An activating CaSR variant with biased signaling reveals a critical residue for Gα11 coupling.

Autosomal dominant hypocalcemia (ADH) is due to enhanced calcium-dependent signaling caused by heterozygous gain-of-function (GOF) variants in the CASR gene (ADH1) or in the GNA11 gene, encoding Gα11 (ADH2). Both ADH1 and ADH2 are associated with hypocalcemia and normal or inappropriately low levels of circulating PTH. ADH1 patients typically manifest hypercalciuria, while ADH2 is associated with short stature in approximately 42% of cases.

The MRAP2 accessory protein directly interacts with melanocortin-3 receptor to enhance signaling.

The central melanocortin system links nutrition to energy expenditure, with melanocortin-4 receptor (MC4R) controlling appetite and food intake, and MC3R regulating timing of sexual maturation, rate of linear growth and lean mass accumulation. Melanocortin-2 receptor accessory protein-2 (MRAP2) is a single transmembrane protein that interacts with MC4R to potentiate it's signalling, and human mutations in MRAP2 cause obesity. Previous studies have been unable to consistently show whether MRAP2 affects MC3R activity.

Endomembrane GPCR signaling: 15 years on, the quest continues.

G-protein-coupled receptors (GPCRs) are the largest family of cell receptors. They mediate the effects of a multitude of endogenous and exogenous cues, are deeply involved in human physiology and disease, and are major pharmacological targets. Whereas GPCRs were long thought to signal exclusively at the plasma membrane, research over the past 15 years has revealed that they also signal via classical G-protein-mediated pathways on membranes of intracellular organelles such as endosomes and the Golgi complex.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor.

Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells.

ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.

Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling.

Design, Synthesis, and Characterization of New δ Opioid Receptor-Selective Fluorescent Probes and Applications in Single-Molecule Microscopy of Wild-Type Receptors.

The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time.

Design, Synthesis, and Evaluation of a New Chemotype Fluorescent Ligand for the P2Y Receptor.

The P2Y receptor (P2YR) is a target for diseases including cancer, idiopathic pulmonary fibrosis, and atherosclerosis. However, there are insufficient P2YR antagonists available for validating P2YR function and future drug development. Evaluation of how ()-5-(7-chloro-2-((2-ethoxyethyl)amino)-4-benzo[5,6]cyclohepta[1,2-]thiazol-4-yl)-1-methyl-4-thioxo-3,4-dihydropyrimidin-2(1)-one, a previously published thiazole-based analogue of AR-C118925, binds in a P2YR homology model was used to design new P2YR antagonist scaffolds.

Enantioselective synthesis of 14-hydroxy-6-oxomorphinans.

The enantioselective synthesis of pharmacologically important 14-hydroxy-6-oxomorphinans is described. 4,5-Desoxynaltrexone and 4,5-desoxynaloxone were prepared using this route and their biological activities against the opioid receptors were measured.

Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice.

Background: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury, supporting platelet activation and thrombosis.

Objectives: To investigate the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced platelet activation and arterial thrombosis.

Methods: The effect of hydroxychloroquine on hemin-induced platelet activation and hemolysis-induced platelet recruitment and aggregation was measured in washed platelets and hemolyzed blood, respectively.

S1PR3 agonism and S1P lyase inhibition rescue mice in the severe state of experimental sepsis.

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care.

Microbially conjugated bile salts found in human bile activate the bile salt receptors TGR5 and FXR.

Background: Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation.

Methods: N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized.

Comprehensive functional characterization of a novel ANO6 variant in a new patient with Scott syndrome.

Background: Scott syndrome is a mild platelet-type bleeding disorder, first described in 1979, with only 3 unrelated families identified through defective phosphatidylserine (PS) exposure and confirmed by sequencing. The syndrome is distinguished by impaired surface exposure of procoagulant PS on platelets after stimulation. To date, platelet function and thrombin generation in this condition have not been extensively characterized.

Arrestin-centred interactions at the membrane and their conformational determinants.

More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation.

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as μ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach.

Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein 1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation.

A time-resolved Förster resonance energy transfer assay to investigate drug and inhibitor binding to ABCG2.

The human ATP-binding cassette (ABC) transporter, ABCG2, is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2.