Protein Gsα Research Articles

1568-P: Liver-specific Expression of Activated G Protein Gsα Leads to Hyperglycemia with Reduced Pancreatic α-Cell Mass and Function

1568-P: Liver-specific Expression of Activated G Protein Gsα Leads to Hyperglycemia with Reduced Pancreatic α-Cell Mass and Function

Abstract CT079: The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei

Abstract Background: Pseudomyxoma peritonei (PMP) is a rare cancer originating from appendiceal mucinous tumors, which may spread to encompass the entire peritoneal cavity. Complete surgical removal and hyperthermic intraperitoneal chemotherapy cures approximately half of the patients, but patients with recurrent or non-resectable disease have no efficacious treatment options. These patients have a high unmet need for novel treatment options, and in the Pseudovax trial we will explore a novel therapeutic concept. A main molecular driver of PMP (in up to 90% of cases) is otherwise rare mutations in the GNAS oncogene, coding for the Gsα protein. We hypothesized that mutated Gsα would be a tumor neoantigen, and in T cells isolated from peripheral blood of PMP patients, a strong T cell proliferative response was observed. In the corresponding tumors, T cells with up-regulated immune checkpoint molecules PD-1 and TIGIT were detected. Study hypotheses: Patients with GNAS-mutated PMP has a preexisting, attenuated immune response directed against mutated Gsα protein. Vaccination with mutated Gsα peptides can be combined with immune checkpoint inhibition (ICI) with acceptable toxicity. The vaccine will reactivate the immune response and ICI will restore measurable anticancer immunity in patients. Study design: The Pseudovax trial is a small (10 patients) first-in-human, proof-of-concept, prospective, open label, phase I trial. Patients with recurrent or non-resectable PMP are eligible for inclusion. During a treatment period of 12 months, a mutated Gsα peptide vaccine + adjuvant (GM-CSF) will be administered as intradermal injections. After 12 weeks, treatment with ICI will be initiated. The primary endpoints are toxicity and circulating immune response against the vaccine, measured as increase in interferon-gamma production or proliferation of circulating vaccine-specific T cells compared to pre-vaccination baseline. Secondary endpoints are progression-free survival (abdominopelvic CT scans, blood levels of carcinoembryonic antigen (CEA) and/or detection of GNAS mutation by ctDNA analysis). Citation Format: Kjersti Flatmark, Annette Torgunrud, Fleten G. Karianne, Ben Davidson, Hedvig V. Juel, Nadia Mensali, Christin Lund Andersen, Else Marit Inderberg. The Pseudovax trial: A novel peptide vaccine targeting mutated GNAS for pseudomyxoma peritonei [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT079.

Microbially conjugated bile salts found in human bile activate the bile salt receptors TGR5 and FXR.

Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation. N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery. MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts. MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR and are substrates for intestinal (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined.

The Regulatory Roles of Cerebellar Glycosphingolipid Microdomains/Lipid Rafts.

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. Cerebellar lipid rafts are cell surface ganglioside microdomains for the attachment of GPI-anchored neural adhesion molecules and downstream signaling molecules such as Src-family kinases and heterotrimeric G proteins. In this review, we summarize our recent findings on signaling in ganglioside GD3 rafts of cerebellar granule cells and several findings by other groups on the roles of lipid rafts in the cerebellum. TAG-1, of the contactin group of immunoglobulin superfamily cell adhesion molecules, is a phosphacan receptor. Phosphacan regulates the radial migration signaling of cerebellar granule cells, via Src-family kinase Lyn, by binding to TAG-1 on ganglioside GD3 rafts. Chemokine SDF-1α, which induces the tangential migration of cerebellar granule cells, causes heterotrimeric G protein Goα translocation to GD3 rafts. Furthermore, the functional roles of cerebellar raft-binding proteins including cell adhesion molecule L1, heterotrimeric G protein Gsα, and L-type voltage-dependent calcium channels are discussed.

