G Protein Research Articles

Despite advancements in screening and treatment, the incidence of breast cancer (BC) and associated mortality are projected to increase. Therefore, developing a companion diagnostic for BC remains important. Herein, we explore the urinary proteome for biomarkers of BC: 130 urine samples from (1) newly diagnosed breast cancer (BC), n = 46, (2) benign breast disease (BBD), n = 36, (3) symptom control (SC), n = 30, and (4) healthy control (HC), n = 18. The BC class included preinvasive: ductal carcinoma in situ (DCIS) (n = 3), invasive ductal carcinoma (IDC) (n = 23), and IDC accompanied by DCIS (n = 8) classes. Protein profiling was performed using ThermoScientific ProteomeDiscoverer and analyzed using MetaboAnalyst v6.0, DAVID, and STRING v12.0. Analyses identified 346 significantly (p < 0.05) differentially expressed proteins (DEP) across BC, BBD, SC, and HC. Multivariate Receiver Operating Characteristic curves (five proteins) suggested Area Under the Curve values of 0.985, 0.989, and 0.999 distinguishing BC from BBD, SC, and HC, respectively. DEP elevated in BC included beta-glucuronidase isoform 1, fibrinogen gamma chain, alpha-actinin-1, peptidase inhibitor 16, cysteine-rich C-terminal protein 1 isoform X1, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1, vascular cell adhesion protein 1, ATP-dependent translocase ABCB1, and tumor protein p63-regulated gene 1 isoform X1. BC types were differentiated based on calpain-2 and cystatin-C expression (p < 0.05). Thus, BC has distinct urinary-protein profiles based on clinical diagnosis, which could be used in real-time noninvasive BC monitoring.

The C-terminal region regulates the constitutive signaling activity of GPR119: G protein recruitment.

Background/Objectives: Compartmentalized glucose metabolism in the brain contributes to neuro-metabolic stability and shapes hypothalamic control of glucose homeostasis. Glucose transporter-2 (GLUT2) is a plasma membrane glucose sensor that exerts sex-specific control of hypothalamic astrocyte glucose and glycogen metabolism. Aging causes counterregulatory dysfunction. Methods: The current research used Western blot and HPLC–electrospray ionization–mass spectrometry to investigate whether aging affects the GLUT2-dependent hypothalamic astrocyte metabolic sensor, glycogen enzyme protein expression, and glycogen mass according to sex. Results: The data document GLUT2-dependent upregulated glucokinase (GCK) protein in glucose-deprived old male and female astrocyte cultures, unlike GLUT2 inhibition of this protein in young astrocytes. Glucoprivation of old male and female astrocytes caused GLUT2-independent downregulation of 5′-AMP-activated protein kinase (AMPK) protein, indicating loss of GLUT2 stimulation of this protein with age. This metabolic stress also caused GLUT2-dependent suppression of phospho-AMPK profiles in each sex, differing from GLUT2-mediated glucoprivic enhancement of activated AMPK in young male astrocytes and phospho-AMPK insensitivity to glucoprivation in young female cultures. GS and GP isoform proteins were refractory to glucoprivation of old male cultures, contrary to downregulation of these proteins in young glucose-deprived male astrocytes. Aging elicited a shift from GLUT2 inhibition to stimulation of male astrocyte glycogen accumulation and caused gain of GLUT2 control of female astrocyte glycogen. Conclusions: The outcomes document sex-specific, aging-related alterations in GLUT2 control of hypothalamic astrocyte glucose and ATP monitoring and glycogen mass and metabolism. These results warrant future initiatives to assess how these adjustments in hypothalamic astrocyte function may affect neural operations that are shaped by astrocyte–neuron metabolic partnership.

Nitric oxide regulates phagocytosis through S-nitrosylation of Rab5.

