Molecular mechanisms of WNT signaling and receptor-transducer coupling

Wingless and int-1 (WNT) signaling is a group of evolutionarily conserved signal transduction pathways that are vital for fundamental cellular processes. The signals are transduced via receptors at the cell membrane, Frizzleds (FZDs), which belong to class F of GPCRs. In this thesis, the focus is on WNT signaling, the binding of WNT-3A to FZDs, and their coupling to their primary intracellular transducer, Dishevelled (DVL), constitutively, i.e. in the absence of ligands.

WNT-3A has been extensively studied for its role in beta-catenin dependent signaling. In this work, eGFP-WNT-3A was used in combination with HiBiT-tagged FZD1-10 to determine the binding kinetics in a live cell nanoBRET assay. The kinetic parameters were determined from both kinetic and saturation binding curves. Among all FZDs, except for FZD3 and FZD9 for which binding could not be detected, the determined affinities range from single to double digit nanomolar concentrations. Additionally, the utility of this assay for other experimental modes was shown, including competition binding with commercial preparations of WNT- 3A, WNT-5A, WNT-5B, WNT-10B, WNT-11 and WNT-16B.

In addition to ligand induced signaling, constitutive activity, which is defined as coupling of the receptor to its transducer in the absence of stimulation by a ligand can have important functional consequences. To this end, G protein tri-cistronic activity sensors (G-CASE) were designed and in addition to measuring ligand induced G protein coupling, were validated specifically for determining constitutive coupling in GPCRs.

FZDs couple constitutively to DVL. To this end, a FZD6-DVL3 complex was formed and stabilized for cryogenic electron microscopy (cryoEM) experiments, which enabled the determination of the structure at an overall resolution of 3.6 Å. The structure consists of FZD6 and residues 409-495 of DVL3 (DEP domain). The receptor exhibits a typical GPCR-like organization with seven transmembrane helices. The interaction of FZD6 with DEP is characterized by two sites: 1) a lipophilic cavity formed by residues L3935.69, V3965.72, V3995.75 and I4005.76 on FZD6 and residues L434 and I436 on DVL3 and 2) a polar site formed by residues R22612.49, K4126.28, R4166.32 and K4988.49 on FZD6 and K435 on DVL3, surrounding an unassigned density. Single, alanine point mutations in R22612.49, R4166.32, K4988.49, as well as the molecular switch W4937.55 in the vicinity, reduce the relative affinity of FZD6 to DVL3 (amino acids 243-496). The moiety in this unassigned density may play a central role in coordinating the polar residues surrounding this site.

Overall, the work presented in this thesis adds to our knowledge on WNT signaling and receptor-transducer coupling methodologically with new approaches to study WNT-FZD kinetics and constitutive GPCR coupling, as well as mechanistically with novel information on the molecular interaction of FZD6-DVL3, with the first structure of a FZD-DVL complex reconstituted in a lipid environment.

List of scientific papers

I. Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBIT/BRET Assessments. Paweł Kozielewicz, Rawan Shekhani, Stefanie Moser, Carl-Fredrik Bowin, Janine Wesslowski, Gary Davidson, Gunnar Schulte. ACS Pharmacol Transl Sci. 2021 May 11;4(3):1235-1245.
https://doi.org/10.1021/acsptsci.1c00084

II. Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors. Hannes Schihada, Rawan Shekhani, Gunnar Schulte. Sci Signal. 2021 Sep 7;14(699):eabf1653.
https://doi.org/10.1126/scisignal.abf1653

III. Structural insight into the functional interaction between Frizzled 6 and the transducer protein Dishevelled. Rawan Shekhani, Magdalena M Scharf, Jan Hendrik Voss, Jia Yu Ho, Fanny Peysson, Shi Min Tan, Allan H. Pang, Jitender Kumar, Vítězslav Bryja, Konstantinos Tripsianes, Sébastien Granier, Rémy Sounier, Yong Zi Tan, Gunnar Schulte. [Manuscript]