Chemokines orchestrate immune cell trafficking through receptor-mediated signalling and are implicated in inflammatory, autoimmune and neuropathic disorders. The XCL1-XCR1 axis is of particular interest because XCR1 is selectively expressed on mature conventional type 1 dendritic cells (cDC1s), where it supports communication with activated CD8+ T cells and NK cells and promotes antigen cross-presentation. This selectivity has made XCR1 an attractive target for dendritic cell-based cancer vaccines, while emerging evidence also links XCL1-XCR1 signalling to neuroinflammation and pain. Despite its therapeutic potential, the mechanisms governing XCR1 activation and trafficking remain understudied. Here, we characterize XCR1 expression, membrane trafficking, and basal internalization to define mechanisms that may influence therapeutic targeting. We show that XCR1 undergoes constitutive internalization through a β-arrestin-independent but adaptor protein 2 (AP2)-dependent pathway, distinguishing it from other chemokine receptors with constitutive endocytosis. Furthermore, we identify specific sequence motifs critical for its subcellular localization and intracellular trafficking. These findings provide new mechanistic insights into XCR1 regulation and may inform the development of targeted therapeutics and antigen-delivery strategies in cancer and inflammation.

Thioredoxin-interacting protein (TXNIP) is a multifunctional regulatory protein of the α-arrestin family that acts as a redox sensor, an energy-responsive molecule, and a transcription-associated regulator. TXNIP has emerged as a key regulator of skeletal muscle homeostasis and has been implicated in the pathogenesis of multiple muscle disorders. Skeletal muscle is a principal site of systemic glucose disposal, accounting for more than 70% of insulin-stimulated glucose uptake in the human body. TXNIP has been implicated in sarcopenia, obesity-related insulin resistance, limb ischemia-induced muscle injury, disuse muscle atrophy, and hereditary muscular dystrophy through multiple mechanisms, including glutathione metabolic regulation, ferroptosis, NLRP3 inflammasome activation, impaired GLUT4 membrane translocation, and altered mitochondrial substrate oxidation. Recent advances in gene editing, single-cell sequencing, metabolomics, and human clinical research have enabled a more detailed characterization of TXNIP-related molecular mechanisms in skeletal muscle. This review provides a systematic synthesis of recent evidence on TXNIP in skeletal muscle, organized around seven themes: its molecular characteristics, expression and regulatory patterns in skeletal muscle, physiological functions, disease-specific pathogenic mechanisms, intervention-related regulatory networks, current limitations, and future perspectives. This review aims to provide a mechanistic rationale for targeting TXNIP as a potential strategy for the prevention and treatment of skeletal muscle-related diseases.

Open reading frame 74 (ORF74) is the single G protein-coupled receptor (GPCR) encoded in the genome of Kaposi's sarcoma-associated herpesvirus (KSHV). Due to its high constitutive activity and broad chemokine binding profile, ORF74 contributes to KSHV-associated diseases through autocrine and paracrine mechanisms that promote cell proliferation, inflammation and angiogenesis. Previous studies showed that ELR+ CXC chemokines (i.e., CXCL1-3 and CXCL5-8), which contain a conserved Glu-Leu-Arg motif, bind ORF74 with high affinity. Among these chemokines, CXCL1-3 and CXCL6-8 act as full or partial agonists in ORF74-mediated G protein signaling and β-arrestin recruitment, whereas CXCL5 behaves as a neutral ligand. However, whether these chemokines preferentially engage specific intracellular transducers remains unclear. We therefore applied a unified NanoLuc Binary Technology (NanoBiT) platform in HEK293A cells to systematically compare how ELR+ CXC chemokines modulate ORF74-transducer proximity across the G protein-GRK-β-arrestin axis. CXCL1-3 and CXCL6-8, but not CXCL5, induced concentration-dependent ORF74-specific responses involving miniGi recruitment, Gβ1 proximity, GRK2/3/5/6 proximity and β-arrestin1/2 recruitment. Using CXCL1 as the reference ligand, bias analysis showed that the active ELR+ CXC chemokines engage ORF74 in a largely balanced manner across these proximal transducer readouts.

