results show that positioning of the anterior neuroectoderm (ANE) to a territory around the anterior pole during early AP formation is spatially and temporally similar between indirect developing hemichordates and sea urchins. Furthermore, we show that the expression of wnt8 and frizzled5/8, two known drivers of ANE patterning in sea urchins, is similar in hemichordate embryos. Lastly, our results highlight divergence in embryonic expression of several early expressed Wnt genes (wnt1, wnt2 and wnt4). These results suggest that expression of the sea urchin AP Wnt signaling network is largely conserved in indirect developing hemichordates setting the foundation for future functional studies in S. californicum.

The Frizzled family of transmembrane receptors (FZD1-10) belongs to the class F of G protein-coupled receptors (GPCRs). FZDs bind to and are activated by Wingless/Int1 (WNT) proteins. The WNT/FZD signaling system regulates crucial aspects of developmental biology and stem-cell regulation. Dysregulation of WNT/FZD communication can lead to developmental defects and diseases such as cancer and fibrosis. Recent insight into the activation mechanisms of FZDs has underlined that protein dynamics and conserved microswitches are essential for FZD-mediated information flow and build the basis for targeting these receptors pharmacologically. In this review, we summarize recent advances in our understanding of FZD activation, and how novel concepts merge and collide with existing dogmas in the field.

The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.

Systemic toxicity is a major challenge in the development of therapeutics. Consequently, cell-type-specific targeting is needed to improve on-target efficacy while reducing off-target toxicity. Here, we describe a cell-targeting system we have termed BRAID (BRidged Activation by Intra/intermolecular Division) whereby an active molecule is divided into two inactive or less active parts that are subsequently brought together via a so-called \'bridging receptor\' on the target cell. This concept was validated using the WNT/β-catenin signaling system, demonstrating that a multivalent WNT agonist molecule divided into two inactive components assembled from different epitopes via the hepatocyte receptor βKlotho induces signaling specifically on hepatocytes. These data provide proof of concept for this cell-specific targeting strategy, and in principle, this may also allow activation of multiple signaling pathways where desirable. This approach has broad application potential for other receptor systems.

The core planar polarity pathway consists of six proteins that form asymmetric intercellular complexes that segregate to opposite cell ends in developing tissues and specify polarized cell structures or behaviors. Within these complexes, the atypical cadherin Flamingo localizes on both sides of intercellular junctions, where it interacts homophilically in trans via its cadherin repeats, whereas the transmembrane proteins Frizzled and Strabismus localize to the opposite sides of apposing junctions. However, the molecular mechanisms underlying the formation of such asymmetric complexes are poorly understood. Using a novel tissue culture system, we determine the minimum requirements for asymmetric complex assembly in the absence of confounding feedback mechanisms. We show that complexes are intrinsically asymmetric and that an interaction of Frizzled and Flamingo in one cell with Flamingo in the neighboring cell is the key symmetry-breaking step. In contrast, Strabismus is unable to promote homophilic Flamingo trans binding and is only recruited into complexes once Frizzled has entered on the opposite side. This interaction with Strabismus requires intact intracellular loops of the seven-pass transmembrane domain of Flamingo. Once recruited, Strabismus stabilizes the intercellular complexes together with the three cytoplasmic core proteins. We propose a model whereby Flamingo exists in a closed conformation and binding of Frizzled in one cell results in a conformational change that allows its cadherin repeats to interact with a Flamingo molecule in the neighboring cell. Flamingo in the adjacent cell then undergoes a further change in the seven-pass transmembrane region that promotes the recruitment of Strabismus.

Wnts are lipid-modified proteins rich in cysteine, regulating developmental processes, and are involved in various pathological conditions. Wnts structure resembles a hand, with a palmitoleylated thumb and an index finger-like domain interacting with frizzled (FZ) receptors. Previous research shows the palmitoleyl group and the disulfides importance in Wnt folding, secretion, and function, but the structural basis is not fully understood. Here, we utilized classical molecular dynamics simulation (800-ns in total) to investigate how the thumb palmitoleyl and its close conserved disulfides (183-190, 181-195) regulated Wnt-FZ interaction and structural dynamics. Using Steered molecular dynamics experiment followed by a relaxing procedure, we also explored if these disulfides are important in Wnt-FZ complex formation. According to our results, the palmitoleyl group contributes significantly to stabilize Wnt-FZ interaction, and the disulfides modulate this contribution. We also demonstrated that disulfide 183-190 regulates the Wnt thumb fluctuation, hydrogen bond network, and secondary structure. The DCCM analysis depicted disulfide 183-190 roles in regulating native-like collective movement in the palmitoleylated loop, which changed after this disulfide removal. The pulling-relaxing experiment showed that both the disulfides, and especially, the disulfide 183-190, are highly important for long-range salt-bridge interaction establishment between Wnt Lys182 and FZ Glu64, led palmitoleyl group appropriate positioning to FZ, suggested this disulfide essential role in Wnt-FZ complex formation. Together, our findings provide new insights to how thumb-positioned disulfides contribute to Wnt-FZ complex formation, structural dynamics, and stability, introducing disulfide 183-190 as a consequential element to target in drug design and development against Wnt signalling.

Proper localization of receptors for synaptic organizing factors is crucial for synapse formation. Wnt proteins promote synapse assembly through Frizzled (Fz) receptors. In hippocampal neurons, the surface and synaptic localization of Fz5 is regulated by neuronal activity, but the mechanisms involved remain poorly understood. Here, we report that all Fz receptors can be post-translationally modified by S-acylation and that Fz5 is S-acylated on three C-terminal cysteines by zDHHC5. S-acylation is essential for Fz5 localization to the cell surface, axons, and presynaptic sites. Notably, S-acylation-deficient Fz5 is internalized faster, affecting its association with signalosome components at the cell surface. S-acylation-deficient Fz5 also fails to activate canonical and divergent canonical Wnt pathways. Fz5 S-acylation levels are regulated by the pattern of neuronal activity. In vivo studies demonstrate that S-acylation-deficient Fz5 expression fails to induce presynaptic assembly. Our studies show that S-acylation of Frizzled receptors is a mechanism controlling their localization and function.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/β-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics\' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.