Search Results (176)

Osteoclastogenesis—the activation and differentiation of osteoclasts—is one of the pivotal processes of bone remodeling and is regulated by RANKL/RANK signaling, the decoy function of osteoprotegerin (OPG), and a cascade of pro- and anti-inflammatory cytokines. The disruption of this balance leads to pathological bone loss in diseases such as osteoporosis and rheumatoid arthritis. FFAR4 (Free Fatty Acid Receptor 4), a G protein-coupled receptor for long-chain omega-3 fatty acids, has been confirmed as a key mediator of metabolic and anti-inflammatory effects. This review focuses on how FFAR4 acts as the selective receptor for the omega-3 fatty acid docosahexaenoic acid (DHA). It activates two divergent signaling pathways. The Gαq-dependent cascade facilitates intracellular calcium mobilization and ERK1/2 activation. Meanwhile, β-arrestin-2 recruitment inhibits NF-κB. These collective actions reshape the cytokine environment. In macrophages, DHA–FFAR4 signaling lowers the levels of TNF-α, interleukin-6 (IL-6), and IL-1β while increasing IL-10 secretion. Consequently, the activation of NFATc1 and NF-κB p65 is profoundly suppressed under TNF-α or RANKL stimulation. Additionally, DHA modulates the RANKL/OPG axis in osteoblastic cells by suppressing RANKL expression, thereby reducing osteoclast differentiation in an inflammatory mouse model. Full article

Spurred by the enormous therapeutic success of glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP1-RAs) against diabetes and obesity, glucagon family receptor pharmacology has garnered a tremendous amount of interest. Glucagon family receptors, e.g., the glucagon receptor itself (GCGR), the GLP-1R, and the glucose-dependent insulinotropic peptide receptor (GIPR), belong to the incretin receptor superfamily, i.e., receptors that increase blood glucose-dependent insulin secretion. All incretin receptors are class B1 G protein-coupled receptors (GPCRs), coupling to the G_s type of heterotrimeric G proteins which activates adenylyl cyclase (AC) to produce cyclic adenosine monophosphate (cAMP). Most GPCRs undergo desensitization, i.e., uncouple from G proteins and internalize, thanks to interactions with the βarrestins (arrestin-2 and -3). Since the βarrestins can also mediate their own G protein-independent signaling, any given GPCR can theoretically signal (predominantly) either via G proteins or βarrestins, i.e., be a G protein- or βarrestin-“biased” receptor, depending on the bound ligand. A plethora of experimental evidence suggests that the GLP-1R does not undergo desensitization in physiologically relevant tissues in vivo, but rather, it produces robust and prolonged cAMP signals. A particular property of constant cycling between the cell membrane and caveolae/lipid rafts of the GLP-1R may underlie its lack of desensitization. In contrast, GIPR signaling is extensively mediated by βarrestins and the GIPR undergoes significant desensitization, internalization, and downregulation, which may explain why both agonists and antagonists of the GIPR exert the same physiological effects. Here, we discuss this evidence and make a case for the GLP-1R being a phenotypically or functionally G_s-selective receptor. We also discuss the implications of this for the development of GLP-1R poly-ligands, which are increasingly pursued for the treatment of obesity and other diseases. Full article

Sleep paralysis (SP), an REM parasomnia, can be characterized as one of the symptoms of narcolepsy. The SP phenomenon involves regaining meta-consciousness by the dreamer during REM, when the physiological atonia of skeletal muscles is accompanied by visual and auditory hallucinations that are perceived as vivid and distressing nightmares. Sensory impressions include personification of an unknown presence, strong chest pressure sensation, and intense fear resulting from subjective interaction with the unfolding nightmare. While the mechanism underlying skeletal muscle atonia is known, the physiology of hallucinations remains unclear. Their complex etiology involves interactions among various membrane receptor systems and neurotransmitters, which leads to altered neuronal functionality and disruptions in sensory perception. According to current knowledge, serotonergic activation of 5-hydroxytryptamine-receptor-2A (5-HT2A)-associated pathways plays a critical role in promoting hallucinogenesis during SP. Furthermore, they share similarities with psychedelic-substance-induced ones (i.e., LSD, psilocybin, and 2,5-dimethoxy-4-iodoamphetamine). These compounds also target the 5-HT2A receptor; however, their molecular mechanism varies from serotonin-induced ones. The current review discusses the intracellular signaling pathways responsible for promoting hallucinations in SP, highlighting the critical role of β-arrestin-2. We propose that the β-arrestin-2 signaling pathway does not directly induce hallucinations but creates a state of network susceptibility that facilitates their abrupt emergence in sensory areas. Understanding the molecular basis of serotonergic hallucinations and gaining better insight into 5-HT2A-receptor-dependent pathways may prove crucial in the treatment of multifactorial neuropsychiatric disorders associated with the dysfunctional activity of serotonin receptors. Full article

