Intron retention of an adhesion GPCR generates 1TM isoforms required for 7TM-GPCR function.

Adhesion G protein-coupled receptors (aGPCRs) are expressed in all organs and are involved in various mechanobiological processes. They are heavily alternatively spliced, forecasting an extraordinary molecular structural diversity. Here, we uncovered the existence of unconventional single-transmembrane (1TM)-containing ADGRL/Cirl proteins devoid of the conventional GPCR layout (i.

Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling.

The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially regarding the early dynamics of downstream TLR pathways. Here, we present a label-free optical biosensor-based assay as a method for detecting TLR activation in a native and label-free environment and defining the dynamics of TLR pathway activation.

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.

Pharmacological Gq inhibition induces strong pulmonary vasorelaxation and reverses pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease with limited survival. Herein, we propose the pharmacological inhibition of Gq proteins as a novel concept to counteract pulmonary vasoconstriction and proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in PAH. We demonstrate that the specific pan-Gq inhibitor FR900359 (FR) induced a strong vasorelaxation in large and small pulmonary arteries in mouse, pig, and human subjects ex vivo.

GRK specificity and Gβγ dependency determines the potential of a GPCR for arrestin-biased agonism.

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent β-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein βγ-subunits to translocate to the membrane.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.

The marine sponge-derived fungus 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid -methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E () and F ().

Transactivation of Met signaling by oncogenic Gnaq drives the evolution of melanoma in Hgf-Cdk4 mice.

Recent pan-cancer genomic analyses have identified numerous oncogenic driver mutations that occur in a cell-type and tissue-specific distribution. For example, oncogenic mutations in Braf and Nras genes arise predominantly in melanocytic neoplasms of the epidermis, while oncogenic mutations in Gnaq/11 genes arise mostly in melanocytic lesions of the dermis or the uvea. The mechanisms promoting cell-type and tissue-specific oncogenic events currently remain poorly understood.

A human obesity-associated MC4R mutation with defective Gq/11α signaling leads to hyperphagia in mice.

Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice.

Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2.

Herein, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of CXC chemokine receptor 2 (CXCR2), a G protein-coupled receptor (GPCR) that has been pursued as a drug target in oncology and inflammation. Starting from the cocrystallized intracellular CXCR2 antagonist 00767013 (), tetramethylrhodamine (TAMRA)-labeled CXCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CXCR2. By means of these studies, we developed Mz438 () as a high-affinity and selective fluorescent CXCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and high-throughput manner.

The Bacterial G Signal Transduction Inhibitor FR900359 Impairs Soil-Associated Nematodes.

The cyclic depsipeptide FR900359 (FR) is derived from the soil bacterium Chromobacterium vaccinii and known to bind G proteins of mammals and insects, thereby abolishing the signal transduction of their G protein-coupled receptors, a process that leads to severe physiological consequences. Due to their highly conserved structure, G family of proteins are a superior ecological target for FR producing organisms, resulting in a defense towards a broad range of harmful organisms. Here, we focus on the question whether bacteria like C.

Glucose-stimulated insulin secretion depends on FFA1 and Gq in neonatal mouse islets.

Aims/hypothesis: After birth, the neonatal islets gradually acquire glucose-responsive insulin secretion, a process that is subjected to maternal imprinting. Although NEFA are major components of breastmilk and insulin secretagogues, their role for functional maturation of neonatal beta cells is still unclear. NEFA are the endogenous ligands of fatty acid receptor 1 (FFA1, encoded by Ffar1 in mice), a Gq-coupled receptor with stimulatory effect on insulin secretion.

IL-6 in the infarcted heart is preferentially formed by fibroblasts and modulated by purinergic signaling.

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model.

Platelet P2Y receptor exhibits constitutive G protein signaling and β-arrestin 2 recruitment.

Background: Purinergic P2Y and P2Y receptors (P2Y-R and P2Y-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y-R is the major target of antiplatelet drugs, no P2Y-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding.

Giving ERK a jERK from the endosome.

Endosomes, long considered as sorting stations for downregulation and recycling of cell surface G protein-coupled receptors (GPCRs), are now well-established sites of signal transduction. Recent work from the groups of Jin Zhang and Roger Sunahara features endosomes as signaling hubs and physical platforms for noncanonical activation of ERK by GPCRs.

Oxytocin-Modulated Ion Channel Ensemble Controls Depolarization, Integration and Burst Firing in CA2 Pyramidal Neurons.

Oxytocin (OXT) and OXT receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current-clamp and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity.

How Carvedilol activates β-adrenoceptors.

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β-adrenoceptors, arrestin-biased signalling via β-adrenoceptors is a molecular mechanism proposed to explain the survival benefits.

Humanized zebrafish as a tractable tool for in vivo evaluation of pro-myelinating drugs.

Therapies that promote neuroprotection and axonal survival by enhancing myelin regeneration are an unmet need to prevent disability progression in multiple sclerosis. Numerous potentially beneficial compounds have originated from phenotypic screenings but failed in clinical trials. It is apparent that current cell- and animal-based disease models are poor predictors of positive treatment options, arguing for novel experimental approaches.

Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching.

Objective: To determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc).

Methods: C57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4 or CD8 T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope.

Selective optogenetic control of G signaling using human Neuropsin.

G proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of G signaling in vitro and in vivo with high spatio-temporal resolution.

Adipocyte G signaling is a regulator of glucose and lipid homeostasis in mice.

Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte G signaling in vitro and in vivo (in mice).