A frizzled quest to dissect the molecular pharmacology of WNT signaling : from biology to signaling mechanism(s)

The wingless/int1 (WNT)/Frizzled (FZD) family of signal transduction pathways is highly conserved across species and controls essential physiological functions important for embryonic development, stem cell renewal, proliferation, differentiation, and cell polarity. Dysregulation of these signaling pathways leads to developmental abnormalities or other conditions such as inflammation, cancer, or neurological disorders. In mammals, 19 different WNTs can bind to and interact with ten isoforms of FZD in a plethora of combinations. These seven transmembrane-spanning receptors are categorized in the Class Frizzled within the superfamily of G protein-coupled receptors (GPCRs). Several important co-factors are known to aid in the activation of WNT/FZD signaling, such as Disheveled (DVL) or low density lipoprotein receptor related protein 5 and 6 (LRP5/6). In addition, interactions of FZDs with heterotrimeric G proteins have continuously been reported. Upon ligand binding, activation of β-catenin-dependent and/or β-catenin-independent downstream signaling pathways takes place.

The overall aim of this thesis was to shed light on mechanistics of WNT/FZD signaling and pharmacology from different angles: In paper I, we investigated the presence and role of WNT-5A in human glioblastomas, a WNT important for neurological functions in the central nervous system (CNS) and found to be dysregulated in many cancers. In this study, we describe the correlative nature of high WNT-5A expression with upregulation of genes involved in immunological processes as well as increased microglia infiltration in the tumor microenvironment. In paper II and III, we focus on FZD4, a FZD isoform important for retinal vascularization. We provide functional evidence for the interaction of FZD4 with heterotrimeric Gα12/13, which is independent of DVL and LRP5/6, and show activation of downstream signaling events. We further describe a novel signaling route through NorrinFZD4-Gα12/13, which exerts an inhibitory effect on the classical Norrin-FZD4-β-catenin signaling pathway known to be important in angiogenesis, thus arguing for a concept of cross-talk and feedback inhibition from the same FZD isoform, a notion that is as of yet completely unappreciated.

In addition, this thesis tries to point out the current limitations and struggles in the field of studying WNT/FZD signaling and the need for further studies identifying crucial links to signal specification, which would aid in future drug development targeting this pathway.

List of scientific papers

I. High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes. Dijksterhuis J, Arthofer E, Marinescu V, Nelander S, Uhlén M, Pontén F, Mulder J, Schulte G. Experimental Cell Research. 2015 Dec;339(2):280-8.
https://doi.org/10.1016/j.yexcr.2015.10.022

II. WNT stimulation dissociates a Frizzled 4 inactive state complex with Gα12/13. Arthofer E, Hot B, Petersen J, Strakova K, Jäger S, Grundmann M, Kostenis E, Gutkind JS, Schulte G. Molecular Pharmacology. 2016 Oct 1;90 (4) 447-459.
https://doi.org/10.1124/mol.116.104919

III. Gα12/13 as a central regulator of FZD4 signaling. Arthofer E, Zhang C, Junge H, Gutkind JS, Balla T, Schulte G. [Manuscript]