A patient with pseudohypoparathyroidism type 1A previously misdiagnosed as hereditary multiple exostosis: A case report

Pseudohypoparathyroidism type 1A (PHP1A), a rare hereditary disorder, is featured by end-organ resistance to parathyroid hormone and Albright's hereditary osteodystrophy. Heterozygous mutation of guanine nucleotide-binding protein α stimulating (GNAS) gene causes the half decreased bioactivity of the Gsα protein levels. Due to the diverse early clinical manifestations of PHP1A, a diagnosis of PHP1A is often easily overlooked and misdiagnosis or missed diagnosis is common. The present study described a girl who was initially diagnosed with hereditary multiple exostoses, but was afterwards confirmed with PHP1A. Moreover, genetic analysis indicated a new mutation (c2277deIC) of the gene.

A novel GNAS variant presents with disorders of GNAS inactivation and cardiomyopathy

The GNAS gene (OMIM#139320), located on chromosome 20q13.2, encodes for the alpha-subunit of the stimulatory signaling protein, Gsα protein. GNAS variants with inactivating properties are associated with Albright's hereditary osteodystrophy (AHO) and when maternally inherited, pseudohypoparathyroidism 1a (OMIM#103580), which includes multiple hormone resistance. In this clinical report we describe a novel GNAS variant, c.159A>G, p.K53N, in an individual with features consistent with AHO and pseudohypoparathyroidism 1a and its segregation through multiple maternal relatives, including two genotype positive maternal first cousins who also display features classic for AHO. The proband developed unique features including cardiomyopathy which required a heart transplant at 5 years old and immune dysregulation resulting in multisystem organ failure and ultimately, death at the age of 18 years. Additional investigations exploring alternative explanations for the proband's presentation were pursued including whole genome sequencing which was negative. We postulate that the atypical features seen in the proband may have resulted from dysregulated Gsα signaling in cardiac tissue. Future studies are needed to explore the properties of the K53N GNAS variant and this proposed mechanism.

Liver-Specific Expression of Constitutively Active Gsα Leads to Hyperglycemia With Impaired Insulin Secretion

Liver-specific Expression of Constitutively Active Gsα Leads to Hyperglycemia With Impaired Insulin SecretionThe ubiquitously-expressed G protein Gsα couples hormone receptors to the stimulation of intracellular cAMP generation. We previously showed that mice with liver-specific Gsα deficiency (LGsKO) have improved glucose tolerance and enlarged pancreatic islets1. In the present study, we have generated mice with liver-specific expression of a constitutively activated Gsα (LGsR201C) by breeding mice containing a Lox-STOP-Lox-GsαR201C transgene within the Hipp11 locus with albumin-Cre mice. Male LGsR201C mice had normal survival but reduced body weight and increased liver weight. Compared to control littermates, LGsR201C mice had significantly increased hepatic cAMP levels and enhanced hyperglycemic response to glucagon, confirming the activation of liver Gsα/cAMP signaling in LGsR201C mice. As a consequence, LGsR201C mice showed elevated blood glucose levels during both fed and fasting states, as well as enhanced hepatic gluconeogenesis evidenced by pyruvate tolerance test. Serum levels of insulin, glucagon, free fatty acids and triglycerides were comparable between control and LGsR201C mice in the fed state. Results of glucose and insulin tolerance tests showed that LGsR201C mice had severe glucose intolerance with normal insulin sensitivity. Unlike control mice, when given a high dose of glucose (3mg/g ip.), LGsR201C mice had completely blunted first- and second-phase insulin secretory responses to glucose. Since LGsR201C mice exhibited normal pancreatic islet size and insulin content examined with immunohistochemistry, the impaired glucose tolerance in LGsR201C mice probably resulted from an impaired insulin secretion. Results of RNA-seq analysis revealed that an array of genes was oppositely regulated in the liver of LGsKO mice vs. LGsR201C mice. Thus, our data indicate possible organ-to-organ communication between liver and pancreatic β-cells that is regulated by liver Gsα signaling. 1.Chen M., et al., JCI 115:3217, 2005

Molecular Definition of Pseudohypoparathyroidism Variants.

