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1.
Cell Calcium ; 94: 102340, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33601151

RESUMO

The recent paper by Pfeil et al., "Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs", opens another path from biochemical in vitro reconstitution to understanding the complex regulation of calcium signaling inside the cell.


Assuntos
Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Cálcio/metabolismo , Sinalização do Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fosfolipase C beta/metabolismo
2.
Nat Commun ; 12(1): 144, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420046

RESUMO

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.


Assuntos
Proteínas de Bactérias/farmacologia , Chromobacterium/metabolismo , Depsipeptídeos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Depsipeptídeos/biossíntese , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Esterases/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Hemípteros , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
3.
Mol Cell ; 80(6): 933-934, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33338407

RESUMO

Pfeil et al., (2020) examine the mechanism of Gi-stimulated Ca2+ release in cells and find an unexpected role for Gαq in Gßγ-dependent activation of phospholipase Cß (PLCß).


Assuntos
Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Transdução de Sinais
4.
Nat Commun ; 11(1): 4752, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958754

RESUMO

Growth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Gigantismo/metabolismo , Hormônio do Crescimento/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Acromegalia/metabolismo , Acromegalia/patologia , Animais , Composição Corporal , Linhagem Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Gigantismo/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Hipófise/metabolismo , Proteína Quinase C/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/genética
5.
Proc Natl Acad Sci U S A ; 117(34): 20874-20880, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32764144

RESUMO

Maintaining energy homeostasis requires coordinating physiology and behavior both on an acute timescale to adapt to rapid fluctuations in caloric intake and on a chronic timescale to regulate body composition. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are acutely activated by caloric need, and this acute activation promotes increased food intake and decreased energy expenditure. On a longer timescale, AgRP neurons exhibit chronic hyperactivity under conditions of obesity and high dietary fat consumption, likely due to leptin resistance; however, the behavioral and metabolic effects of chronic AgRP neuronal hyperactivity remain unexplored. Here, we use chemogenetics to manipulate Gq signaling in AgRP neurons in mice to explore the hypothesis that chronic activation of AgRP neurons promotes obesity. Inducing chronic Gq signaling in AgRP neurons initially increased food intake and caused dramatic weight gain, in agreement with published data; however, food intake returned to baseline levels within 1 wk, and body weight returned to baseline levels within 60 d. Additionally, we found that, when mice had elevated body weight due to chronic Gq signaling in AgRP neurons, energy expenditure was not altered but adiposity and lipid metabolism were both increased, even under caloric restriction. These findings reveal that the metabolic and behavioral effects of chronic Gq signaling in AgRP neurons are distinct from the previously reported effects of acute Gq signaling and also of leptin insensitivity.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Obesidade/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal , Restrição Calórica , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Energia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Feminino , Homeostase/efeitos dos fármacos , Hipotálamo/metabolismo , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Obesidade/fisiopatologia , Transdução de Sinais , Ganho de Peso/efeitos dos fármacos
6.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(4): 521-525, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32691561

RESUMO

Objective: To quantitatively detect GNAQ/11 mutations in uveal melanoma (UM) by droplet digital PCR (ddPCR). Methods: Formaldehyde-fixed paraffin-embedded (FFPE) tumor samples were taken from 78 UM patients with enucleation in West China Hospital between 2009 and 2015. None of the patients received radiotherapy or chemotherapy before enucleation. A retrospective study was conducted to detect GNAQ/11 mutation in UM by ddPCR. To compare the consistency of the results of the two detection methods, DNA sequencing was performed on the target gene by Sanger sequencing. 78 patients with UM were studied retrospectively. GNAQ/11 mutations in uveal melanoma was detected by ddPCR. The consistency of the results of the two detection methods was analyzed. Results: GNAQ/11 mutations frequency was 91.9%. The consistency test between Sanger sequencing and ddPCR of GNAQ/11 mutations in 74 patients with UM was conducted. Kappa coefficient=0.436, P=0.001. The error rate of Sanger sequencing results was significantly higher in the heterogeneous group than in the homogeneous group (12/37 vs. 3/16, P=0.53), but the difference was not statistically significant. Conclusion: The results of ddPCR and Sanger sequencing showed good consistency, and the mutation ratio of GNAQ/11 in UM was significantly different. GNAQ/11 mutation frequency in UM patients detected by ddPCR was close to the reported frequency. It is more recommended to use ddPCR with high sensitivity to detect gene mutations in samples of tumor tissue DNA derived from FFPE. Sanger sequencing is prone to false negative results.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Subunidades alfa de Proteínas de Ligação ao GTP , Melanoma , Mutação , Reação em Cadeia da Polimerase , Neoplasias Uveais , China , DNA/química , Análise Mutacional de DNA , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Melanoma/diagnóstico , Melanoma/genética , Estudos Retrospectivos , Neoplasias Uveais/diagnóstico , Neoplasias Uveais/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-32244061

