FASEB J

Dishevelled (Dvl) is a multifunctional effector of different Wnt cascades. Both canonical Wnt3a and noncanonical Wnt5a stimulate casein-kinase-1 (CK1) -mediated phosphorylation of Dvl, visualized as electrophoretic mobility shift [phosphorylated and shifted Dvl (ps-Dvl)]. However, the role of this phosphorylation remains obscure. Here we report the functional interaction of ps-Dvl with the receptor tyrosine kinase Ror2, which is an alternative Wnt receptor and is able to inhibit canonical Wnt signaling. We demonstrate interaction between Ror2 and ps-Dvl at the cell membrane after Wnt3a or Wnt5a stimulus dependent on CK1. Ps-Dvl interacts with the C-terminal proline-serine-threonine-rich domain of Ror2, which is required for efficient inhibition of canonical Wnt signaling. We further show that the Dvl C terminus, which seems to be exposed in ps-Dvl and efficiently binds Ror2, is an intrinsic negative regulator of the canonical Wnt pathway downstream of beta-catenin. The Dvl C terminus is necessary and sufficient to inhibit canonical Wnt/beta-catenin signaling, which is dependent on the presence of Ror2. Furthermore, both the Dvl C terminus and CK1epsilon can inhibit the Wnt5a/Ror2/ATF2 pathway in mammalian cells and Xenopus explant cultures. This suggests that phosphorylation of Dvl triggers negative feedback regulation for different branches of Wnt signaling in a Ror2-dependent manner

Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs

The seven-transmembrane-spanning receptors of the FZD(1–10) class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD(2), FZD(4), or FZD(5), we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD(2/4/5) as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs

Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>WNT-5A signaling in the central nervous system is important for morphogenesis, neurogenesis and establishment of functional connectivity; the source of WNT-5A and its importance for cellular communication in the adult brain, however, are mainly unknown. We have previously investigated the inflammatory effects of WNT/β-catenin signaling in microglia in Alzheimer's disease. WNT-5A, however, generally recruits β-catenin-independent signaling. Thus, we aim here to characterize the role of WNT-5A and downstream signaling pathways for the inflammatory transformation of the brain's macrophages, the microglia.</p> <p><b>Methods</b></p> <p>Mouse brain sections were used for immunohistochemistry. Primary isolated microglia and astrocytes were employed to characterize the WNT-induced inflammatory transformation and underlying intracellular signaling pathways by immunoblotting, quantitative mRNA analysis, proliferation and invasion assays. Further, measurements of G protein activation by [γ-³⁵ S]GTP binding, examination of calcium fluxes and cyclic AMP production were used to define intracellular signaling pathways.</p> <p><b>Results</b></p> <p>Astrocytes in the adult mouse brain express high levels of WNT-5A, which could serve as a novel astroglia-microglia communication pathway. The WNT-5A-induced proinflammatory microglia response is characterized by increased expression of inducible nitric oxide synthase, cyclooxygenase-2, cytokines, chemokines, enhanced invasive capacity and proliferation. Mapping of intracellular transduction pathways reveals that WNT-5A activates heterotrimeric G_i/o proteins to reduce cyclic AMP levels and to activate a G_i/o protein/phospholipase C/calcium-dependent protein kinase/extracellular signal-regulated kinase 1/2 (ERK1/2) axis. We show further that WNT-5A-induced ERK1/2 signaling is responsible for distinct aspects of the proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation.</p> <p><b>Conclusions</b></p> <p>Thus, WNT-5A-induced and G protein-dependent signaling to ERK1/2 is important for the regulation of proinflammatory responses in mouse primary microglia cells. We show for the first time that WNT-5A/G protein signaling mediates physiologically important processes in primary mammalian cells with natural receptor and G protein stochiometry. Consequently, WNT-5A emerges as an important means of astrocyte-microglia communication and we, therefore, suggest WNT-5A as a new player in neuroinflammatory conditions, such as neurodegenerative disease, hypoxia, stroke, injury and infection.</p