Small-molecule glucagon-like peptide-1 receptor agonists (GLP-1RAs) represent an innovative advancement in oral therapeutics, addressing key limitations associated with injectable peptide-based incretin therapies. These nonpeptidic agents exert their actions primarily through non-canonical binding orthosteric sites within the GLP-1 receptor transmembrane domain, enabling selective G protein (Gs)-biased signaling with reduced β-arrestin-mediated adverse effects. Orforglipron has notably advanced through Phase 3 clinical development, demonstrating significant reductions in hemoglobin A1c and body weight (up to 7.9%) with favorable tolerability. Conversely, promising candidates such as danuglipron and lotiglipron were discontinued due to hepatotoxicity, underscoring critical safety concerns intrinsic to small-molecule GLP-1RA development. Current clinical candidates, including GSBR-1290, CT-996, and ECC5004, continue to offer substantial potential due to their oral bioavailability, simplified dosing regimens, and favorable gastrointestinal tolerability. Nevertheless, challenges persist regarding hepatic safety, pharmacodynamic variability, and limited long-term outcome data. This review integrates current structural, pharmacological, and clinical evidence, highlights key mechanistic innovations—including biased agonism, covalent binding strategies, and allosteric modulation—and discusses future directions for this rapidly evolving therapeutic class in metabolic disease management. Full article

CB₁ and CB₂ cannabinoid receptors are members of the GPCR superfamily that modulate the effects of endocannabinoids. CB₁ is the most abundant CB receptor in the central nervous system, while CB₂ is present both peripherally and in the brain. CB₂ plays a role in inflammation, as well as neurodegenerative and psychiatric disorders. To identify new ligands for CB₂, we screened a library of FDA-approved drugs for activity at the receptor using a thallium flux assay, resulting in the discovery of the immunosuppressant mycophenolate mofetil as a potent, selective activator of CB₂. Further characterization of the compound confirmed agonist activity in a variety of complementary assays, including PI hydrolysis, cAMP inhibition, and β-arrestin recruitment. Radioligand binding assays established a non-competitive interaction with the site occupied by [³H]CP55,940. CB₂ agonists GW-842,166X and MDA7 were also profiled, revealing that GW-842,166X exhibits a similar activity profile to mycophenolate mofetil, whereas MDA7 presents a distinct profile. These differences provide insight into the complex CB₂ pharmacology impacting preclinical and clinical studies, and ultimately, new treatment strategies for brain disorders. Full article

Background: Autism spectrum disorder (ASD) involves complex interactions between genetic and environmental factors. Recent studies suggest that dysregulation of β-arrestin2 (Arrb2) in the central nervous system is linked to ASD. However, its specific mechanisms remain unknown. Methods: This study employs a systems genetics approach to comprehensively investigate Arrb2 in multiple brain tissues, including the amygdala, cerebellum, hippocampus, and prefrontal cortex, using BXD recombinant inbred (RI) strains. In addition, genetic variance analysis, correlation analysis, expression quantitative trait loci (eQTL) mapping, and functional annotation were used to identify the key downstream targets of Arrb2, validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting (WB). Results: Arrb2 exhibited expression variations across the four brain regions in BXD mice. eQTL mapping revealed that Arrb2 is cis-regulated, and increased Arrb2 expression levels were significantly correlated with ASD-like symptoms, such as impaired social interactions and abnormal learning and memory. Furthermore, protein–protein interaction (PPI) network analysis, tissue correlation, functional relevance to autism, and differential expression identified eight downstream candidate genes regulated by Arrb2. The experimental results demonstrated that deletion of Arrb2 led to the downregulation of Myh9, Dnmt1, and Brd4 expression, along with protein kinase A (PKA)-induced hyperactivation of Synapsin I. These findings suggest that Arrb2 may contribute to the pathogenesis of autism by modulating the expression of these genes. Conclusions: This study highlights the role of Arrb2 in ASD pathogenesis and identifies Myh9, Dnmt1, and Brd4 as key downstream regulators. These findings provide new insights into the molecular mechanisms of ASD and pave the way for novel therapeutic targets. Full article