RESUMO

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-ß and the thromboxane A2 receptor-ß (TPß receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPß receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCß, WDR36 augmented TPß receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPß receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPß towards PLC and SphK1, most likely by scavenging Gαq and PLCß. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.


Assuntos
Proteínas do Olho/metabolismo , Fosfolipase C beta/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Tromboxanos/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Proteínas do Olho/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Mioblastos/metabolismo
8.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118718, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32289337

RESUMO

Alternative splicing enables G protein-coupled receptor (GPCR) genes to greatly increase the number of structurally and functionally distinct receptor isoforms. However, the functional role and relevance of the individual GPCR splice variants in regulating physiological processes are still to be assessed. A naturally occurring alternative splice variant of Bombyx CAPA-PVK receptor, BomCAPA-PVK-R1-Δ341, has been shown to act as a dominant-negative protein to regulate cell surface expression and function of the canonical CAPA-PVK receptor. Herein, using functional assays, we identify the splice variant Δ341 as a specific receptor for neuropeptide CAPA-PK, and upon activation, Δ341 signals to ERK1/2 pathway. Further characterization demonstrates that Δ341 couples to Gαi/o, distinct from the Gαq-coupled canonical CAPA-PVK receptor, triggering ERK1/2 phosphorylation through Gßγ-PI3K-PKCζ signaling cascade. Moreover, our ELISA data show that the ligand-dependent internalization of the splice variant Δ341 is significantly impaired due to lack of GRKs-mediated phosphorylation sites. Our findings highlight the potential of this knowledge for molecular, pharmacological and physiological studies on GPCR splice variants in the future.


Assuntos
Bombyx/genética , Bombyx/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Processamento de RNA/fisiologia , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Processamento Alternativo , Animais , Clonagem Molecular , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva , Sistema de Sinalização das MAP Quinases , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Transdução de Sinais , Transcriptoma
9.
Mol Pharmacol ; 97(5): 304-313, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32132133

RESUMO

Free-fatty acid receptor-4 (FFA4), previously termed GPR120, is a G protein-coupled receptor (GPCR) for medium and long-chained fatty acids, agonism of which can regulate a myriad of metabolic, sensory, inflammatory, and proliferatory signals. Two alternative splice isoforms of FFA4 exist that differ by the presence of an additional 16 amino acids in the longer (FFA4-L) transcript, which has been suggested to be an intrinsically ß-arrestin-biased GPCR. Although the shorter isoform (FFA4-S) has been studied more extensively, very little is known about mechanisms of regulation or signaling of the longer isoform. Because ß-arrestin recruitment is dependent on receptor phosphorylation, in the current study, we used the endogenous agonist docosahexaenoic acid (DHA) to examine the mechanisms of FFA4-L phosphorylation, as well as DHA-dependent ß-arrestin recruitment and DHA-dependent extracellular-signal regulated kinase-1/2 (ERK1/2) signaling in human embryonic kidney 293 cells. Our results reveal differences in basal phosphorylation of the two FFA4 isoforms, and we show that DHA-mediated phosphorylation of FFA4-L is primarily regulated by G protein-coupled receptor kinase 6, whereas protein kinase-C can also contribute to agonist-induced and heterologous phosphorylation. Moreover, our data demonstrate that FFA4-L phosphorylation occurs on the distal C terminus and is directly responsible for recruitment and interactions with ß-arrestin-2. Finally, using CRISPR/Cas9 genome-edited cells, our data reveal that unlike FFA4-S, the longer isoform is unable to facilitate phosphorylation of ERK1/2 in cells that are devoid of ß-arrestin-1/2. Together, these results are the first to demonstrate phosphoregulation of FFA4-L as well as the effects of loss of phosphorylation sites on ß-arrestin recruitment and ERK1/2 activation. SIGNIFICANCE STATEMENT: Free-fatty acid receptor-4 (FFA4) is a cell-surface G protein-coupled receptor for medium and long-chained fatty acids that can be expressed as distinct short (FFA4-S) or long (FFA4-L) isoforms. Although much is known about FFA4-S, the longer isoform remains virtually unstudied. Here, we reveal the mechanisms of docosahexaenoic acid-induced phosphorylation of FFA4-L and subsequent ß-arrestin-2 recruitment and extracellular-signal regulated kinase-1/2 activity.