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor (GPCR) with an important role in calcium homeostasis, activating mutations of which cause hypocalcemia. Despite the receptors clinical importance and evidence suggesting β-arrestin-1/2 can modulate CaSR signaling, the residues within CaSR required for β-arrestin recruitment and the role of β-arrestin in desensitization, trafficking and signaling are ill defined. We confirmed that β-arrestin-1 and β-arrestin-2 are recruited to CaSR upon receptor activation and that depletion of GPCR kinases impairs β-arrestin recruitment. Deletion of the distal CaSR C-terminal tail, which replicates several mutations identified in individuals with hypocalcemia, reduced β-arrestin-1/2 recruitment and enhanced receptor signaling. Examination of the receptor cytoplasmic region (intracellular loops and C-terminal tail) identified three regions of serine and threonine residues that resemble phosphorylation codes in other GPCRs. Mutation of each of these residues to alanine demonstrated one region between amino acids 1003-1011 is important for both β-arrestin-1 and β-arrestin-2 recruitment, while disruption of a region between residues 976-981 impaired β-arrestin-2 recruitment. Alanine mutagenesis of these residues also enhanced CaSR signaling and impaired receptor internalization suggesting an important role in receptor desensitization. Three rare-coding variants reported in disorders of calcium homeostasis were identified in these residues and two, T1006M and T1008P, were shown to reduce β-arrestin recruitment and CaSR internalization and enhance CaSR signaling. Thus, we have identified two regions within the CaSR C-terminal tail that are important for β-arrestin recruitment, receptor desensitization and internalization. Mutation of residues within these regions may represent another mechanism contributing to hypocalcemia.

Nonvisual arrestins, also known as Beta-arrestins, are known for their diverse role in GPCR signal transduction. Here, we present evidence that Beta-Arrestin-2, a protein whose nuclear concentration is actively kept low, binds to Nucleolar Organizing Regions (NORs) on the metaphase chromosomes at the onset of cell division, after the disappearance of the nuclear membrane and stays bound therein in the daughter cells. After reformation of the nucleoli, it stays bound to the nucleoli until actively transported out of the reformed nucleus during interphase. This binding is unique for Beta-Arrestin-2, as the closely associated Beta-Arrestin-1 does not show this behavior at all. We also observed an intranuclear accumulation of Beta-Arrestin-2 after P2X7 receptor stimulation, similar to what has been described for Beta-Arrestin-2 after TRPV receptor stimulation. Present data identify a cyclic and well-organized shuttling between NORs and the cytoplasm of Beta-Arrestin-2 during cell division, indicating a novel and unknown role for Beta-Arrestin-2 in cell physiology. Our data may also indicate a common intracellular signaling mechanism for a diverse group of receptors currently believed to function only as ligand-gated channels (P2X7 and TRPV).

Biased signaling in G protein-coupled receptors offers therapeutic promise, yet rational design of biased ligands remains challenging due to limited mechanistic understanding. Here, we report a molecular basis for controlling signaling bias at the immunometabolic receptor GPR84. We identify three structurally-matched ligands (OX04529, OX04954, and OX04539) with varying steric profiles that exhibit comparable Gi protein activation but markedly different β-arrestin recruitment capacities. A high-resolution cryo-EM structure of GPR84-Gi in complex with OX04529, complemented by molecular dynamics simulations and targeted mutagenesis, reveals that steric interactions between ligand substituents and Leu336[6.52] and Phe187[5.47] indirectly disrupt a critical polar network involving Tyr332[6.48], Asn104[3.36] and Asn362[7.45] essential for β-arrestin recruitment. Based on these insights, we develop a steric-dependent model that enables rational design of G protein-biased agonists with predictable β-arrestin recruitment profiles. This mechanistic framework provides the means to design biased agonists with customized signaling profiles at GPR84 and potentially other class A GPCRs.

Binding of biased ligands to a G protein-coupled receptor (GPCR) stabilizes specific receptor conformations, leading to distinct physiological responses. Recently, intra-transducer bias has emerged as an additional mechanism that fine-tunes receptor function by stabilizing specific architectures of the receptor-transducer complex. This mechanism was demonstrated for the angiotensin receptor (AT1R) and its β-arrestin-biased ligand TRV026, which selectively promotes β-arrestin-1 engagement via the receptor C terminus while preventing interaction with the receptor core. Here, we present a computational protocol integrating double electron-electron resonance (DEER) data with deep learning-based structure prediction and molecular dynamics simulations to reconstruct all-atom models of TRV026-bound AT1R. Using a new clustering method, we identify key ligand-receptor interactions underlying high-affinity binding, localized receptor flexibility, and ligand bias. Our results provide a framework for validating neural-network predictions and physics-based simulations to capture rare signaling states of membrane proteins and identify target conformations for designing more functionally selective, efficacious therapeutics.