Background/Objectives: D4R antagonists have recently been suggested as potential therapeutic alternatives to the standard treatments of glioblastoma (GBM). In this study, new piperidine-based ligands, analogs of the potent and selective D4R compounds 77-LH-28-1 (7) and its 4-benzyl analog 8, were synthesized and studied to investigate the effects produced by variations in the distances between the pharmacophoric features on the D4R affinity and selectivity. Methods: All the new compounds 9–20 were evaluated for their radioligand binding affinity at D2-like receptor subtypes and the results were rationalized by docking studies and molecular dynamics (MD) simulations. The functional profiles of the most interesting derivatives were assessed at D4R Go and Gi protein and β-arrestin by BRET assay and their potential anticancer activity was determined in GBM cell lines. Results: Radioligand binding results highlighted that the derivatives bearing a terminal butyl chain showed structure–activity relationships different from those with a benzyl terminal. From functional studies performed on the best derivatives 12 and 16, the response profiles of both compounds were more robust in antagonist mode, with derivative 16 showing higher antagonist potency than 12 across all three transducers. Interestingly, 12 and 16 dose-dependently decreased the cell viability of GBM cells, inducing cell death and cell cycle arrest, promoting an increase in ROS production, causing mitochondrial dysfunction, and significantly inhibiting colony formation. Conclusions: The promising biological profiles of 12 and 16 make them new lead candidates that warrant further investigation to gain a better understanding of the mechanism behind their antitumor activity and better evaluate their potential for GBM treatment. Full article

The seven transmembrane-spanning lutropin/chorionic gonadotropin receptors (LH/CGRs) trigger extracellular signal-related kinases (ERK1/2) via a noticeable network dependent on either G protein (Gαs) or β-arrestins. LH/CGRs are highly conserved, with the largest region within the transmembrane helices and common N-glycosylation sites in the extracellular domain. We aimed to determine the glycosylation sites that play crucial roles in cAMP and pERK1/2 regulation by constructing four mutants (N49Q, N201Q, N306Q, and N312Q). The cAMP response in cells expressing the N201Q mutant was completely impaired, despite high-dose agonist treatment. The cell-surface expression level was lowest in transiently transfected cells, but normal surface loss of the receptor occurred in cells expressing the wild-type and other mutant proteins. However, the N201Q mutant was only slightly reduced after 5 min of agonist stimulation. All mutants showed a peak in cAMP signaling 5 min after stimulation with a pERK1/2 agonist. Of note, cAMP activity was completely impaired in the N201Q mutant; however, this mutant still displayed a pERK1/2 response. These data show that the specific N-linked glycosylation site in eel LH/CGR is clearly distinguished by its differential responsiveness to cAMP signaling and pERK1/2 activity. Thus, we suggest that the cAMP and pERK1/2 signaling pathways involving eel LH/CGRs represent pleiotropic signal transduction induced by agonist treatment. Full article

Upon activation of certain G protein-coupled receptors, Mdm2 promotes the ubiquitination of both GRK2 and arrestin3. Similar to arrestin3, GRK2 ubiquitination was associated with its endocytic activity and proteasomal degradation. Ubiquitination of GRK2 was essential for arrestin3 ubiquitination, and vice versa. Cellular components involved in arrestin3 ubiquitination, including Gβγ, clathrin, and 14-3-3η, were also necessary for GRK2 ubiquitination. Additionally, the arrestin-biased signaling pathway contributed to the ubiquitination of both GRK2 and arrestin3. By employing Mdm2-knockdown cells alongside GRK2 and arrestin3 mutants deficient in ubiquitination sites, as well as receptors lacking phosphorylation sites, we established that the ubiquitinated forms of GRK2 and arrestin3 facilitate clathrin-dependent endocytosis, whereas non-ubiquitinated GRK2 and arrestin3 are responsible for caveolar and a distinct third endocytic pathway, respectively. In the context of clathrin-mediated endocytosis, arrestin3’s interaction with clathrin and GRK2’s interaction with the β2-adaptin subunit of adaptor protein complex 2 were critical. These findings suggest that GRK2 and arrestin3 ubiquitination are mutually dependent, with their ubiquitination states determining their roles in distinct endocytic pathways. Full article

