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

Kowalski-Jahn, Maria; Schihada, Hannes; Turku, Ainoleena; Huber, Thomas; Sakmar, Thomas P.; Schulte, Gunnar

doi:10.1126/sciadv.abj7917

Cited by 23 publications

(14 citation statements)

References 72 publications

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“…Further work in this direction is required to clarify mechanistic details. Nevertheless, the emerging concept of conformational dynamics of FZDs is in agreement with general concepts of GPCR activation ( 42 ) and is further supported by previously reported WNT-induced dissociation of FZD dimers ( 43 ), WNT-induced changes in FRET-based FZD conformational sensors ( 20 , 38 ), WNT concentration-response curves for fluorescence changes in circularly permuted GFP (cpGFP)–tagged FZD conformational sensors ( 3 ), and the WNT-induced dynamics of the CRD of FZDs that precede alterations in core conformation ( 44 ). Thus, WNT-induced pathway selection and signal initiation of FZDs are not only determined by their proximity to different coreceptors but also appear to be, at least in part, determined by a conformational selection of WNT-FZD-DVL interaction.…”

Section: Discussionsupporting

confidence: 88%

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction

et al. 2023

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Frizzleds (FZDs) are G protein–coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD 5 -DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD 5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD 5 -DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD 5 -DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD 5 . The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.

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Section: Discussionsupporting

confidence: 88%

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction

et al. 2023

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“…We tested this model and concluded that WNTs induce FZD-DVL conformational dynamics independently of LRP5/6, clearly suggesting that (i) WNT-FZD interactions can occur independently of LRP5/6, that (ii) agonist-induced FZD-DVL dynamics potentially precede LRP5/6 signalosome formation and that (iii) WNT-induced conformational changes of FZDs regulate the initial communication with DVL in a receptor complex reminiscent of the ternary complex described for agonist- and effector-bound GPCRs ( 4, 22 ). The emerging concept of conformational dynamics of FZDs is in agreement with general concepts of GPCR activation ( 41 ) and is further supported by previously reported WNT concentration response curves for fluorescence changes in cpGFP-tagged FZD conformational sensors ( 3 ) and the WNT-induced dynamics of the cysteine-rich domain of FZDs that precede alterations in core conformation ( 42 ). Thus, WNT-induced pathway selection and signal initiation of FZDs is not only determined by their proximity to different co-receptors but appears to be – at least in part – determined by a conformational selection of WNT-FZD-DVL interaction.…”

Section: Discussionsupporting

confidence: 78%

WNT stimulation induced conformational dynamics in the Frizzled-Dishevelled interaction

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Kozielewicz

Grätz

et al. 2022

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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.

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“…Ye et al first employed the strategy for studies of GPCRs ( Ye et al, 2008 ). Specific unnatural amino acids can be introduced to facilitate “targeted photo-crosslinking” or bio-orthogonal covalent labeling with small-molecule fluorophores to create FRET or BRET conformational sensors ( Koole et al, 2017 ; Kowalski-Jahn et al, 2021 ). Simms and colleagues employed a targeted photo-cross-linking strategy to study the ECL2 of CALCRL and identified two major contact points for CGRP, I284 and L291 ( Simms et al, 2018 ).…”

Section: Molecular Characterization Of Gpcr–ramp Interactionsmentioning

confidence: 99%

Elucidating the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins

et al. 2022

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G protein-coupled receptors (GPCRs) are known to interact with several other classes of integral membrane proteins that modulate their biology and pharmacology. However, the extent of these interactions and the mechanisms of their effects are not well understood. For example, one class of GPCR-interacting proteins, receptor activity-modifying proteins (RAMPs), comprise three related and ubiquitously expressed single-transmembrane span proteins. The RAMP family was discovered more than two decades ago, and since then GPCR–RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several secretin (class B) GPCRs, most notably the calcitonin receptor-like receptor. Recent bioinformatics and multiplexed experimental studies suggest that GPCR–RAMP interactions might be much more widespread than previously anticipated. Recently, cryo-electron microscopy has provided high-resolution structures of GPCR–RAMP–ligand complexes, and drugs have been developed that target GPCR–RAMP complexes. In this review, we provide a summary of recent advances in techniques that allow the discovery of GPCR–RAMP interactions and their functional consequences and highlight prospects for future advances. We also provide an up-to-date list of reported GPCR–RAMP interactions based on a review of the current literature. Significance Statement Receptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR)biology and pharmacology. The application of new methodologies to study membrane protein–protein interactions suggests that RAMPs interact with many more GPCRs than had been previously known. These findings, especially when combined with structural studies of membrane protein complexes, have significant implications for advancing GPCR-targeted drug discovery and the understanding of GPCR pharmacology, biology, and regulation.

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

Abstract: WNTs induce conformational changes in the extracellular CRD of Frizzleds irrespective of their signaling profile.

Cited by 23 publications

References 72 publications

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction

WNT stimulation induced conformational dynamics in the Frizzled-Dishevelled interaction

Elucidating the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins

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