title: WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13.
abstract: 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.
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Elisa
authors.initials: E
authors.lastname: Arthofer
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Belma
authors.initials: B
authors.lastname: Hot
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Julian
authors.initials: J
authors.lastname: Petersen
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Katerina
authors.initials: K
authors.lastname: Strakova
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Stefan
authors.initials: S
authors.lastname: Jäger
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Manuel
authors.initials: M
authors.lastname: Grundmann
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.).
authors.forename: J Silvio
authors.initials: JS
authors.lastname: Gutkind
authors.affiliation: Section of Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (E.A., B.H., J.P., K.S., S.J., G.S.); Section on Molecular Signal Transduction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (E.A.); Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic (K.S., G.S.); Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany (M.G., E.K.); Department of Pharmacology, Moores Cancer Center, University of California, San Diego, La Jolla, California (J.S.G.) gunnar.schulte@ki.se.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Schulte
mesh_terms: D000072261:Dishevelled Proteins / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D051157:Frizzled Receptors / Q000737:chemistry / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D000072417:Protein Domains; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051153:Wnt Proteins / Q000494:pharmacology*
chemical_list: D000072261:Dishevelled Proteins; C402930:FZD4 protein, human; D051157:Frizzled Receptors; D064067:Rho Guanine Nucleotide Exchange Factors; D051153:Wnt Proteins; D049452:Green Fluorescent Proteins; D044365:GTP-Binding Protein alpha Subunits, G12-G13