Opioid withdrawal remains a significant barrier to cessation, particularly in treatment-experienced populations who have made repeated unsuccessful attempts using established pharmacotherapies. SR-17018 is a μ-opioid receptor agonist with preclinical evidence of reduced β-arrestin 2 recruitment and altered receptor regulatory dynamics relative to conventional opioids. Whether these properties translate into clinically meaningful differences in humans remains unknown. This report presents a descriptive analysis of a self-reported observational registry (N = 55) characterizing SR-17018 exposure patterns, perceived outcomes, and adverse effects. Most participants (69.1%) reported three or more prior detoxification attempts, characterizing the cohort as treatment-experienced and refractory. During SR-17018 exposure, 54.5% of participants reported full cessation of opioid use and 32.7% reported meaningful reduction in use; smaller proportions reported no change (5.5%) or relapse (7.3%). Collectively, 87.3% reported improvement. These outcomes are self-reported and were not independently verified. Most participants reported dosing SR-17018 three or more times daily (76.4%), with perceived effects lasting approximately 4-8 h per dose (54.5%). Participants reported a mean withdrawal relief score of 8.20/10 (SD and distribution reported in Results), with 90.9% rating relief at seven or higher. Adverse effects were predominantly mild to moderate and included anxiety, sweating, sedation, insomnia, gastrointestinal symptoms, and subjective breathing-related concerns in a minority of respondents (7.3%); the nature of these breathing reports could not be medically verified. Because the recruitment channel selects against participants who discontinued for adverse effects or inefficacy, and because approximately half the cohort reported concurrent use of other substances, these tolerability figures-including the low discontinuation rate (1.8%) - are biased toward a favorable safety impression and cannot be read as evidence that SR-17018 is well tolerated. No objective safety monitoring was performed. These findings provide preliminary descriptive data characterizing real-world SR-17018 exposure in a treatment-experienced population. The uncontrolled, self-reported, cross-sectional design precludes any conclusion regarding efficacy or safety, and the sample is likely biased toward favorable experiences. The principal value of these data is to motivate and prioritize controlled human investigation: prospective, objectively monitored studies are required before any clinical inference about SR-17018 can be drawn.

We have investigated the signal transduction mechanisms linking β2-adrenoceptor activation to extracellular signal-regulated kinase1/2 (ERK) activity and cell migration in vascular smooth muscle, with a focus on the roles played by G protein-coupled receptor kinases (GRKs) and arrestin proteins in these processes. Activation of β2-adrenoceptors in rat aortic smooth muscle (RASM) cells caused a time- and concentration-dependent suppression of basal phospho-ERK (pERK), and this effect was recapitulated by direct activation of adenylyl cyclase activity by forskolin. Activation of β2-adrenoceptors also inhibited the abilities of vasoconstrictors (angiotensin II, endothelin-1 and uridine 5'-triphosphate) to stimulate ERK phosphorylation. Using a variety of pharmacological and genetic approaches it was shown that the ability of β2-adrenoceptor activation to suppress ERK phosphorylation was exchange protein directly activated by cAMP (EPAC) (and not Protein kinase A, PKA), GRK5 and arrestin3-dependent. We have also shown that the ability of β2-adrenoceptor activation to attenuate vasoconstrictor-induced RASM cell migration is mediated by similar (EPAC-, GRK5- and arrestin3-dependent) signal transduction pathways. Finally, we report that the ability of β2-adrenoceptor activation to suppress ERK phosphorylation and to attenuate vasoconstrictor-induced cell migration is lost in RASM cells isolated from hypertensive spontaneously hypertensive rats (SHR), but not normotensive Wistar-Kyoto (WKY) rats. Our studies reveal an unexpected pathway linking the β2-adrenoceptor to the regulation of extracellular-signal regulated kinase (ERK) activity and cell migration in aortic smooth muscle cells.

Glucagon-Like Peptide-1 (GLP-1) is an incretin family peptide hormone with significant therapeutic applications. This study evaluated multiple functional methodologies for assessing GLP-1 agonist biological activity, encompassing in vitro cell-based bioassays and in vivo systems. We developed and comparatively analyzed cell-based bioassays measuring distinct cellular signaling events, such as β-arrestin recruitment, cyclic adenosine monophosphate (cAMP) elevation, and calcium (Ca[2+]) mobilization. Each assay was systematically evaluated for robustness and quality control applicability. All assay systems demonstrated functionality independent of the cellular mechanism employed. Real-time Ca[2+] flux measurements confirmed the cell-based assay endpoint data, while cross-verification analyses using the oral glucose tolerance test in mice confirmed the representativeness of the cAMP formats. Initial validation studies revealed inter-assay variations, leading to the selection of the cAMP HTRF assay as the preferred quality control methodology. Further assay refinement and subsequent validation studies confirmed the cAMP HTRF assay's suitability for quality control applications. This methodology has been successfully implemented in routine release testing of a commercially available GLP-1 agonist.