Purpose To assess the predictive value of guanine nucleotide-binding protein, alpha-stimulating activity polypeptide (GNAS) and cerebral perfusion in various vascular regions for the severity of ischemic white matter lesions (WMLs). Methods Patients hospitalized at the Neurology Department of the Affiliated Hospital of Chengde Medical University between April and November 2023 were evaluated for ischemic cerebral WMLs using magnetic resonance imaging. In this retrospective cohort study, patients were classified into two groups: mild and severe, based on Fazekas scores. White matter perfusion was assessed using image segmentation of arterial spin labeling sequence images. Predictive variables were identified via machine learning (ML). GNAS levels in peripheral blood were measured to explore their association with WML severity. Results Among 85 patients (43 mild [24 males and 19 females], 42 severe [27 males and 15 females]), significant differences were observed in age (64.00 ± 8.47 years vs. 68.38 ± 10.85 years, p = 0.041), cerebral atrophy (37.2% vs. 71.4%, p = 0.002), and history of hypertension (41.7% vs. 77.0%, p = 0.002). Corpus callosum perfusion was lower in the severe group (35.84 ± 6.34 vs. 31.73 ± 8.60 mL/[min·100 g], p = 0.037). ML yielded 77.27% model accuracy. Although no significant difference in GNAS levels was observed ( p = 0.375), a significant difference was noted in the Fazekas scores ( p &amp;lt; 0.001). Conclusion In patients with ischemic WMLs, factors such as age, sex, history of cerebral infarction, GNAS levels, and specific perfusion metrics are predictive of WML progression. Advanced imaging and ML improve detection. GNAS levels correlated with Fazekas scores, indicating their downregulation in the hypoperfused white matter.

ABSTRACTPrevious studies have demonstrated that overexpression of DRG2 enhances the osteoclastic activity. However, its impact on osteoblastic differentiation remains unexplored. This study investigates the relationship between DRG2 and osteoblastic activity through in vitro and in vivo studies. DRG2 expression was inhibited in mouse MC3T3‐E1 cells, and its effects on the expression of bone formation markers and osteoblast‐related transcription factors were evaluated. The capacity for osteoblastic differentiation was assessed in mouse preosteoblasts following DRG2 gene knockdown using a short hairpin RNA (shRNA) plasmid. Female C57BL/6N strain DRG2 knockout (KO) mice were generated, and bone phenotypes of the vertebrae and femurs were analysed. Additionally, osteoblastic differentiation capacity was evaluated in bone marrow mesenchymal stem cells (BM‐MSCs) isolated from these mice under physiological and ovariectomized conditions. Inhibition of DRG2 in MC3T3‐E1 cells resulted in upregulation of bone formation markers and osteoblast transcription factors. BM‐MSCs from DRG2 KO mice exhibited significantly higher osteoblastogenesis than wild‐type (WT) mice. DRG2 KO mice demonstrated significantly increased percent bone volume and bone mineral density (BMD) in the vertebra and femur compared to WT mice under physiological and ovariectomized conditions. The inhibition of DRG2 promotes osteoblastic differentiation and is associated with increased BMD, suggesting its potential role in enhancing bone formation.

The α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) in G protein-coupled receptor (GPCR) pathways operate as gate switches by cycling through inactive GDP-bound and active GTP-bound states. Three flexible regions (switches I to III) of Gα subunits undergo the most substantial conformational rearrangements and determine interactions with receptors, signaling regulators, and effectors. Here, we describe three patients with severe pediatric encephalopathy harboring c.723+1G>A or c.723+2T>A variants in the splicing donor site of intron 6 of GNAO1, the gene encoding the major neuronal G protein α subunit Gαo. These substitutions destroyed the conserved GU sequence of the pre-mRNA and rendered the donor site unrecognizable, prompting cryptic splice site engagement and production of the dominant pathogenic Gαo[V234_T241del] variant, which lacked switch III (amino acid residues 232 to 243 in Gαo). Structural, biochemical, and cellular analyses characterized V234_T241del as a strong neomorphic variant that was severely deficient in guanine nucleotide handling and cellular interactions and sensitive to zinc salts, an emerging targeted treatment for a GNAO1 encephalopathy subgroup. Our studies provide pathological and molecular insights into the function and effect of removal of an entire switch region in 1 of the 16 human G protein α subunits that underlie multiple physiological processes and diverse pathologies.

Background and ObjectivesOur aim was to report a novel variant of Guanine nucleotide-binding protein subunit beta-4 gene (GNB4) variants and to report the clinical and molecular spectrum of the GNB4-related neuropathy based on the currently reported cases.MethodsWe performed detailed neurologic and electrophysiologic assessments and segregation analysis in a patient form single family with Charcot-Marie-Tooth (CMT) disease with novel GNB4 variant. Via literature search, we found studies that included neuropathy cases with GNB4 variants; we then reviewed and reclassified these variants in accordance with the ACMG guidelines and latest databases and evidence.ResultsWe identified a novel GNB4 variant NM_021629.4:c.239T>C p.(Ile80Thr) in a single family with CMT. In total, we identified 8 GNB4 variants reported in the literature from 10 unrelated families in addition to the novel variant reported in this study. We compiled standardised clinical and genotypic information for 15 affected individuals from 8 families carrying 6 distinct likely pathogenic or pathogenic (LP/P) GNB4 variants reported in the literature and the variant found in this study. We found pathogenic variants to cluster on 3D in the GNB4 protein.DiscussionWe reinterpreted the 8 previously reported GNB4 gene variants in the literature from 10 unrelated families in addition to the novel variant reported in this study in patients with hereditary neuropathy. We show that GNB4-associated neuropathy is associated with mixed axonal and demyelinating features presenting with usually symmetric lower limb motor weakness and sensory loss and/or upper limb weakness and sensory loss.

5-Hydroxytryptamine receptor type 7 (5-HT7) receptor is a G protein-coupled receptor (GPCR) exhibiting noncanonical signaling properties. It has been shown that 5-HT7 can form stable inactive preassembled complexes with its cognate Gs protein. Structural determinants of such complex formation and the distinction between preassembled and intermediate activated complexes remain unknown. Here, we use molecular modeling and molecular dynamics simulations to determine and characterize the binding interface between this receptor and the Gs protein in both the active and preassembly complexes. Our results show key interaction patterns specific for the different states and pinpoint unique structural features distinguishing active, inactive, and preassembled states of the receptor.

BackgroundThe Rho GTPase-activating protein (RhoGAP) family represents a large and diverse group of proteins that act as key regulators of Rho GTPases, small GTP-binding proteins involved in cellular signaling. Tight regulation of Rho GTPase activity is essential for fundamental biological processes, including cell motility, contractility, growth, differentiation, and development. Despite their biological importance, the roles of RhoGAPs in cancer remain largely undefined.MethodsBioinformatics analysis was performed using data from The Cancer Genome Atlas (TCGA), covering over 10,000 samples across 33 cancer types. The role of ARHGAP11A in the DNA damage response was evaluated through single-cell sequencing, western blotting, and colony formation assays.ResultsPan-cancer analysis of RhoGAP family genes identified ARHGAP11A as the most significantly overexpressed member in tumors compared to adjacent normal tissues. ARHGAP11A expression was found to be elevated in most cancer types and was associated with DNA repair activity. Furthermore, its expression positively correlated with tumor mutational burden (TMB), a recognized predictive biomarker for immunotherapy. Intriguingly, it also correlated with increased infiltration of regulatory T (Treg) cells, which are known to suppress anti-tumor immunity. Consistent with these observations, high ARHGAP11A expression was concurrently linked to poor patient survival outcomes across multiple cancers. We also observed a positive correlation between ARHGAP11A and CHK1, suggesting a functional collaboration. In vitro experiments further demonstrated that ARHGAP11A confers resistance to DNA damage induced by CHK1 inhibitors.ConclusionARHGAP11A is a promising prognostic marker in cancer, with potential therapeutic implications through targeting DNA repair pathways and modulating the tumor immune microenvironment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15106-8.

Disclosure: M.H. Alabdely: None. A.A. Khan: None.Background: Autosomal dominant hypocalcemia (ADH) type 1 and type 2 are rare disorders caused by gain-of-function variants that activate the calcium sensing receptor (CASR) gene, leading to ADH type 1, or its signaling protein Guanine nucleotide-binding protein subunit alpha-11 (GNA11), resulting in ADH type 2. These disorders are inherited in an autosomal dominant pattern and are characterized by hypoparathyroidism, manifesting as hypocalcemia, inappropriately low serum parathyroid hormone (PTH) concentrations and hypercalciuria due to enhanced sensitivity of the CaSR to extracellular calcium concentrations. Clinical features range from asymptomatic to severe. Treatment with calcium and active vitamin D can exacerbate hypercalciuria and nephrocalcinosis. Alternative treatment options that have been used in patients with ADH1 include recombinant human PTH or calcilytic therapy. Here, we describe a male patient with ADH2 harboring a novel GNA11 variant, evaluated at our tertiary center in Canada.Case summary:A 43-year-old male experienced intermittent numbness in hands for 3 years prior to presentation; in association with exercise. Other manifestations include bronchospasm and laryngospasm triggered by wrestling or climbing 2-3 flights of stairs, over the past 15 years. He had no peri oral numbness, muscle spasm, seizures, arrhythmias, brain fog, kidney stones, fractures or osteoporosis. Additionally, he had no clinical features features suggestive of genetic or autoimmune disorders resulting in hypoparathyroidism. There was no history of neck surgery or a family history of endocrine or autoimmune disease. In April 2024, he was incidentally found to have low calcium levels. By July 2024, he was found to have low PTH levels as well as persistently low calcium levels. Medical examination was unremarkable and Chvostek’s sign was negative. Laboratory tests revealed low corrected calcium (1.98 mmol/L, NR: 2.15-2.60), and low or inappropriately normal PTH (2.2 pmol/L, NR:1.6-9.3) on two occasions. The serum phosphorus was (1.03 mmol/L, NR: 0.81-1.45), serum magnesium (0.88 mmol/L, NR: 0.65-1.05), 25OHD (83 nmol/L, NR: 75-250), ALP (56 IU/L, NR: 30-129) and 24 hr urine for calcium collection was inadequate.Genetic testing confirmed a heterozygous variant of uncertain significance (VUS) in GNA11 gene c.1-12C>T pArg338Cys which has not been previously reported. The clinical picture is consistent with ADH2. The patient was started on calcium carbonate 500 mg three times daily with meals with follow up in 4 weeks. Conclusion: We report a male patient with hypoparathyroidism who was found to have a novel VUS in GNA 11 gene. This variant could be pathogenic, as the patient presents with a phenotype consistent with ADH2. To date, six different gain-of-function variants in the GNA11 have been identified. An important contribution to the Literature is identifying several variants in ADH2.Presentation: Saturday, July 12, 2025

Abstract Disclosure: A. Shahatit: None. A. Banka: None. L. De Mattei: None. B. Celik: None. J. Meireles: None. U. Bin Hameed: None. Introduction: Pseudohypoparathyroidism (PHP) is a rare disorder marked by end-organ resistance to parathyroid hormone (PTH), leading to hypocalcemia and hyperphosphatemia despite elevated PTH. Chronic PHP may result in basal ganglia calcifications (BGC), contributing to neurological complications. Case Presentation: A 44-year-old man with hypertension, hypothyroidism, and prior strokes with residual aphasia and right-sided weakness was referred for severe hypocalcemia. On exam, he had short stature. Labs showed: calcium 4.8 mg/dL (normal: 8.5-10.5), ionized calcium 2.53 mg/dL (4.6-5.3), phosphorus 6.7 mg/dL (2.5-4.5), PTH 218 pg/mL (10-65), vitamin D 66.2 ng/mL (30-100), creatinine 0.87 mg/dL (0.6-1.2), and TSH 9.98 µIU/mL (0.4-4.0). Head CT revealed extensive calcifications in the posterior fossa, deep gray nuclei, subcortical frontal and temporal lobes, and subcutaneous scalp tissues. The patient reported lifelong hypocalcemia and prior imaging showing brain calcifications. He was non-adherent to levothyroxine.He was treated with IV calcium gluconate followed by oral calcium citrate, calcitriol, and levothyroxine. Calcium normalized (to 8.0 mg/dL), and he was discharged on calcium carbonate, calcitriol, and levothyroxine with endocrinology and neurology follow-up. Discussion: This case highlights classic PHP features: hypocalcemia, hyperphosphatemia, elevated PTH, and extensive tissue calcifications. BGC in PHP is linked to seizures, movement disorders, and cognitive deficits. Coexisting hypothyroidism may reflect TSH resistance, often seen in PHP Type 1a due to GNAS mutations, which impair Gs alpha protein signaling in multiple hormone pathways. Learning Points:Suspect PHP with hypocalcemia, hyperphosphatemia, and high PTH in the setting of normal renal function.Intracranial and subcutaneous calcifications may signal long-standing disease.PHP Type 1a may include TSH resistance and other hormone resistance syndromes.Lifelong calcium and active vitamin D therapy is essential. Conclusion: This case emphasizes the importance of recognizing PHP early to prevent irreversible neurologic complications and highlights diagnostic clues from labs and imaging. Presentation: Saturday, July 12, 2025

Extracellular vesicles (EVs) have gained prominence as advanced drug delivery systems due to their inherent merits. Recent advancements highlight their utility in transporting therapeutic proteins with significant progress in targeted therapies. To optimize EV efficacy, engineering strategies focus on enhancing cargo encapsulation efficiency. The robust interaction between immunoglobulin G Fc fragments and the cytoplasmic receptor TRIM21 offers a novel framework for stable cargo loading. This study identifies Ras-related GTP-binding protein D (RRAGD) as a critical regulator of lysosomal biogenesis and function. Leveraging Fc-TRIM21 interactions, engineered EVs encapsulating RRAGD were developed. Surface modification with CAP peptides enhanced nucleus pulposus (NP) cell-targeting specificity of EVs. In vitro and in vivo experiments demonstrated that engineered EVs ameliorated lysosomal dysfunction and suppressed apoptosis in NP cells, and slowed intervertebral disc degeneration (IDD) progression. The mechanistic analysis revealed the functional co-localization of RRAGD with lysosomal marker LAMP1 and lysosomal regeneration transcription factor TFEB, indicating lysosomal targeting. This study establishes CAP-modified engineered EVs based on the Fc/TRIM21 platform as a therapeutic strategy to restore lysosomal homeostasis and counteract IDD pathogenesis, underscoring the potential of EV-based therapies for degenerative disc diseases.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s12951-025-03772-6.

The prostaglandin D2 receptor 1 (DP1), a member of the prostanoid G protein-coupled receptor (GPCR) family, plays critical roles in allergic responses, sleep regulation, immune modulation, and vasodilation. Here, we present five high-resolution cryo-electron microscopy (cryo-EM) structures of the human DP1 receptor, including an apo structure, two inactive state structures bound to two different inverse agonists developed by ONO Pharmaceutical, and two active state structures in complex with the Gs protein and bound to the endogenous agonist PGD2 and its selective derivative BW245C. Structural analysis, complemented by molecular dynamics simulations and site-directed mutagenesis, reveals key residues involved in ligand recognition and suggests a distinct activation mechanism for DP1, which lacks most of the conserved class A GPCR motifs. Notably, the unique residue K76 within the conserved sodium pocket acts as a major activation switch, while amphiphilic helix 8 adopts an unconventional orientation essential for receptor function. These findings offer valuable insights into the structure and function of prostanoid receptors and may facilitate the development of therapeutics targeting DP1.

The small GTP-binding Ras-like protein DIRAS2 promotes cell proliferation in oral leukoplakia via the RAF/MEK/MAPK pathway.

Physiological functions and structural features of Gα12/13 proteins.

The G Protein Inhibitor YM-254890 is an Allosteric Glue.

Pharmacological characterization of vampire bat-derived CGRP at the human CGRP receptor in the Xenopus oocyte system.

Evolving perspectives on lipid mediated cell-to-cell communication: The adenylyl cyclase signaling system.

Small fiber neuropathy (SFN) affects small-diameter sensory and autonomic nerve fibers, leading to chronic pain and autonomic dysfunction. While SFN can be associated with diabetes and autoimmune diseases, a significant proportion of cases are idiopathic. Although immune-mediated mechanisms are being recognized increasingly in SFN, their precise role remains unclear. This study investigates the presence of autoantibodies against interferon-induced GTP-binding protein MX (MX1) in SFN patients and explores their potential pathogenic role. A total of 59 patients with skin biopsy-confirmed SFN and 20 healthy controls were recruited. Serum samples were analyzed for the presence of anti-MX1 autoantibodies using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed on rat sciatic nerves to assess the localization of patient IgG to unmyelinated nerve fibers, and immunocytochemistry and flow cytometry confirmed specific binding to MX1. Functional characterization of MX1 was conducted using whole-cell patch-clamp recordings in dorsal root ganglion (DRG) neurons overexpressing MX1. Additionally, protein interactions between MX1 and transient receptor potential cation channel subfamily C member 6 (TRPC6) were assessed using co-immunoprecipitation and surface biotinylation assays. Anti-MX1 autoantibody levels were significantly elevated in SFN patients compared to controls (p = 0.0278), particularly in the autoimmune SFN subgroup. Patient sera exhibited IgG binding to unmyelinated nerve fibers, with idiopathic and autoimmune SFN cases showing similar staining patterns, suggesting a similar immune-mediated mechanism. Immunocytochemistry showed binding to HEK293-MX1 cells and flow cytometry revealed higher MX1/WT fluorescence intensity ratios in patient sera, further confirming specific immune recognition of MX1. Patch-clamp recordings demonstrated that MX1 overexpression in DRG neurons led to significant membrane depolarization and increased action potential firing frequency (p < 0.0001), indicating heightened neuronal excitability. However, MX1 did not directly interact with TRPC6 or alter its function, suggesting an alternative pathway for its effects. The addition of anti-MX1 IgG did not further modify DRG electrophysiology, implying that the autoimmune component may contribute to SFN pathogenesis through indirect mechanisms. Our findings support the hypothesis that MX1 influences neuronal excitability and plays a role in SFN pathophysiology. Future studies should validate these findings in larger cohorts and explore potential therapeutic strategies targeting MX1-associated pathways in SFN.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03557-6.