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several differentially methylated regions (DMRs). GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. PHP type Ia (PHP1A) is caused by heterozygous inactivating mutations involving the maternal exons 1-13. Heterozygosity of these maternal GNAS mutations cause PTH-resistant hypocalcemia and hyperphosphatemia because paternal Gsα expression is suppressed in certain organs thus leading to little or no Gsα protein in the proximal renal tubules and other tissues. Besides biochemical abnormalities, PHP1A patients show developmental abnormalities, referred to as Albright’s hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who carry paternal Gsα-specific mutations and typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss of methylation at the A/B DMR alone or at all maternally methylated GNAS exons. Loss of methylation of exon A/B and the resulting biallelic expression of A/B transcript reduces Gsα expression thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, which is the most frequent PHP1B variant. However, this disease variant remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

Targeted Long-Read Sequencing Identifies a Retrotransposon Insertion as a Cause of Altered GNAS Exon A/B Methylation in a Family With Autosomal Dominant Pseudohypoparathyroidism Type 1b (PHP1B).

Pseudohypoparathyroidism type Ib (PHP1B) is characterized predominantly by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia. These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A/B, which reduces in cis expression of the stimulatory G protein α-subunit (Gsα). Paternal Gsα expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A/B reduces further Gsα protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200 bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation. © 2022 American Society for Bone and Mineral Research (ASBMR).

A Signature of Circulating miRNAs Associated With Fibrous Dysplasia of Bone: the mirDys Study.

Fibrous dysplasia (FD) is a rare bone disease caused by activating mutations of GNAS encoding the Gsα protein, enhancing cyclic adenosine monophosphate (cAMP) production by overstimulation of adenylyl cyclase and impairing osteoblastic differentiation. The clinical presentation ranges from asymptomatic to polyostotic forms with severe disability, explained by the mosaic distribution of the GNAS mutation. Physicians have to deal with the gap of knowledge in FD pathogenesis, the absence of prognostic markers and the lack of specific treatment. The identification of specific biomarkers for FD is an important step to improve the clinical and therapeutic approaches. An epigenetic regulation driven by microRNAs (miRNAs), known as promising biomarkers in bone disease, could be involved in FD. We have sought circulating miRNAs that are differentially expressed in FD patients compared to controls and would reflect dysregulations of osteogenesis-related genes and bone disorder. The global miRNA profiling was performed using Next Generation Sequencing in patient serum collected from a discovery cohort of 20 patients (10 polyostotic and 10 monostotic) and 10 controls. From these, we selected 19 miRNAs for a miRNA validation phase from serum of 82 patients and 82 controls, using real-time qPCR. Discovery screening identified 111 miRNAs differentially expressed in patient serum, after adjusting for the false discovery rate (FDR). Among the 82 patients, 55% were polyostotic, and 73% were women with a mean age of 42 years. Six miRNAs (miR-25-3p, miR-93-5p, miR-182-5p, miR-324-5p, miR-363-3p, and miR-451a) were significantly overexpressed in serum, with FDR <0.05. The expression level of these six miRNAs was not associated with the FD severity. In conclusion, we identified a signature of circulating miRNAs associated with FD. These miRNAs are potential negative regulators of gene expression in bone cell progenitors, suggesting their activity in FD by interfering with osteoblastic and osteoclastic differentiation to impair bone mineralization and remodeling processes. © 2020 American Society for Bone and Mineral Research.

SUN-368 Wnt Inhibition Decreases Trabecular Bone in a Mouse Model of Fibrous Dysplasia