G protein-coupled receptors (GPCRs) are best known for initiating signaling at the plasma membrane, yet a substantial and functionally distinct subset of these receptors localizes to the primary cilium, a highly specialized cell-surface organelle. Over the past decade, the primary cilium has emerged as a privileged signaling compartment that concentrates GPCRs, signaling effectors, and second messengers within a physically constrained and biochemically unique environment. In this review, we synthesize current knowledge of GPCR signaling from a primary cilium perspective, with emphasis on receptor repertoire, trafficking mechanisms, and compartment-specific signal transduction. We discuss how ciliary entry and exit are regulated by dedicated transport systems, how canonical GPCR signaling components-G proteins, GPCR kinases, β-arrestins, regulators of G protein signaling, and A-kinase anchoring proteins-are organized within cilia, and how the ciliary geometry, membrane composition, voltage, pH, and cytoskeletal architecture shape signaling outcomes. We further highlight how cilia support autonomous and highly sensitive microdomains of cAMP and calcium (Ca[2+]) signaling, enabling integration and hierarchical control of multiple GPCR inputs. Finally, we examine the physiological consequences of ciliary GPCR signaling in neural, renal, adipose, and pancreatic and developmental tissues, where defects in ciliary signaling contribute to metabolic disease, impaired morphogenesis, and tissue homeostasis, and ciliopathies-a range of human disorders arising from primary cilium dysfunction. Together, these studies establish the primary cilium as a dynamic and modular GPCR signaling hub and underscore the importance of subcellular localization in determining receptor function and signaling specificity. SIGNIFICANCE STATEMENT: Beyond their traditional role at the plasma membrane, many GPCRs signal from primary cilia, specialized cellular compartments that create unique signaling environments. This review synthesizes advances in ciliary GPCR trafficking, organization, and signal transduction, showing how subcellular localization determines receptor function and how disrupted ciliary signaling contributes to human diseases, including ciliopathies, metabolic disorders, and developmental defects.

Allosteric modulators offer opportunities for pathway-selective G protein-coupled receptor (GPCR) signaling, but the mechanisms enabling biased allosteric modulation remain poorly understood. We identify a helix 8-proximate allosteric site in the immune-metabolic receptor GPR84 and define how it achieves Gi-biased signaling. Cryo-electron microscopy structures of the GPR84-Gi complexes bound to the orthosteric agonist OX04539 alone or with the positive allosteric modulator PSB-16671 reveal a helix 8-proximate allosteric site for PSB-16671 formed between transmembrane helices 1 (TM1) and TM7. Molecular dynamics simulations and mutagenesis uncover a polar interaction network linking orthosteric and allosteric sites through conserved residues including Asp66[2.50], Asn104[3.36], and Asn362[7.45], and disrupting this network enhances allosteric cooperativity. PSB-16671 stabilizes a receptor conformation with pronounced TM6 displacement that favors Gi coupling while disfavoring β-arrestin recruitment, sustaining macrophage phagocytosis of cancer cells without the desensitization induced by balanced agonists. Sequence analysis suggests that receptor-specific helix 8-proximate allosteric sites may be broadly targetable across class A GPCRs. These findings establish mechanistic principles for biased allosteric modulation applicable beyond GPR84.

Mammalian neurokinin 1 receptor (NK1R) exists as full-length (NK1L) and truncated (NK1S) splice variants. However, the functional role of NK1S remains controversial; therefore, we investigated their functional interplay. Using NanoBiT and co-immunoprecipitation, we demonstrate that NK1L and NK1S form heterodimeric complexes. Through this interaction, NK1S negatively modulates NK1L-mediated G protein signaling, specifically impairing Gαq coupling and Ca[2+] mobilization. Conversely, NK1S enhances β-arrestin1 recruitment to NK1L, altering receptor trafficking. In A549 cells, NK1S suppressed NK1L-mediated cell migration despite sustaining ERK phosphorylation. These findings provide mechanistic evidence that NK1S can regulate NK1L through dimerization and may shift signaling bias from G proteins toward β-arrestin-linked pathways to influence cellular outcomes.