A Putative Frizzled 7-Targeting Compound Acts as a Firefly Luciferase Inhibitor.

The Frizzled family (FZD) of G protein-coupled receptors regulates WNT signaling mediating proliferative input. Dysregulation of FZD and exaggerated WNT/β-catenin signaling is frequently observed in intestinal cancers. Therefore, it is attractive to develop therapeutics targeting FZD for cancer treatment.

A molecular mechanism to diversify Ca signaling downstream of Gs protein-coupled receptors.

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca. By combining CRISPR/Cas9 genome editing to delete Gα, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca release module.

Class-Wide Analysis of Frizzled-Dishevelled Interactions Using BRET Biosensors Reveals Functional Differences among Receptor Paralogs.

Wingless/Int-1 (WNT) signaling is mediated by WNT binding to 10 Frizzleds (FZD), which propagate the signal inside the cell by interacting with different transducers, most prominently the phosphoprotein Dishevelled (DVL). Despite recent progress, questions about WNT/FZD selectivity and paralog-dependent differences in the FZD/DVL interaction remain unanswered. Here, we present a class-wide analysis of the FZD/DVL interaction using the DEP domain of DVL as a proxy in bioluminescence resonance energy transfer (BRET) techniques.

Structural basis of frizzled 7 activation and allosteric regulation.

Frizzleds (ten paralogs: FZD) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD, one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract.

Measurement of Atmospheric Mercury: Current Limitations and Suggestions for Paths Forward.

Mercury (Hg) researchers have made progress in understanding atmospheric Hg, especially with respect to oxidized Hg (Hg) that can represent 2 to 20% of Hg in the atmosphere. Knowledge developed over the past ∼10 years has pointed to existing challenges with current methods for measuring atmospheric Hg concentrations and the chemical composition of Hg compounds. Because of these challenges, atmospheric Hg experts met to discuss limitations of current methods and paths to overcome them considering ongoing research.

Traceable Calibration of Atmospheric Oxidized Mercury Measurements.

Most previous measurements of oxidized mercury were collected using a method now known to be biased low. In this study, a dual-channel system with an oxidized mercury detection limit of 6-12 pg m was deployed alongside a permeation tube-based automated calibrator at a mountain top site in Steamboat Springs Colorado, USA, in 2021 and 2022. Permeation tubes containing elemental mercury and mercury halides were characterized via an International System of Units (SI)-traceable gravimetric method and gas chromatography/mass spectrometry before deployment in the calibrator.

Illuminating Neuropeptide Y Y Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y receptor (YR), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The YR is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, YR binding affinities have been mostly studied in radiochemical binding assays.

Frizzleds act as dynamic pharmacological entities.

The Frizzled family of transmembrane receptors (FZD) belongs to the class F of G protein-coupled receptors (GPCRs). FZDs bind to and are activated by Wingless/Int1 (WNT) proteins. The WNT/FZD signaling system regulates crucial aspects of developmental biology and stem-cell regulation.

Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD.

The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition.

Atmospheric Dry and Wet Deposition of Total Phosphorus to the Great Lakes.

Quantifying atmospheric loadings of total phosphorus (TP) to freshwater environments is essential to improve understanding of its fate and transport, and to mitigate the effects of excessive levels in freshwater ecosystems. To date, atmospheric deposition of TP in the U.S.

Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive.

NanoBiT- and NanoBiT/BRET-based assays allow the analysis of binding kinetics of Wnt-3a to endogenous Frizzled 7 in a colorectal cancer model.

Background And Purpose: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt-FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment.

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

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-DVL2 interaction elicited by WNT-3A and WNT-5A.

Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs.

Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand.

Development of a Neurotensin-Derived Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS Receptor-Expressing Tumors.

Overexpression of the neurotensin receptor type 1 (NTSR), a peptide receptor located at the plasma membrane, has been reported for a variety of malignant tumors. Thus, targeting the NTSR with F- or Ga-labeled ligands is considered a straightforward approach towards in vivo imaging of NTSR-expressing tumors via positron emission tomography (PET). The development of suitable peptidic NTSR PET ligands derived from neurotensin is challenging due to proteolytic degradation.

Feather mercury concentrations in omnivorous and granivorous terrestrial songbirds in Southeast Michigan.

Sublethal exposure to methylmercury (MeHg) can have consequences for the reproductive, neurological, and physiological health of birds. Songbirds, regardless of trophic position, are often exposed to mercury (Hg) and may be at risk for health effects - especially if they inhabit a place that is subject to high Hg atmospheric deposition and/or have local conditions that are prone to methylation. This study investigates Hg concentrations in terrestrial songbirds of Southeast Michigan, where historical and present-day anthropogenic emissions of heavy metals are elevated.

N-Terminus to Arginine Side-Chain Cyclization of Linear Peptidic Neuropeptide Y Y Receptor Ligands Results in Picomolar Binding Constants.

The family of neuropeptide Y (NPY) receptors comprises four subtypes (YR, YR, YR, YR), which are addressed by at least three endogenous peptides, i.e., NPY, peptide YY, and pancreatic polypeptide (PP), the latter showing a preference for YR.

Addressing a Trapped High-Energy Water: Design and Synthesis of Highly Potent Pyrimidoindole-Based Glycogen Synthase Kinase-3β Inhibitors.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC value of our co-crystallized glycogen synthase kinase-3β (GSK-3β) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor ()-3-(3-((7-ethynyl-9-pyrimido[4,5-]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (-, IC value of 6 nM).

Differently fluorescence-labelled dibenzodiazepinone-type muscarinic acetylcholine receptor ligands with high MR affinity.

A series of fluorescent dibenzodiazepinone-type muscarinic acetylcholine M receptor (MR) ligands was synthesized using various fluorescent dyes (5-TAMRA, / ≈ 547/576 nm; BODIPY 630/650, / ≈ 625/640 nm; pyridinium dye Py-1, / ≈ 611/665 nm and pyridinium dye Py-5, / ≈ 465/732 nm). All fluorescent probes exhibited high MR affinity (p (radioligand competition binding): 8.75-9.

BRET- and fluorescence anisotropy-based assays for real-time monitoring of ligand binding to M muscarinic acetylcholine receptors.

BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M muscarinic acetylcholine receptor (MR) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments.