Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-to-cell communication during embryonic development and adult tissue homeostasis through canonical and non-canonical signaling pathways. Wnt signaling is intensively studied due to its central role, and therapeutic implications, in the development and progression of cancer, tissue renewal, and differentiation of stem cells. Wnt binding to Frizzled, which has structural similarities to G-protein coupled receptors, and the co-receptors Lrp5/6, is indispensable for Wnt/-catenin signaling, and is counterbalanced by a variety of endogenous antagonists. In this proposal we address the complete lack of extracellular structural information on Wnts, the manner in which Wnts complex with Frizzled receptors, as well as Wnt and Frizzled interactions with endogenous inhibitory proteins such as WIF, Dkk and Kremen. Given the central importance of Wnt signaling for a variety of human diseases, structural information has now become critical in order to clearly delineate the basis of receptor- ligand specificity and mechanisms of receptor activation. A current confusion in this field is that Wnts, Fz and Lrp6 are highly cross-reactive, complicating the attribution of specific biological phenotypes to specific Wnt-Fz receptor pairs. Structures of Wnt-receptor complexes could help solve this problem by visualizing degenerate versus ligand-specific contacts, as well as the architectures of the higher order signaling complexes. However, a major technical obstacle to obtaining structural data has been that Wnt proteins contain a hydrophobic lipid modification that complicates Wnt over-expression and crystallization. In this proposal we utilize a variety of novel (yeast display) and traditional (X-ray crystallography) methodologies to obtain three-dimensional structures of extracellular Wnt complexes involved in receptor activation, and inhibition.
We aim to reconstitute several recombinant Wnt-Frizzled, Wnt-Frizzled-Lrp5/6 and Wnt-antagonist complexes, in order to characterize their assemblies, affinities and stoichiometries. We will also attempt a highly innovative experiment to produce water-soluble, non-palmitoylated, bioactive Wnts by in vitro evolution. Using X-ray crystallography, we hope to elucidate the structural principles of Wnt-Frizzled-Lrp5/6 recognition, and inhibition of Wnt-Frizzled-Lrp5/6 complex formation by the antagonists WIF, DKK and Kremen, and together with established collaborators in the Wnt field, translate the structural data into functionally insightful experiments. Ultimately the results of these ambitious studies will be valuable for revealing new protein engineering strategies to interrogate and therapeutically target Wnt signaling.

Public Health Relevance

Wnts are secreted growth factors that exert their actions in embryonic development and adult tissue regeneration through interactions with Frizzled G-protein coupled receptors, in concert with a variety of co- receptors and antagonists. Wnt signaling represents an important new frontier for therapeutic intervention in cancer, tissue renewal, and differentiation of stem cells. We wish to acquire information about the assembly and three-dimensional structures of Wnt-receptor complexes in order to understand Wnt signaling mechanisms, and reveal new protein engineering strategies to clinically target Wnt-associated diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097015-02
Application #
8245015
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Flicker, Paula F
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$300,200
Indirect Cost
$110,200
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Janda, Claudia Y; Dang, Luke T; You, Changjiang et al. (2017) Surrogate Wnt agonists that phenocopy canonical Wnt and ?-catenin signalling. Nature 545:234-237
Yan, Kelley S; Janda, Claudia Y; Chang, Junlei et al. (2017) Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal. Nature 545:238-242
Luca, Vincent C; Kim, Byoung Choul; Ge, Chenghao et al. (2017) Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355:1320-1324
Janda, Claudia Y; Garcia, K Christopher (2015) Wnt acylation and its functional implication in Wnt signalling regulation. Biochem Soc Trans 43:211-6
Luca, Vincent C; Jude, Kevin M; Pierce, Nathan W et al. (2015) Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science 347:847-53
Spiess, Katja; Jeppesen, Mads G; Malmgaard-Clausen, Mikkel et al. (2015) Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo. Proc Natl Acad Sci U S A 112:8427-32
Burg, John S; Ingram, Jessica R; Venkatakrishnan, A J et al. (2015) Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Science 347:1113-7
Janda, Claudia Y; Waghray, Deepa; Levin, Aron M et al. (2012) Structural basis of Wnt recognition by Frizzled. Science 337:59-64
Bazan, J Fernando; Janda, Claudia Y; Garcia, K Christopher (2012) Structural architecture and functional evolution of Wnts. Dev Cell 23:227-32