Background: G protein-coupled receptor (GPCR) signaling mediates a wide spectrum of physiological functions, including bone development and remodeling. Fibrous dysplasia (FD) is a common skeletal dysplasia where normal bone and bone marrow are replaced by fibrous tissue and expansile trabecular bone lesions. The craniofacial bones are often involved, leading to pain and facial deformities. FD is a mosaic disease caused by a somatic mutation in the GNAS gene encoding the G-protein alpha subunit (Gsα) that leads to constitutive activation of the Gs signaling pathway. Unfortunately, FD has no effective medical treatments.Major challenges have hampered the development of pharmacologic strategies that specifically target GNAS or the Gsα protein. We previously developed the Col1(2.3)/Rs1 mouse model (Rs1) in which the Gs signaling pathway is activated specifically in bone by an engineered GPCR protein. These mice showed increased trabecular bone formation with loss of marrow space and cortical bone, which strongly resembles human FD (1–4). There was also a dramatic increase in the number of immature osteoblasts present in the FD lesions, suggesting that activation of Gs signaling caused an accumulation of these cells. Our prior studies showed increased Wnt signaling, which may be a major driver of this effect. Furthermore, blocking the Gs signaling could reverse the bone phenotype, providing proof-of-concept for finding drugs that could reverse the phenotype. Therefore, we administered the Wnt inhibitor LGK974, currently used in human clinical trials, to the Rs1 mice to test if the FD lesions could be pharmacologically reversed. Methods: We administered LGK974 in 4-week-old Rs1 and non-Rs1 mice. We used a low dose (5mg/kg) for 8 weeks or high dose (30mg/kg) for 4 weeks. The mice were evaluated by histology and micro computed tomography (micro-CT) for mineral density (mg/cm3), bone volume (mm3), and trabecular thickness (um). Results: LGK974 decreased β-catenin levels in bone on western blots. In the low-dose group, the histology and micro-CT showed no statistically significant differences between drug and control groups. In the high-dose group, the micro-CT showed significantly decreased trabecular bone thickness (p=0.0364, n=3) in the drug-treated group (22±2µm) compared with controls (17±2µm). Furthermore, histology showed resorption of the abnormal bone; however, the fibrocellular infiltrate in the Rs1 mice was still present. Conclusions: Wnt inhibition can lead to decreased fibrous dysplastic bone, but separates abnormal bone formation from the fibrocellular infiltrate. These results provide new insight into understanding interactions between the Wnt and Gs signaling pathways in FD pathogenesis and bone formation.

OR04-06 G Protein GSα in Muscle Is Essential for Survival During Cold Adaptation in the Absence of Thermogenesis of Brown Adipose Tissue

Adaptive thermogenesis is important for the control of body temperature (Tb) and maintenance of body weight, and it is primarily regulated by sympathetically-driven brown adipose tissue (BAT). Studies indicate that muscle is also involved in thermogenic regulation. Gsα couples to ligands and receptors, including β-adrenergic receptors, to increase intracellular cAMP. Our previous studies have showed that mice with adipose-specific Gsα deficiency had inactive BAT and impaired cold tolerance. To determine whether Gsα/cAMP signaling in skeletal muscle compensates for loss of BAT thermogenesis, we generated mice with Gsα deficiency in adipocyte tissue alone (AdipGsKO), in skeletal muscle alone (SkMGsKO) or in both (AdipSkMGsKO). Compared to control mice, AdipGsKO and SkMGsKO mice had normal body weight, while AdipSkMGsKO showed reduced body weight with normal food intake and energy expenditure. Both AdipGsKO and AdipSkMGsKO mice had elevated fasting glucose levels, but similar glucose tolerance to control or SkMGsKO mice. SkMGsKO mice displayed reduced insulin sensitivity. When acutely exposed to 6oC for 3 hours, AdipGsKO and AdipSkMGsKO mice rapidly decreased their Tb, indicating that they are sensitive to acute cold exposure, consistent with their inactive BAT. To assess adaptation to chronic cold, mice were exposed to gradually declining ambient temperature from 22oC to 6oC with a daily decrease of 2oC and were then kept at 6oC for 5 days. As expected, both AdipGsKO and AdipSkMGsKO mice failed to stimulate BAT UCP1 by cold adaptation. Unexpectedly, AdipGsKO mice maintained normal Tb similar to control and SkMGsKO mice. However, AdipSkMGsKO mice started to rapidly drop their Tb when ambient temperature declined to 14oC and 85% of SkMGsKO mice (11/13) died before the end of experiment. These results suggest that when there is a lack of BAT function, Gsα/cAMP signaling in muscle plays an essential role for mice to survive in response to chronic cold challenge.

Shortened Fingers and Toes: GNAS Abnormalities are Not the Only Cause.

