ABSTRACT
WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.
Subject(s)
Dishevelled Proteins/metabolism , Drug Discovery/methods , Frizzled Receptors/agonists , Protein Interaction Domains and Motifs/drug effects , Pyridines/chemistry , Thiophenes/chemistry , Wnt Signaling Pathway/drug effects , Cell Membrane/metabolism , Frizzled Receptors/chemistry , Frizzled Receptors/metabolism , HEK293 Cells , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Targeted Therapy/methods , Morpholines/pharmacology , Proof of Concept Study , Purines/pharmacology , Pyridines/pharmacology , Smoothened Receptor/agonists , Structure-Activity Relationship , Thiophenes/pharmacologyABSTRACT
Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 É (CK1É) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1É-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1É in DVL conformational dynamics.
Subject(s)
Casein Kinase 1 epsilon/metabolism , Dishevelled Proteins/metabolism , PDZ Domains/physiology , Wnt Signaling Pathway/physiology , Animals , Biosensing Techniques , Casein Kinase 1 epsilon/genetics , Dishevelled Proteins/genetics , Enzyme Assays/methods , Fluorescence Resonance Energy Transfer , Frizzled Receptors/metabolism , Gene Knockout Techniques , HEK293 Cells , Humans , Microscopy, Fluorescence/methods , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Oocytes , Phosphorylation/physiology , Single-Cell Analysis/methods , Xenopus laevisABSTRACT
Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the ß-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.
Subject(s)
Cell Proliferation , Frizzled Receptors/metabolism , Pancreatic Neoplasms/pathology , Wnt-5a Protein/metabolism , Calcium/metabolism , Diglycerides/metabolism , Frizzled Receptors/chemistry , HEK293 Cells , Humans , Pancreatic Neoplasms/metabolism , Protein Conformation , Protein Kinase C/metabolism , Signal Transduction , Tumor Cells, Cultured , Wnt-5a Protein/chemistryABSTRACT
The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of Mâ receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M1 receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes.
