Kowalski-Jahn, M. | Search bioRxiv

bioRxiv2022

WNT stimulation induced conformational dynamics in the Frizzled-Dishevelled interaction

Frizzleds (FZD1-10) are G protein-coupled receptors containing an extracellular cysteine-rich-domain (CRD) that presents the orthosteric binding site of the 19 mammalian WNTs, the endogenous agonists of FZDs. FZDs signal via a diverse set of effector proteins, of which Dishevelled (DVL1-3) is the most well studied and which acts as a hub for several FZD-mediated signaling pathways. However, the mechanistic details of how FZD-DVL interaction mediate pathway initiation and provide pathway selectivity remain an enigma. Here, we use bioluminescence resonance energy transfer-based assays employing Venus-tagged DVL2 together with NanoLuciferase-tagged FZD5 to investigate the WNT-3A- and WNT-5A-induced dynamics of the FZD-DVL interaction. Our biophysical assessment suggests that the ligand-induced BRET changes over time originate from conformational dynamics in the FZD5-DVL2 complex rather than recruitment dynamics of DVL2 to FZD5. Thus, we suggest that extracellular agonist and intracellular transducers could cooperate with each other through allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs. One Sentence SummaryAnalysis of the interaction of FZD5 and DVL2 uncovers WNT-induced conformational dynamics of a WNT-FZD5-DVL2 complex.

pharmacology and toxicology↗

bioRxiv2021

Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/{beta}-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

cell biology↗