The PTH/PTHrP receptor (PTHR1) mediates the actions of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) by coupling this G protein-coupled receptor (GPCR) to the alpha-subunit of the heterotrimeric stimulatory G protein (Gsα) and thereby to the formation of cAMP. In growth plates, PTHrP-dependent activation of the cAMP/PKA second messenger pathway prevents the premature differentiation of chondrocytes into hypertrophic cells resulting in delayed growth plate closure. Heterozygous mutations in GNAS, the gene encoding Gsα, lead to a reduction in cAMP levels in growth plate chondrocytes that is sufficient to cause shortening of metacarpals and/or -tarsals, i. e. typical skeletal aspects of Albright's Hereditary Osteodystrophy (AHO). However, heterozygous mutations in other genes, including those encoding PTHrP, PRKAR1A, PDE4D, and PDE3A, can lead to similar or even more pronounced acceleration of skeletal maturation that is particularly obvious in hands and feet, and reduces final adult height. Genetic mutations other than those resulting in Gsα haploinsufficiency thus reduce intracellular cAMP levels in growth plate chondrocytes to a similar extent and thereby accelerate skeletal maturation.

First two cases of craniomaxillofacial fibrous dysplasia from Nepal - case series.

Fibrous dysplasia is a benign fibro-osseous disease of the bone, which is most commonly associated with congenital mutations in cAMP regulating protein Gsα coded by GNAS-1 gene. Often it is seen involving the craniofacial skeleton and can range from an asymptomatic monostotic form to polyostotic variant involving almost all the bones of the skull, thereby leading to functional and esthetic problems. This requires a continuous monitoring of the involved region throughout the life of the patient, even after the surgical interventions. We are presenting two cases of craniofacial form of fibrous dysplasia. One case shows monostotic form, while the other case shows features of polyostotic form of disease. To the best of our knowledge, these are the first two cases of craniomaxillofacial fibrous dysplasia from Nepal, which will be reported and published.

The stimulatory G protein Gsα is required in melanocortin 4 receptor–expressing cells for normal energy balance, thermogenesis, and glucose metabolism

Central melanocortin 4 receptors (MC4Rs) stimulate energy expenditure and inhibit food intake. MC4Rs activate the G protein Gsα, but whether Gsα mediates all MC4R actions has not been established. Individuals with Albright hereditary osteodystrophy (AHO), who have heterozygous Gsα-inactivating mutations, only develop obesity when the Gsα mutation is present on the maternal allele because of tissue-specific genomic imprinting. Furthermore, evidence in mice implicates Gsα imprinting within the central nervous system (CNS) in this disorder. In this study, we examined the effects of Gsα in MC4R-expressing cells on metabolic regulation. Mice with homozygous Gsα deficiency in MC4R-expressing cells (MC4RGsKO) developed significant obesity with increased food intake and decreased energy expenditure, along with impaired insulin sensitivity and cold-induced thermogenesis. Moreover, the ability of the MC4R agonist melanotan-II (MTII) to stimulate energy expenditure and to inhibit food intake was impaired in MC4RGsKO mice. MTII failed to stimulate the secretion of the anorexigenic hormone peptide YY (PYY) from enteroendocrine L cells, a physiological response mediated by MC4R-Gsα signaling, even though baseline PYY levels were elevated in these mice. In Gsα heterozygotes, mild obesity and reduced energy expenditure were present only in mice with a Gsα deletion on the maternal allele in MC4R-expressing cells, whereas food intake was unaffected. These results demonstrate that Gsα signaling in MC4R-expressing cells is required for controlling energy balance, thermogenesis, and peripheral glucose metabolism. They further indicate that Gsα imprinting in MC4R-expressing cells contributes to obesity in Gsα knockout mice and probably in individuals with Albright hereditary osteodystrophy as well.

Comparison Between the Structure‐Function Relationships in Oncogenic and Wild‐Type Gα Subunits

Guanine nucleotide binding proteins (G‐proteins), in conjunction with their receptors (GPCR), are one of the most prevalent signaling systems and are involved in the regulation of an array of cellular processes. Upon binding of an extracellular stimulus to the GPCR, the activated α‐subunit (Gα) exchanges GDP for GTP, releases both the βγ‐complex and the GPCR, and associates with an effector that further relays the signal. The signal terminates with the hydrolysis of GTP to GDP, causing the α‐subunit to reunite with the βγ‐subunits and the GPCR. G‐protein signaling must be tightly regulated to ensure the appropriate responses to extracellular stimuli. Any misregulation caused by mutations can be detrimental. Gsα and Giα1 are G‐protein subunits that, respectively, stimulate or inhibit adenylyl cyclase and regulate the production of the secondary messenger, cAMP. We studied two mutations observed in cancers of the intestine (R231H mutation in Gsα) and the colon (R208Q mutation in Giα1). Spectroscopic and crystallographic approaches were used to further understand these disease states.Fluorescence spectroscopy, circular dichroism, and UV/Vis spectroscopy showed lower melting temperature in the active conformation of the oncogenic mutants, indicating disruptions in the noncovalent interactions. Using malachite green and fluorescence GTP assays, we found that the kcat values for oncogenic Giα1 were lower than for the WT protein; the trend was the opposite for the WT and oncogenic Gsα proteins. When comparing the x‐ray structure of the R208Q mutant with that of the WT Giα1, the oncogenic point mutations do not show significant differences in the structure of the protein as a whole. However, molecular dynamics simulations modeling the interactions of T181 in Giα1 and T204 in Gsα (key residues in the hydrolysis of GTP) show drastically different interaction energies between mutants and WT proteins. Taken together, these findings suggest that the altered rates of GTP hydrolysis lead to an imbalance of cAMP in tumor cells.Support or Funding InformationNIH R15GM112025This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structural insights into positive and negative allosteric regulation of a G protein-coupled receptor through protein-lipid interactions

Lipids are becoming known as essential allosteric modulators of G protein-coupled receptor (GPCRs). However, how they exert their effects on GPCR conformation at the atomic level is still unclear. In light of recent experimental data, we have performed several long-timescale molecular dynamics (MD) simulations, totalling 24 μs, to rigorously map allosteric modulation and conformational changes in the β2 adrenergic receptor (β2AR) that occur as a result of interactions with three different phospholipids. In particular, we identify different sequential mechanisms behind receptor activation and deactivation, respectively, mediated by specific lipid interactions with key receptor regions. We show that net negatively charged lipids stabilize an active-like state of β2AR that is able to dock Gsα protein. Clustering of anionic lipids around the receptor with local distortion of membrane thickness is also apparent. On the other hand, net-neutral zwitterionic lipids inactivate the receptor, generating either fully inactive or intermediate states, with kinetics depending on lipid headgroup charge distribution and hydrophobicity. These chemical differences alter membrane thickness and density, which differentially destabilize the β2AR active state through lateral compression effects.

Elevated expression of Gsα in intrahepatic cholangiocarcinoma associates with poor prognosis

The stimulatory G protein a subunit (Gsα) plays important roles in diverse cell processes including tumorigenesis. Activating mutations in Gsα gene (GNAS) have been reported to be associated with poor prognosis in various human carcinomas. Furthermore, Gsα signaling is crucial in promoting liver regeneration by interacting with growth factor signaling, indicating that Gsα might play a promoting role in cancer development. However, little is known about the correlation between Gsα levels and clinicopathological parameters in intrahepatic cholangiocarcinoma (ICC). We performed immunoblotting to examine the expression levels of Gsα and Ki67 proteins in tumor tissues and the corresponding adjacent tissues. A total of 74 pair of specimens resected from 74 ICC patients were examined. The association between Gsα levels and clinicopathological findings and prognosis of the patients was evaluated. Western blotting demonstrated that the expression of Gsα was significantly higher in ICC tissues compared with that in their corresponding adjacent tissues. Gsα protein was highly expressed in about half of ICC tissues (48.6%, 36/74) while only 28.4% (21/74) of tumor adjacent tissues showed Gsα high expression (P=0.011). High Gsα expression in ICC was significantly associated with the numbers of tumor nodules (P=0.037) and lymph node metastases (P=0.010). Moreover, the level of Gsα was significantly and positively correlated with Ki67 expression (P<0.001). In addition, the recurrence-free survival rate and overall survival rate in the Gsα high group were significantly lower than those in the Gsα low group (P=0.004 and P=0.005, respectively). High Gsα expression is correlated with poor prognosis in ICC patients. Gsα might serve as a potential prognostic indicator of ICC.

Current Nomenclature of Pseudohypoparathyroidism: Inactivating Parathyroid Hormone/Parathyroid Hormone-Related Protein Signaling Disorder.

