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J Biol Chem ; 297(3): 101099, 2021 09.
Article En | MEDLINE | ID: mdl-34418434

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB1 cannabinoid receptor G-protein coupling in part by altering the selectivity for Gαi subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel ß-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The ß-barrel has a gap between strands ß8 and ß10, which deviates from ß-sandwich fatty acid-binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the ß-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing ß-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gαi N terminus. However, CRIP1a could not bind the nonmyristolyated Gαi peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB1 receptor.

Carrier Proteins/metabolism , Carrier Proteins/ultrastructure , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Cannabinoids/metabolism , Carrier Proteins/genetics , Endocannabinoids , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/ultrastructure , Membrane Proteins/metabolism , Mice , Peptides/metabolism , Protein Binding , Protein Conformation , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB1/ultrastructure , Receptors, Cannabinoid/metabolism , Receptors, Cannabinoid/ultrastructure
Am J Physiol Heart Circ Physiol ; 321(3): H496-H508, 2021 09 01.
Article En | MEDLINE | ID: mdl-34270373

Angiotensin II (ANG II) plays an important role in the regulation of various physiological functions including proliferation, hypertrophy of vascular smooth muscle cells (VSMCs) through the overexpression of Giα proteins. Sirtuin 1 (Sirt1), a class III histone deacetylase and epigenetic regulator is implicated in a wide range of cellular functions, including migration and growth of VSMCs and in ANG II-induced hypertension. The present study was undertaken to examine the role of Sirt1 in ANG II-induced overexpression of Giα proteins and hyperproliferation of aortic VSMCs. We show that ANG II treatment of VSMCs increased the expression of Sirt1, which was attenuated by AT1 and AT2 receptor antagonists, losartan, and PD123319, respectively. In addition, the knockdown of Sirt1 by siRNA attenuated ANG II-induced overexpression of Giα-2 and Giα-3 proteins, hyperproliferation of VSMCs and the overexpression of cell cycle proteins, cyclin D1, Cdk4, and phosphorylated retinoblastoma proteins. Furthermore, ANG II-induced increased levels of superoxide anion (O2-) and NADPH oxidase activity and increased phosphorylation of ERK1/2 and Akt that are implicated in enhanced expression of Giα proteins and hyperproliferation of VSMCs were also attenuated to control levels by silencing of Sirt1. In addition, depletion of Sirt1 by siRNA also attenuated ANG II-induced enhanced phosphorylation of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), and insulin-like growth factor receptor (IGFR) in VSMCs. In summary, our results demonstrate that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated mitogen-activated protein (MAP) kinase/Akt signaling pathway enhances the expression of Giα proteins and cell cycle proteins and results in the hyperproliferation of VSMCs.NEW & NOTEWORTHY ANG II regulates various physiological functions including proliferation of VSMCs through the overexpression of Giα proteins. Sirt1, a class III histone deacetylase, is implicated in several cellular functions, including VSMC growth and ANG II-induced hypertension. We showed for the first time that ANG II increased the expression of Sirt1, which through oxidative stress, growth factor receptor-mediated MAP kinase/Akt signaling pathway enhances the levels of Giα and cell cycle proteins resulting in the hyperproliferation of VSMCs.

Angiotensin II/pharmacology , Cell Proliferation , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Sirtuin 1/genetics , Angiotensin II Type 1 Receptor Blockers/pharmacology , Animals , Aorta/cytology , Cells, Cultured , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Imidazoles/pharmacology , Losartan/pharmacology , Male , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/physiology , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptors, Platelet-Derived Growth Factor/genetics , Receptors, Platelet-Derived Growth Factor/metabolism , Sirtuin 1/metabolism
PLoS One ; 16(7): e0241939, 2021.
Article En | MEDLINE | ID: mdl-34292976

For sustainable function, each pancreatic islet ß cell maintains thousands of insulin secretory granules (SGs) at all times. Glucose stimulation induces the secretion of a small portion of these SGs and simultaneously boosts SG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, young insulin SGs are more likely secreted during glucose-stimulated insulin secretion (GSIS) for unknown reasons, while older SGs tend to lose releasability and be degraded. Here, we examine the roles of microtubule (MT) and Gαo-signaling in regulating the preferential secretion of young versus old SGs. We show that both MT-destabilization and Gαo inactivation results in more SGs localization near plasma membrane (PM) despite higher levels of GSIS and reduced SG biosynthesis. Intriguingly, MT-destabilization or Gαo-inactivation results in higher secretion probabilities of older SGs, while combining both having additive effects on boosting GSIS. Lastly, Gαo inactivation does not detectably destabilize the ß-cell MT network. These findings suggest that Gαo and MT can modulate the preferential release of younger insulin SGs via largely parallel pathways.

GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Insulin Secretion , Microtubules/metabolism , Secretory Vesicles/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Cellular Senescence , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Glucose/pharmacology , Insulin Secretion/drug effects , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Mice , Mice, Inbred ICR , Mice, Knockout , Nocodazole/pharmacology , Signal Transduction/drug effects
Biochem Biophys Res Commun ; 569: 100-105, 2021 09 10.
Article En | MEDLINE | ID: mdl-34237428

Follicle Stimulating Hormone (FSH) acts via FSH-Receptor (FSH-R) by employing cAMP as the dominant secondary messenger in testicular Sertoli cells (Sc) to support spermatogenesis. Binding of FSH to FSH-R, results the recruitment of the intracellular GTP binding proteins, either stimulatory Gαs or inhibitory Gαi that in turn regulate cAMP production in Sc. The cytosolic concentration of cAMP being generated by FSH-R thereafter critically determines the downstream fate of the FSH signalling. The pleiotropic action of FSH due to differential cAMP output during functional maturation of Sc has been well studied. However, the developmental and cellular regulation of the Gα proteins associated with FSH-R is poorly understood in Sc. In the present study, we report the differential transcriptional modulation of the Gα subunit genes by FSH mediated cAMP signalling in neonatal and pubertal rat Sc. Our data suggested that unlike in neonatal Sc, both the basal and FSH/forskolin induced expression of Gαs, Gαi-1, Gαi-2 and Gαi-3 transcripts was significantly (p < 0.05) up-regulated in pubertal Sc. Further investigations involving treatment of Sc with selective Gαi inhibitor pertussis toxin, confirmed the elevated expression of Gi subunits in pubertal Sc. Collectively our results indicated that the high level of Gαi subunits serves as a negative regulator to optimize cAMP production in pubertal Sc.

Cyclic AMP/metabolism , Follicle Stimulating Hormone/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Sertoli Cells/drug effects , Signal Transduction/drug effects , Animals , Animals, Newborn , Cells, Cultured , Colforsin/pharmacology , Follicle Stimulating Hormone/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Gene Expression Regulation/drug effects , Male , Pertussis Toxin/pharmacology , Protein Binding , Rats, Wistar , Receptors, FSH/genetics , Receptors, FSH/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sertoli Cells/cytology , Sertoli Cells/metabolism , Sexual Maturation/physiology , Spermatogenesis/drug effects , Spermatogenesis/genetics
Nat Commun ; 12(1): 4590, 2021 07 28.
Article En | MEDLINE | ID: mdl-34321466

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Signal Transduction/physiology , Stearic Acids/metabolism , Acylation , Cell Membrane/metabolism , Cell Proliferation , Fatty Acids/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Lipoylation , MCF-7 Cells , Oleic Acids/metabolism
Commun Biol ; 4(1): 662, 2021 06 02.
Article En | MEDLINE | ID: mdl-34079054

Pathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of Gi-signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among Gi-coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest Gi-coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders.

GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Gyrus Cinguli/cytology , Gyrus Cinguli/physiology , Pyramidal Cells/physiology , Animals , Clozapine/analogs & derivatives , Clozapine/pharmacology , Female , GTP-Binding Protein alpha Subunits, Gi-Go/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Gene Expression/drug effects , Gyrus Cinguli/drug effects , Humans , Impulsive Behavior/drug effects , Impulsive Behavior/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Pyramidal Cells/cytology , Pyramidal Cells/drug effects , Receptors, Metabotropic Glutamate/drug effects , Receptors, Metabotropic Glutamate/genetics , Receptors, Metabotropic Glutamate/physiology , Signal Transduction
Mol Pharmacol ; 100(3): 217-223, 2021 09.
Article En | MEDLINE | ID: mdl-34135098

Regulators of G protein signaling (RGS) proteins modulate signaling by G protein-coupled receptors. Using a knock-in transgenic mouse model with a mutation in Gαo that does not bind RGS proteins (RGS-insensitive), we determined the effect of RGS proteins on presynaptic µ opioid receptor (MOR)-mediated inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). The MOR agonists [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and met-enkephalin (ME) inhibited evoked inhibitory postsynaptic currents (eIPSCs) in the RGS-insensitive mice compared with wild-type (WT) littermates, respectively. Fentanyl inhibited eIPSCs similarly in both WT and RGS-insensitive mice. There were no differences in opioid agonist inhibition of spontaneous GABA release between the genotypes. To further probe the mechanism underlying these differences between opioid inhibition of evoked and spontaneous GABA release, specific myristoylated Gα peptide inhibitors for Gαo1 and Gαi1-3 that block receptor-G protein interactions were used to test the preference of agonists for MOR-Gα complexes. The Gαo1 inhibitor reduced DAMGO inhibition of eIPSCs, but Gαi1-3 inhibitors had no effect. Both Gαo1 and Gαi1-3 inhibitors separately reduced fentanyl inhibition of eIPSCs but had no effects on ME inhibition. Gαi1-3 inhibitors blocked the inhibitory effects of ME and fentanyl on miniature postsynaptic current (mIPSC) frequency, but both Gαo1 and Gαi1-3 inhibitors were needed to block the effects of DAMGO. Finally, baclofen-mediated inhibition of GABA release is unaffected in the RGS-insensitive mice and in the presence of Gαo1 and Gαi1-3 inhibitor peptides, suggesting that GABAB receptor coupling to G proteins in vlPAG presynaptic terminals is different than MOR coupling. SIGNIFICANCE STATEMENT: Presynaptic µ opioid receptors (MORs) in the ventrolateral periaqueductal gray are critical for opioid analgesia and are negatively regulated by RGS proteins. These data in RGS-insensitive mice provide evidence that MOR agonists differ in preference for Gαo versus Gαi and regulation by RGS proteins in presynaptic terminals, providing a mechanism for functional selectivity between agonists. The results further define important differences in MOR and GABAB receptor coupling to G proteins that could be exploited for new pain therapies.

GTP-Binding Protein alpha Subunit, Gi2/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Presynaptic Terminals/physiology , Receptors, Opioid, mu/physiology , gamma-Aminobutyric Acid/metabolism , Analgesics, Opioid/pharmacology , Animals , Baclofen/pharmacology , Female , GTP-Binding Protein alpha Subunit, Gi2/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Inhibitory Postsynaptic Potentials/drug effects , Inhibitory Postsynaptic Potentials/physiology , Male , Mice , Mice, Transgenic , Models, Animal , RGS Proteins/metabolism , Receptors, GABA-B/metabolism , Receptors, Opioid, mu/agonists
Nat Commun ; 12(1): 2861, 2021 05 17.
Article En | MEDLINE | ID: mdl-34001891

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

Epithelium/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Hair Cells, Auditory/metabolism , Homeodomain Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Transcription Factors/metabolism , Animals , Cell Polarity/genetics , Female , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Hair Cells, Auditory/cytology , Homeodomain Proteins/genetics , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal/methods , Receptors, G-Protein-Coupled/genetics , Transcription Factors/genetics , Zebrafish/metabolism
Theranostics ; 11(10): 4894-4909, 2021.
Article En | MEDLINE | ID: mdl-33754034

IL-4 induces Akt activation in macrophages, required for full M2 (alternative) polarization. We examined the roles of Gαi1 and Gαi3 in M2 polarization using multiple genetic methods. Methods and Results: In MEFs and primary murine BMDMs, Gαi1/3 shRNA, knockout or dominant negative mutations attenuated IL-4-induced IL4Rα endocytosis, Gab1 recruitment as well as Akt activation, leaving STAT6 signaling unaffected. Following IL-4 stimulation, Gαi1/3 proteins associated with the intracellular domain of IL-4Rα and the APPL1 adaptor, to mediate IL-4Rα endosomal traffic and Gab1-Akt activation in BMDMs. In contrast, gene silencing of Gαi1/3 with shRNA or knockout resulted in BMDMs that were refractory to IL-4-induced M2 polarization. Conversely, Gαi1/3-overexpressed BMDMs displayed preferred M2 response with IL-4 stimulation. In primary human macrophages IL-4-induced Akt activation and Th2 genes expression were inhibited with Gαi1/3 silencing, but augmented with Gαi1/3 overexpression. In Gαi1/3 double knockout (DKO) mice, M2 polarization, by injection of IL-4 complex or chitin, was potently inhibited. Moreover, in a murine model of asthma, ovalbumin-induced airway inflammation and hyperresponsiveness were largely impaired in Gαi1/3 DKO mice. Conclusion: These findings highlight novel and essential roles for Gαi1/3 in regulating IL-4-induced signaling, macrophage M2 polarization and allergic asthma response.

Asthma/immunology , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Interleukin-4/immunology , Macrophages/immunology , Respiratory Hypersensitivity/genetics , Animals , GTP-Binding Protein alpha Subunits, Gi-Go/immunology , Mice , Mice, Knockout , Ovalbumin , Proto-Oncogene Proteins c-akt/metabolism , Respiratory Hypersensitivity/immunology , TOR Serine-Threonine Kinases/metabolism
Am J Med Genet A ; 185(4): 1204-1210, 2021 04.
Article En | MEDLINE | ID: mdl-33442900

Feingold Syndrome type 1 (FS1) is an autosomal dominant disorder due to a loss of function mutations in the MYCN gene. FS1 is generally clinically characterized by mild learning disability, microcephaly, short palpebral fissures, short stature, brachymesophalangy, hypoplastic thumbs, as well as syndactyly of toes, variably associated with organ abnormalities, the most common being gastrointestinal atresia. In current literature, more than 120 FS1 patients have been described, but diagnostic criteria are not well agreed upon, likewise the genotype-phenotype correlations are not well understood. Here, we describe 11 FS1 patients, belonging to six distinct families, where we have identified three novel MYCN mutations along with three pathogenetic variants, the latter which have already been reported. Several patients presented a mild phenotype of the condition and they have been diagnosed as being affected only after segregation analyses of the MYCN mutation identified in the propositus. We also describe here the first ever FS1 patient with severe intellectual disability having a maternally inherited MYCN variant together with an additional GNAO1 mutation inherited paternally. Mutations in the GNAO1 gene are associated with a specific form of intellectual disability and epilepsy, thus the finding of two different rare diseases in the same patient could explain his severe phenotype. Therein, a thorough investigation is merited into the possibility that additional variants in patients with a MYCN mutation and severe phenotype do exist. Finally, in order to guarantee a more reliable diagnosis of FS1, we suggest using both major and minor clinical-molecular diagnostic criteria.

Eyelids/abnormalities , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Genetic Predisposition to Disease , Intellectual Disability/genetics , Limb Deformities, Congenital/genetics , Microcephaly/genetics , N-Myc Proto-Oncogene Protein/genetics , Tracheoesophageal Fistula/genetics , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Adolescent , Child , Child, Preschool , Eyelids/pathology , Female , Genetic Association Studies , Genetic Testing , Genotype , Humans , Infant , Infant, Newborn , Intellectual Disability/complications , Intellectual Disability/pathology , Limb Deformities, Congenital/complications , Limb Deformities, Congenital/pathology , Male , Microcephaly/complications , Microcephaly/pathology , Phenotype , Syndactyly/complications , Syndactyly/genetics , Syndactyly/pathology , Tracheoesophageal Fistula/complications , Tracheoesophageal Fistula/pathology
Mol Cell Neurosci ; 110: 103585, 2021 01.
Article En | MEDLINE | ID: mdl-33358996

Olfactory GPCRs (ORs) in mammalian olfactory receptor neurons (ORNs) mediate excitation through the Gαs family member Gαolf. Here we tentatively associate a second G protein, Gαo, with inhibitory signaling in mammalian olfactory transduction by first showing that odor evoked phosphoinositide 3-kinase (PI3K)-dependent inhibition of signal transduction is absent in the native ORNs of mice carrying a conditional OMP-Cre based knockout of Gαo. We then identify an OR from native rat ORNs that are activated by octanol through cyclic nucleotide signaling and inhibited by citral in a PI3K-dependent manner. We show that the OR activates cyclic nucleotide signaling and PI3K signaling in a manner that reflects its functionality in native ORNs. Our findings lay the groundwork to explore the interesting possibility that ORs can interact with two different G proteins in a functionally identified, ligand-dependent manner to mediate opponent signaling in mature mammalian ORNs.

GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Olfactory Receptor Neurons/metabolism , Animals , Cells, Cultured , Female , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction
Brain Dev ; 43(4): 576-579, 2021 Apr.
Article En | MEDLINE | ID: mdl-33358199

BACKGROUND: Mutations in GNAO1 typically result in neurodevelopmental disorders, including involuntary movements. They may be improved using calcium-channel modulators. CASE: The patient visited our hospital at age 2 years because of moderate global developmental delay. Her intermittent, generalized involuntary movements started at age 8 years. A de novo GNAO1 mutation, NM_020988.2:c.626G > A, (p.Arg209Cys), was identified by whole exome sequencing. At age 9 years, she experienced severe, intermittent involuntary movements, which led to rhabdomyolysis. She needed intensive care with administration of midazolam, dantrolene sodium hydrate, and plasma exchange. We started treating her with gabapentin (GBP), after which she recovered completely. At age 11 years, she developed continuous, generalized involuntary movements. This prompted us to increase the GBP dose, which again resolved the involuntary movements completely. CONCLUSION: In the case of movement disorders associated with GNAO1 mutations, GBP treatment may be attempted before more invasive procedures are performed.

Anticonvulsants/therapeutic use , Dyskinesias/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Gabapentin/therapeutic use , Mutation , Child , Child, Preschool , Dyskinesias/drug therapy , Female , Humans , Treatment Outcome
Blood ; 137(9): 1181-1191, 2021 03 04.
Article En | MEDLINE | ID: mdl-32898863

Leukemogenesis is characterized by chromosomal rearrangements with additional molecular disruptions, yet the cooperative mechanisms are still unclear. Using whole-exome sequencing of a pair of monozygotic twins who were discordant for childhood acute lymphoblastic leukemia (ALL) with ETV6-RUNX1 (E/R) gene fusion successively after birth, we identified the R209C mutation of G protein subunit α o1 (GNAO1) as a new ALL risk loci. Moreover, GNAO1 missense mutations are recurrent in ALL patients and are associated with E/R fusion. Ectopic expression of the GNAO1 R209C mutant increased its GTPase activity and promoted cell proliferation and cell neoplastic transformation. Combined with the E/R fusion, the GNAO1 R209C mutation promoted leukemogenesis through activating PI3K/Akt/mTOR signaling. Reciprocally, activated mTORC1 phosphorylated p300 acetyltransferase, which acetylated E/R and thereby enhanced the E/R transcriptional activity of GNAO1 R209C. Thus, our study provides clinical evidence of the functional cooperation of GNAO1 mutations and E/R fusion, suggesting GNAO1 as a therapeutic target in human leukemia.

Carcinogenesis/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Animals , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/genetics , Female , HEK293 Cells , Humans , Male , Mice , Models, Molecular , Mutation , Mutation, Missense , Oncogene Proteins, Fusion/genetics , Point Mutation
Molecules ; 25(21)2020 Nov 06.
Article En | MEDLINE | ID: mdl-33172018

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Cell Differentiation , Cell Line , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Guanine Nucleotide Dissociation Inhibitors/genetics , HEK293 Cells , Humans , Neural Stem Cells , Up-Regulation
J Cell Biol ; 219(10)2020 10 05.
Article En | MEDLINE | ID: mdl-32805026

In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gßγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.

Carrier Proteins/genetics , Cochlea/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Heterotrimeric GTP-Binding Proteins/genetics , Animals , Cell Polarity/genetics , Hair Cells, Auditory/metabolism , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Mechanotransduction, Cellular/genetics , Mice , Microtubules/genetics , Phosphatidylinositol 3-Kinases/genetics , Wnt Signaling Pathway/genetics
Nat Commun ; 11(1): 2995, 2020 06 12.
Article En | MEDLINE | ID: mdl-32532984

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes. Adipocyte function is regulated by receptor-mediated activation of heterotrimeric G proteins. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this question, we generated mice lacking functional Gi proteins selectively in adipocytes. Here we report that these mutant mice displayed significantly impaired glucose tolerance and reduced insulin sensitivity when maintained on an obesogenic diet. In contrast, using a chemogenetic strategy, we demonstrated that activation of Gi signaling selectively in adipocytes greatly improved glucose homeostasis and insulin signaling. We also elucidated the cellular mechanisms underlying the observed metabolic phenotypes. Our data support the concept that adipocyte Gi signaling is essential for maintaining euglycemia. Drug-mediated activation of adipocyte Gi signaling may prove beneficial for restoring proper glucose homeostasis in type 2 diabetes.

Adipocytes/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Insulin Resistance/genetics , Signal Transduction/genetics , Adipocytes/cytology , Animals , Blood Glucose/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Gene Expression Profiling/methods , Glucose Intolerance/genetics , Homeostasis/genetics , Insulin/blood , Insulin/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Obesity/blood , Obesity/genetics , Obesity/metabolism
J Genet ; 992020.
Article En | MEDLINE | ID: mdl-32366735

Goat is the most preferred domesticated animal in Indian subcontinent. However, the climatic change-induced heat stresscauses a formidable challenge for maintaining optimum productivity. G protein subunit alpha i3 (GNAI3) is one of the genes that may have significant role in heat tolerance mechanism in goats. The caprine GNAI3 gene was searched for homology analysis and its three dimensional protein structure was predicted followed by its validation through in silico approach. Nucleotide sequence-based phylogenetic tree analysis showed that the caprine GNAI3 gene has close evolutionary relationship with that of Ovis aries. Homology modelling of caprine GNAI3 protein was done in MODELLER 9.18 (P1), PHYRE2 (P2), GENO3D (P3) and SWISS MODEL (P4). The modelled structures were further validated after observing the Ramachandran and hydrophobicity plots. In the best of three dimensional protein structure (P4 as produced by SWISS MODEL), 330 (98.8%), three (0.9%) and one (0.3%) amino acid residues were found in favoured region, allowed region and outlier region, respectively. Degree of hydrophobicity of the generated protein structures revealed the presence of alternate hydrophobic and hydrophilic regions. The ligand receptor interaction site of the predicted 3D model was traced out using Discovery Studio 3.5. STRING database revealed protein interactions with Plcb1, Plcb2, Plcb3 and other proteins of G family such as Gnb1, Gnb2, Gnb3,Gnb4, Gng2, Gng4 and Gpsm1. KEGG pathway maps revealed interaction with eNOS, iNOS, VEGF and MAPK, which are reported to be transcribed in response to heat stress. Thus, caprine GNAI3 can be used as a possible biomarker for studying heattolerance mechanism in goats.

GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Goats/genetics , Thermotolerance/genetics , Amino Acid Sequence , Animals , Computational Biology , Computer Simulation , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Goats/metabolism , Hydrophobic and Hydrophilic Interactions , Ligands , Models, Molecular , Phylogeny , Protein Conformation , Protein Interaction Mapping , Sequence Homology, Amino Acid
Epilepsy Res ; 163: 106323, 2020 07.
Article En | MEDLINE | ID: mdl-32247221

BACKGROUND: Early-onset epileptic encephalopathies with burst suppression (EOEE-BS) are a group of neonatal epileptic syndromes characterized by intractable epilepsy and severe psychomotor delay with structural and metabolic factors accounting for major etiologies. However, recent advances in gene sequencing have identified that genetic factors might also play a significant role in the development of EOEE-BS. Herein, we used various genetic tests to identify pathogenic genetic variants in EOEE-BS irrespective of structural malformations and analyzed the clinical features associated with each different etiology. METHODS: A total of 48 patients with EOEE-BS were included. Except for patients with severe hypoxic damage, patients with structural malformations were included in our patient cohort. Clinical features of the patients were reviewed, and etiological diagnoses were made based on several genetic tests, metabolic studies, and radiological findings. RESULT: A genetic diagnosis was made in 31 (64.6 %) patients, with the most commonly diagnosed gene being STXBP1 (n = 13, 27.1 %), followed by KCNQ2 (n = 5, 10.4 %), SCN2A (n = 5, 10.4 %), DEPDC5 (n = 3, 6.3 %), CASK (n = 1, 2.1 %), CDKL5 (n = 1, 2.1 %), GNAO1 (n = 1, 2.1 %), SLC6A8 (n = 1, 2.1 %), and LIS1 deletion (n = 1, 2.1 %). Other than the classification of epilepsy syndrome, no clinical features were associated with the genetically diagnosed group. Among eight patients with structural malformations, genetic diagnosis was achieved in five (62.5 %), and those patients had pathogenic mutations in DEPDC5 and CASK or LIS1 deletion, indicating the significance of gene sequencing irrespective of structural abnormalities. Treatment responses to a variety of medications and the ketogenic diet differed by etiology, and surgical resection proved to be effective in patients with cortical dysplasia. CONCLUSION: Genetic etiologies are an important factor in EOEE-BS irrespective of structural malformations and the treatment options may differ by etiology.

Brain Diseases/genetics , Brain/pathology , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Genetic Testing , Brain Diseases/diagnosis , Brain Diseases/etiology , Electroencephalography/methods , Female , Humans , Infant , Male , Mutation/genetics
Neurology ; 94(20): e2148-e2167, 2020 05 19.
Article En | MEDLINE | ID: mdl-32345733

OBJECTIVE: Determining the genetic basis of speech disorders provides insight into the neurobiology of human communication. Despite intensive investigation over the past 2 decades, the etiology of most speech disorders in children remains unexplained. To test the hypothesis that speech disorders have a genetic etiology, we performed genetic analysis of children with severe speech disorder, specifically childhood apraxia of speech (CAS). METHODS: Precise phenotyping together with research genome or exome analysis were performed on children referred with a primary diagnosis of CAS. Gene coexpression and gene set enrichment analyses were conducted on high-confidence gene candidates. RESULTS: Thirty-four probands ascertained for CAS were studied. In 11/34 (32%) probands, we identified highly plausible pathogenic single nucleotide (n = 10; CDK13, EBF3, GNAO1, GNB1, DDX3X, MEIS2, POGZ, SETBP1, UPF2, ZNF142) or copy number (n = 1; 5q14.3q21.1 locus) variants in novel genes or loci for CAS. Testing of parental DNA was available for 9 probands and confirmed that the variants had arisen de novo. Eight genes encode proteins critical for regulation of gene transcription, and analyses of transcriptomic data found CAS-implicated genes were highly coexpressed in the developing human brain. CONCLUSION: We identify the likely genetic etiology in 11 patients with CAS and implicate 9 genes for the first time. We find that CAS is often a sporadic monogenic disorder, and highly genetically heterogeneous. Highly penetrant variants implicate shared pathways in broad transcriptional regulation, highlighting the key role of transcriptional regulation in normal speech development. CAS is a distinctive, socially debilitating clinical disorder, and understanding its molecular basis is the first step towards identifying precision medicine approaches.

Apraxias/genetics , Speech Disorders/genetics , Speech/physiology , Transcription Factors/genetics , Adolescent , Apraxias/diagnosis , Apraxias/physiopathology , Child , Child, Preschool , Female , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Genetic Association Studies , Humans , Male , Speech Disorders/diagnosis , Speech Disorders/physiopathology