Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.
- MeSH
- buněčná membrána metabolismus MeSH
- epitel metabolismus MeSH
- fosfoproteiny imunologie MeSH
- fosforylace MeSH
- frizzled receptory chemie metabolismus MeSH
- HEK293 buňky MeSH
- hmotnostní spektrometrie MeSH
- kasein kinasa I metabolismus MeSH
- lidé MeSH
- protein dishevelled chemie fyziologie MeSH
- protilátky imunologie MeSH
- sekvence aminokyselin MeSH
- serin metabolismus MeSH
- signální transdukce MeSH
- vejcovody metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This themed section of the British Journal of Pharmacology stems from the EMBO Conference: Wnt Meeting 2016 held from the 14th to 16th September 2016 in Brno, Czech Republic. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.
- MeSH
- antitumorózní látky farmakologie MeSH
- buněčná membrána účinky léků metabolismus MeSH
- lidé MeSH
- nádory farmakoterapie metabolismus patologie MeSH
- signální dráha Wnt účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodní články MeSH
- úvodníky MeSH
G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD6) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD6 dimerizes and that the dimer interface of FZD6 is formed by the transmembrane α-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of FZD6 signaling to extracellular signal-regulated kinases1/2. The discovery of agonist-dependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimer-interfering small molecules.Frizzled 6 (FZD6) is a G protein-coupled receptor (GPCR) involved in several cellular processes. Here, the authors use live cell imaging and spectroscopy to show that FZD6 forms dimers, whose association is regulated by WNT proteins and that dimer dissociation is crucial for FZD6 signaling.
- MeSH
- dimerizace MeSH
- frizzled receptory metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- protein Wnt 5a metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both β-catenin-dependent and β-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.
- MeSH
- aminokyselinové motivy MeSH
- embryo nesavčí metabolismus MeSH
- frizzled receptory chemie metabolismus MeSH
- HEK293 buňky MeSH
- heterotrimerní G-proteiny metabolismus MeSH
- konzervovaná sekvence * MeSH
- lidé MeSH
- mutační analýza DNA MeSH
- nádory metabolismus patologie MeSH
- polymerizace MeSH
- protein dishevelled chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- signální dráha Wnt MeSH
- signální transdukce MeSH
- simulace molekulární dynamiky MeSH
- strukturní homologie proteinů MeSH
- tyrosin metabolismus MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Xenopus laevis embryologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both β-catenin-dependent and -independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12, Gαi1, GαoA, Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13-dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13 activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5-E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.
- MeSH
- buněčné linie MeSH
- centrální nervový systém krevní zásobení MeSH
- cévní endotel účinky léků metabolismus MeSH
- frizzled receptory metabolismus MeSH
- fyziologická neovaskularizace * MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- protein dishevelled metabolismus MeSH
- proteiny vázající GTP - alfa-podjednotky G12-G13 metabolismus MeSH
- proteiny Wnt farmakologie MeSH
- signální transdukce * MeSH
- vazba proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.
- MeSH
- autoantigeny metabolismus MeSH
- centrozom metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- kinasy NEK fyziologie MeSH
- lidé MeSH
- protein dishevelled fyziologie MeSH
- proteiny buněčného cyklu metabolismus MeSH
- proteiny nervové tkáně metabolismus MeSH
- signální dráha Wnt MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD6 assembles with Gαi1/Gαq (but not with Gαs, Gαo and Ga12/13), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD4, a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD4 assembles-in a DVL-independent manner-with Gα12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Gαi1, Gαo, Gαs, and Gαq The FZD4-G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD4 green fluorescent protein-transfected cells depend on Gα12/13 Furthermore, expression of FZD4 and Gα12 or Gα13 in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Gα12/13 signaling, underlining the functionality of an FZD4-Gα12/13-RHO signaling axis. In summary, Gα12/13-mediated WNT/FZD4 signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD4 signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.
- MeSH
- faktory zaměňující Rho guanin nukleotidy metabolismus MeSH
- FRAP MeSH
- frizzled receptory chemie metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- protein dishevelled metabolismus MeSH
- proteinové domény MeSH
- proteiny vázající GTP - alfa-podjednotky G12-G13 metabolismus MeSH
- proteiny Wnt farmakologie MeSH
- rezonanční přenos fluorescenční energie MeSH
- signální transdukce účinky léků MeSH
- vazba proteinů účinky léků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5A(high)) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.
- MeSH
- čipová analýza tkání MeSH
- gliom metabolismus patologie MeSH
- lidé MeSH
- mikroglie metabolismus patologie MeSH
- monocyty metabolismus patologie MeSH
- proteiny Wnt biosyntéza genetika metabolismus MeSH
- protoonkogenní proteiny biosyntéza genetika metabolismus MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The seven-transmembrane-spanning receptors of the FZD1-10 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 FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 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.
- MeSH
- beta-katenin metabolismus MeSH
- buněčné linie MeSH
- fosforylace MeSH
- frizzled receptory metabolismus MeSH
- mapování interakce mezi proteiny MeSH
- mapy interakcí proteinů * MeSH
- myši MeSH
- protein - isoformy metabolismus MeSH
- proteiny Wnt metabolismus MeSH
- signální dráha Wnt * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