Disorders related to parathyroid hormone (PTH) resistance and PTH signaling pathway impairment are historically classified under the term of pseudohypoparathyroidism (PHP). The disease was first described and named by Fuller Albright and colleagues in 1942. Albright hereditary osteodystrophy (AHO) is described as an associated clinical entity with PHP, characterized by brachydactyly, subcutaneous ossifications, round face, short stature and a stocky build. The classification of PHP is further divided into PHP-Ia, pseudo-PHP (pPHP), PHP-Ib, PHP-Ic and PHP-II according to the presence or absence of AHO, together with an in vivo response to exogenous PTH and the measurement of Gsα protein activity in peripheral erythrocyte membranes in vitro. However, PHP classification fails to differentiate all patients with different clinical and molecular findings for PHP subtypes and classification become more complicated with more recent molecular characterization and new forms having been identified. So far, new classifications have been established by the EuroPHP network to cover all disorders of the PTH receptor and its signaling pathway. Inactivating PTH/PTH-related protein signaling disorder (iPPSD) is the new name proposed for a group of these disorders and which can be further divided into subtypes - iPPSD1 to iPPSD6. These are termed, starting from PTH receptor inactivation mutation (Eiken and Blomstrand dysplasia) as iPPSD1, inactivating Gsα mutations (PHP-Ia, PHP-Ic and pPHP) as iPPSD2, loss of methylation of GNAS DMRs (PHP-Ib) as iPPSD3, PRKAR1A mutations (acrodysostosis type 1) as iPPSD4, PDE4D mutations (acrodysostosis type 2) as iPPSD5 and PDE3A mutations (autosomal dominant hypertension with brachydactyly) as iPPSD6. iPPSDx is reserved for unknown molecular defects and iPPSDn+1 for new molecular defects which are yet to be described. With these new classifications, the aim is to clarify the borders of each different subtype of disease and make the classification according to molecular pathology. The iPPSD group is designed to be expandable and new classifications will readily fit into it as necessary.

Cardioprotective adaptation to chronic hypoxia stimulates the ROS-dependent/cytosolic phospholipase A2a pathway in rat heart

Adaptation to chronic intermittent hypoxia (CIH) is associated with reactive oxygen species (ROS) generation implicated in the cardiac ischemia-resistant phenotype. Phospholipases A2 (PLA2s) belong to ROS-dependent enzymes and are the key enzymes that take part in the repairing and remodeling of the cell membranes. The aim was to study the effect of CIH on PLA2s isoform expression and β-adrenoceptors (β-AR) signaling in the rat heart. Chronic administration of antioxidant tempol was used to verify the ROS involvement in CIH effect on PLA2s expression. Selected proteins were analysed in the left ventricular (LV) myocardium of adult male Wistar rats adapted to CIH (7,000 m, 8h/day, 5 weeks). CIH increased the total cPLA2α protein in cytosol and membranes (by 191% and 38%, respectively) and p-cPLA2α (by 23%) in membranes. On the other hand, both iPLA2 and sPLA2IIA were down-regulated. Tempol treatment prevented only CIH-induced cPLA2α up-regulation and its phosphorylation on Ser505.The total number of β-AR did not change. Nevertheless, the proportion of β2-AR significantly increased from 30% in normoxic LV to 40% in CIH. Concomitantly, the protein abundance of Gi1,2 and Gi3 increased by 39% and 35%, respectively, by CIH, whereas there was no significant change in the expression of the stimulatory Gsα protein; adenylyl cyclase 5/6 was down-regulated. Furthermore, active/phosphorylated forms of upstream cPLA2 activating signaling molecules (PKCα, ERK1/2 and p38) were increased by CIH. The protein level of cyclooxygenase 2 (COX-2) and the concentration of prostaglandin E2 increased by 36% and 84%, respectively. Our results show that CIH diversely affects myocardial PLA2s and suggest that ROS are responsible for the activation of cPLA2α under these conditions. Moreover, CIH enhances β2-AR-Gi signaling which activates the cPLA2α-COX-2 pathway via ERK/p38. Further experiments should assess the potential role of this pathway in the induction of protected phenotype of CIH hearts.