β-arrestin 2 (ARRB2) is involved in the desensitization and trafficking of G protein-coupled receptors (GPCRs) and plays a critical role in cell proliferation, apoptosis, chemotaxis, and immune response modulation. The role of ARRB2 in the pathogenesis of multiple myeloma (MM) has not been elucidated. This study addressed this question by evaluating the expression of ARRB2 in bone marrow (BM) samples from newly diagnosed MM patients and deriving correlations with key clinical outcomes. In light of recent trends towards the use of immune checkpoint inhibitors across malignancies, the effect of ARRB2 in the regulation of the PD-1/PD-L1 axis was also investigated. The expression of ARRB2 was significantly higher in MM patients resistant to proteosome inhibitor (bortezomib) treatment compared to those who responded. Higher ARRB2 expression in the BM of newly diagnosed MM patients was associated with inferior progression-free survival and overall survival. PD-1 expression was downregulated in CD3 T cells isolated from ARRB2 knockout (KO) mice. Furthermore, knockdown of ARRB2 with siRNA reduced PD-1 expression in murine CD3 T cells and PD-L1 expression in murine myeloid-derived suppressor cells. These findings suggest an important role of ARRB2 in MM pathogenesis, potentially mediated via modulation of immune checkpoints in the tumor microenvironment. Our study provides new evidence that ARRB2 may have non-canonical functions independent of GPCRs with relevance to the understanding of MM pathobiology as well as immunotherapy and checkpoint inhibitor escape/resistance more broadly. Full article

Equine follicle-stimulating hormone receptor (eFSHR) contains four extracellular N-linked glycosylation sites, which play important roles in agonist-induced signal transduction. Glycosylation regulates G protein-coupled receptor mechanisms by influencing folding, ligand binding, signaling, trafficking, and internalization. Here, we examined whether the glycosylated sites in eFSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction and the phosphate extracellular signal-regulated kinase 1/2 (pERK1/2) response. We constructed mutants (N191Q, N199Q, N268Q, and N293Q) of the four N-linked glycosylation sites in eFSHR using site-directed mutagenesis. In wild-type (wt) eFSHR, the cAMP response gradually increased dose-dependently, displaying a strong response at the EC₅₀ and Rmax. Two mutants (N191Q and N199Q) considerably decreased the cAMP response. Both EC₅₀ values were approximately 0.46- and 0.44-fold compared to that of the eFSHR-wt, whereas Rmax levels were 0.29- and 0.45-fold compared to eFSHR-wt because of high-ligand treatment. Specifically, the EC₅₀ and Rmax values in the N268Q mutant were increased 1.23- and 1.46-fold, respectively, by eFSHR-wt. pERK1/2 activity in eFSHR-wt cells was rapid, peaked within 5 min, consistently sustained until 15 min, and then sharply decreased. pERK1/2 activity in the N191Q mutant showed a pattern similar to that of the wild type, despite impaired cAMP responsiveness. The N199Q mutant showed low pERK1/2 activity at 5 and 15 min. Interestingly, pERK1/2 activity in the N268Q and N298Q mutants was similar to that of eFSHR-wt at 5 min, but neither mutant showed any signaling at 15 min, despite displaying high cAMP responsiveness. Overall, eFSHR N-linked glycosylation sites can signal to pERK1/2 via PKA and the other signals, dependent on G protein coupling and β-arrestin-dependent recruitment. Our results provide strong evidence for a new paradigm in which cAMP signaling is not activated, yet pERK1/2 cascade remains strongly induced. Full article

Opioids are the most effective option for severe pain. However, it is well documented that the side effects associated with prolonged opioid use significantly constrain dosage in the clinical setting. Recently, researchers have concentrated on the development of biased opioid receptor agonists that preferentially activate the G protein signaling pathway over β-arrestin signaling. This approach is based on the hypothesis that G protein signaling mediates analgesic effects, whereas β-arrestin signaling is implicated in adverse side effects. Although certain studies have demonstrated that the absence or inhibition of β-arrestin signaling can mitigate the incidence of side effects, recent research appears to challenge these earlier findings. In-depth investigations into biased signal transduction of opioid receptor agonists have been conducted, potentially offering novel insights for the development of biased opioid receptors. Consequently, this review elucidates the contradictory roles of β-arrestin signaling in the adverse reactions associated with opioid receptor activation. Furthermore, a comparative analysis was conducted to evaluate the efficacy of the classic G protein-biased agonists, TRV130 and PZM21, relative to the traditional non-biased agonist morphine. This review aims to inform the development of novel analgesic drugs that can optimize therapeutic efficacy and safety, while minimizing adverse reactions to the greatest extent possible. Full article

Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364–470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8–10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG’s FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment. Full article

The search for effective pain management solutions remains a critical challenge, especially amidst growing concerns over the use of conventional opioids. In the US, opioid-related mortality rates have surged to as many as 80 deaths per 100,000 people in some states, with an estimated economic burden of USD 1.5 trillion annually—exceeding the gross domestic product (GDP) of most US industrial sectors. A remarkable breakthrough lies in the discovery that indole and oxindole alkaloids, produced by several genera within the plant Tribe Naucleeae, act on opioid receptors without activating the beta-arrestin-2 pathway, the primary driver of respiratory depression and overdose deaths. This systematic review explores the pharmacological properties, mechanisms of action, dosing considerations, interactions, and long-term effects of mitragynine and corynoxeine, alkaloids from the Southeast Asian plant Mitragyna speciosa (kratom) and others in the Tribe Naucleeae. Mitragynine, a partial opioid receptor agonist, and corynoxeine, known for its anti-inflammatory and neuroprotective effects, demonstrate significant therapeutic potential for managing diverse pain types—including neuropathic, inflammatory, nociceptive, visceral, and central pain syndromes—with a focus on cancer pain. Unlike traditional opioids, these compounds do not recruit beta-arrestin-2, avoiding key adverse effects such as respiratory depression, severe constipation, and rapid tolerance development. Their distinct pharmacological profiles make them innovative candidates for safer, non-lethal pain relief. However, challenges persist, including the unregulated nature of kratom products, inconsistencies in potency due to crude extract variability, potential for misuse, and adverse drug interactions. Addressing these issues requires establishing standardized quality control protocols, such as Good Manufacturing Practices (GMP), to ensure consistent potency and purity. Clear labeling requirements with dosage guidelines and warnings should be mandated to ensure safe use and prevent misuse. Furthermore, the implementation of regulatory oversight to monitor product quality and enforce compliance is essential. This review emphasizes the urgency of focused research to optimize dosing regimens, characterize the pharmacodynamic profiles of these alkaloids, and evaluate long-term safety. By addressing these gaps, the mitragynine- and corynoxeine-related drug classes can transition from promising plant-derived molecules to validated pharmacotherapeutic agents, potentially revolutionizing the field of pain management. Full article

Kappa opioid receptor (KOR) agonists have well-established antinociceptive effects. However, many KOR agonists have negative side effects, which limit their therapeutic potential. Some researchers have suggested that the development of biased agonists that preferentially stimulate KOR G-protein pathways over β-arrestin pathways may yield drugs with fewer adverse side effects. This was investigated in the current study. We describe the synthesis and characterization of three U50,488 analogues, 1, 2, and 3. We evaluated the acute and chronic antinociceptive effects of these compounds in mice using the warm-water tail flick assay and in a paclitaxel-induced neuropathic pain model. Side effects were investigated using open-field, passive wire hang, rotarod, elevated zero maze, conditioned place aversion, and whole-body plethysmography, with some tests being conducted in KOR or β-arrestin2 knock out mice. All compounds were highly potent, full agonists of the KOR, with varying signaling biases in vitro. In the warm-water tail withdrawal assay, these agonists were ~10 times more potent than U50,488, but not more efficacious. All KOR agonists reversed paclitaxel-induced neuropathic pain, without tolerance. Compound 3 showed no significant side effects on any test. Signaling bias did not correlate with the antinociceptive or side effects of any compounds and knockout of β-arrestin2 had no effect on U50,488-induced sedation or motor incoordination. These findings highlight the therapeutic potential of 3, with its lack of side effects typically associated with KOR agonists, and also suggest that G-protein signaling bias is a poor predictor of KOR agonist-induced side effects. Full article