Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity.

Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel antiosteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of transcriptional reprogramming during human osteoclast differentiation.

Restraining of glycoprotein VI- and integrin α2β1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2β1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2β1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation.

The functional profiles of fentanyl and nitazene analogs at the μ-opioid receptor - high efficacy is dangerous regardless of signaling bias.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the 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 reduced opioid side effects. Here, we characterized the functional profiles of various NSOs at MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach.

Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity.

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties of receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and the resulting effects is important.

G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation.

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment.

PF4 activates the c-Mpl-Jak2 pathway in platelets.

Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet α-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

Background: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase.

Amplified inhibition of atherosclerotic plaque-induced platelet activation by glenzocimab with dual antiplatelet therapy.

Background: Aspirin and platelet P2Y inhibitors, such as ticagrelor, suboptimally inhibit microvascular thrombosis during ST-elevation myocardial infarction. Glycoprotein (GP) IIb/IIIa inhibitors may further inhibit this but cause excessive bleeding.

Objectives: We investigated whether combination of glenzocimab, a GPVI inhibitor, with aspirin and ticagrelor provides additional antithrombotic effects, as GPVI has a critical role in atherothrombosis but minimal involvement in hemostasis.

Validation of agent-based models of surface receptor oligomerisation.

Receptor dimerisation and higher order oligomerisation regulates signalling by a wide variety of transmembrane receptors. We discuss how agent-based modelling (ABM) combined with advanced microscopy and structural studies can provide new insights into the regulation of clustering, including spatial considerations, revealing novel targets for therapeutic intervention.

Pharmacological and Physicochemical Properties Optimization for Dual-Target Dopamine D (DR) and μ-Opioid (MOR) Receptor Ligands as Potentially Safer Analgesics.

A new generation of dual-target μ opioid receptor (MOR) agonist/dopamine D receptor (DR) antagonist/partial agonists with optimized physicochemical properties was designed and synthesized. Combining in vitro cell-based on-target/off-target affinity screening, in silico computer-aided drug design, and BRET functional assays, we identified new structural scaffolds that achieved high affinity and agonist/antagonist potencies for MOR and DR, respectively, improving the dopamine receptor subtype selectivity (e.g.

Antagonistic Roles of Human Platelet Integrin αIIbβ3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

Background: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches.

Methods: Whole-blood perfusion was performed over a collagen surface at arterial shear rate.

Conformational flexibility of β-arrestins - How these scaffolding proteins guide and transform the functionality of GPCRs.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and β-arrestins. To influence specific GPCR signaling behaviors, β-arrestins recruit effectors to form larger signaling complexes.

Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear.

Background: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation.

Objectives: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear.

Methods: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands.

RISING STARS: Targeting G protein-coupled receptors to regulate energy homeostasis.

G protein-coupled receptors (GPCRs) have a critical role in energy homeostasis, contributing to food intake, energy expenditure and glycaemic control. Dysregulation of energy expenditure can lead to metabolic syndrome (abdominal obesity, elevated plasma triglyceride, LDL cholesterol and glucose, and high blood pressure), which is associated with an increased risk of developing obesity, diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular complications. As the prevalence of these chronic diseases continues to rise worldwide, there is an increased need to understand the molecular mechanisms by which energy expenditure is regulated to facilitate the development of effective therapeutic strategies to treat and prevent these conditions.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type.

Background:  In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1.

GIP reduces osteoclast activity and improves osteoblast survival in primary human bone cells.

Objective: Drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are emerging as treatments for type-2 diabetes and obesity. GIP acutely decreases serum markers of bone resorption and transiently increases bone formation markers in short-term clinical investigations. However, it is unknown whether GIP acts directly on bone cells to mediate these effects.

Recent advances in calcium-sensing receptor structures and signaling pathways.

The calcium-sensing receptor (CaSR) is a class C GPCR that has a fundamental role in extracellular calcium homeostasis by regulating parathyroid hormone release and urinary calcium excretion. Germline mutations in the receptor cause disorders of calcium homeostasis and studies of the functional effects of these mutations has facilitated understanding of CaSR signaling and how allosteric modulators affect these responses. In the past year, five cryo-EM structures of the near full-length CaSR have been published, demonstrating how agonist-binding transmits changes in the CaSR extracellular domain to the transmembrane region to activate G proteins, and how allosteric modulators affect these structural dynamics.

Characterizing the binding of glycoprotein VI with nanobody 35 reveals a novel monomeric structure of glycoprotein VI where the conformation of D1+D2 is independent of dimerization.

Background: The platelet-signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site.

Heparin and heparin proteoglycan-mimetics activate platelets via PEAR1 and PI3Kβ.

Background: Platelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease.

Objectives: We have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan.

Dopamine D/D Receptor Ligands Based on Cariprazine for the Treatment of Psychostimulant Use Disorders That May Be Dual Diagnosed with Affective Disorders.

Highly selective dopamine D receptor (DR) partial agonists/antagonists have been developed for the treatment of psychostimulant use disorders (PSUD). However, none have reached the clinic due to insufficient potency/efficacy or potential cardiotoxicity. Cariprazine, an FDA-approved drug for the treatment of schizophrenia and bipolar disorder, is a high-affinity DR partial agonist ( = 0.

Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane.

The γ-aminobutyric acid type B (GABA) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABA receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABA receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments.