A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca2+ 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 Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, 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 Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.

Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I-II (76.9%), 1 tier-III (7.7%), and 2 tier-IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract.

OBJECTIVE: Pseudohypoparathyroidism (PHP) comprises a cluster of heterogeneous diseases characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone (PTH) resistance. PHP type 1B (PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16. STX16 exon 2-6 deletion is commonly observed in autosomal dominant (AD)-PHP1B, while sporadic PHP1B commonly results from methylation abnormalities of maternal differentially methylated regions and remains unclear at the molecular level.
METHODS: A 39-year-old male patient with PHP1B, who had his first seizure at 15 years of age, presented to our hospital. The methylation-specific multiplex ligation-dependent probe amplification results showed a half-reduced copy number of STX16 exon 5-7 and loss of methylation at GNAS exon A/B. His mother also had a half-reduced copy number of STX16 exon 5-7 but with normal methylation of GNAS. His father has a normal copy number of STX16 and normal methylation of GNAS.
CONCLUSIONS: For the recognition and early diagnosis of this kind of disease, here we report the clinical symptoms, auxiliary examinations, genetic testing characteristics, and treatment of the patient.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

Formation and retrieval of remote contextual memory depends on cortical engram neurons that are defined during learning. Manipulation of astrocytic Gq and Gi associated G-protein coupled receptor (GPCR) signaling has been shown to affect memory processing, but little is known about the role of cortical astrocytic Gs-GPCR signaling in remote memory acquisition and the functioning of cortical engram neurons. We assessed this by chemogenetic manipulation of astrocytes in the medial prefrontal cortex (mPFC) of male mice, during either encoding or consolidation of a contextual fear memory, while simultaneously labeling cortical engram neurons. We found that stimulation of astrocytic Gs signaling during memory encoding and consolidation did not alter remote memory expression. In line with this, the size of the mPFC engram population and the recall-induced reactivation of these neurons was unaffected. Hence, our data indicate that activation of Gs-GPCR signaling in cortical astrocytes is not sufficient to alter memory performance and functioning of cortical engram neurons.

Chemoresistance is one of the major causes of poor prognosis in osteosarcoma. Alternative therapeutic strategies for osteosarcoma are limited, indicating that increasing sensitivity to currently used chemotherapies could be an effective approach to improve patient outcomes. Using a kinome-wide CRISPR screen, we identified PRKDC as a critical determinant of doxorubicin (DOX) sensitivity in osteosarcoma. The analysis of clinical samples demonstrated that PRKDC was hyperactivated in osteosarcoma, and functional experiments showed that the loss of PRKDC significantly increased sensitivity of osteosarcoma to DOX. Mechanistically, PRKDC recruited and bound GDE2 to enhance the stability of protein GNAS. The elevated GNAS protein levels subsequently activated AKT phosphorylation and conferred resistance to DOX. The PRKDC inhibitor AZD7648 and DOX synergized and strongly suppressed the growth of osteosarcoma in mouse xenograft models and human organoids. In conclusion, the PRKDC-GDE2-GNAS-AKT regulatory axis suppresses DOX sensitivity and comprises targetable candidates for improving the efficacy of chemotherapy in osteosarcoma. Significance: Targeting PRKDC suppresses AKT activation and increases sensitivity to doxorubicin in osteosarcoma, which provides a therapeutic strategy for overcoming chemoresistance.

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.

UNASSIGNED: The dysregulation of cell fate toward osteoprecursor cells associated with most GNAS-based disorders may lead to episodic de novo extraskeletal or ectopic bone formation in subcutaneous tissues. The bony lesion distribution suggests the involvement of abnormal differentiation of mesenchymal stem cells (MSCs) and/or more committed precursor cells. Data from transgenic mice support the concept that GNAS is a crucial factor in regulating lineage switching between osteoblasts (OBs) and adipocyte fates. The mosaic nature of heterotopic bone lesions suggests that GNAS genetic defects provide a sensitized background for ectopic osteodifferentiation, but the underlying molecular mechanism remains largely unknown.
UNASSIGNED: The effect of GNAS silencing in the presence and/or absence of osteoblastic stimuli was evaluated in the human L88/5 MSC line during osteodifferentiation. A comparison of the data obtained with data coming from a bony lesion from a GNAS-mutated patient was also provided.
UNASSIGNED: Our study adds some dowels to the current fragmented notions about the role of GNAS during osteoblastic differentiation, such as the premature transition of immature OBs into osteocytes and the characterization of the differences in the deposed bone matrix.
UNASSIGNED: We demonstrated that our cell model partially replicates the in vivo behavior results, resulting in an applicable human model to elucidate the pathophysiology of ectopic bone formation in GNAS-based disorders.

Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/β-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/β-catenin signaling pathway.