Assuntos
Processamento Alternativo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestina 2/metabolismo , Processamento Alternativo/efeitos dos fármacos , Sequência de Aminoácidos , Ácidos Docosa-Hexaenoicos/farmacologia , Quinases de Receptores Acoplados a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos
10.
Proc Natl Acad Sci U S A ; 117(13): 7455-7460, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184323

RESUMO

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gßγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-ßarrestin-Gßγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Cultura Primária de Células , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo
11.
J Biomed Sci ; 27(1): 21, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906951

RESUMO

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.


Assuntos
Toxinas Bacterianas/metabolismo , Metilação de DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Melatonina/farmacologia , Mucina-2/biossíntese , Receptor MT2 de Melatonina/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Células HT29 , Humanos , Camundongos , Vibrioses/patologia
12.
Clin Immunol ; 212: 108346, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31954803

RESUMO

Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.


Assuntos
Autoanticorpos/imunologia , Doenças do Sistema Nervoso Autônomo/imunologia , Doença de Chagas/imunologia , Receptor Muscarínico M2/imunologia , Adulto , Idoso , Regulação Alostérica , Autoanticorpos/metabolismo , Autoanticorpos/farmacologia , Doenças do Sistema Nervoso Autônomo/etiologia , Doenças do Sistema Nervoso Autônomo/metabolismo , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Carbacol/farmacologia , Doença de Chagas/complicações , Doença de Chagas/metabolismo , Doença de Chagas/fisiopatologia , Agonistas Colinérgicos/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/metabolismo , beta-Arrestina 1/metabolismo
13.
Am J Physiol Renal Physiol ; 318(3): F660-F672, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984793

RESUMO

Gα12 and Gα13 are ubiquitous members of the heterotrimeric guanine nucleotide-binding protein (G protein) family that play central and integrative roles in the regulation of signal transduction cascades within various cell types in the kidney. Gα12/Gα13 proteins enable the kidney to adapt to an ever-changing environment by transducing stimuli from cell surface receptors and accessory proteins to effector systems. Therefore, perturbations in Gα12/Gα13 levels or their activity can contribute to the pathogenesis of various renal diseases, including renal cancer. This review will highlight and discuss the complex and expanding roles of Gα12/Gα13 proteins on distinct renal pathologies, with emphasis on more recently reported findings. Deciphering how the different Gα12/Gα13 interaction networks participate in the onset and development of renal diseases may lead to the discovery of new therapeutic strategies.


Assuntos
Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Nefropatias/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Nefropatias/genética
14.
J Biol Chem ; 295(8): 2520-2540, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31892516

RESUMO

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.


Assuntos
Receptores de Trombina/metabolismo , Transdução de Sinais , Trombina/metabolismo , Alanina/genética , Substituição de Aminoácidos , Cálcio/metabolismo , Sinalização do Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isomerismo , Sistema de Sinalização das MAP Quinases , Metilação , Simulação de Acoplamento Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Agregação Plaquetária , Receptores de Trombina/agonistas , Homologia Estrutural de Proteína , beta-Arrestinas/metabolismo
15.
Anticancer Res ; 40(1): 213-220, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892569

RESUMO

BACKGROUND/AIM: Kisspeptin produced from the KISS1 gene is secreted from the living cells, binds to endogenous receptor KISS1R (also called G protein-coupled receptor 54, GPR54), and has various functions in normal physiological conditions. Although an anti-metastatic role of kisspeptin in cancer is well known in several cancer types, its role in brain tumors is not yet understood. Herein, we investigated a the role of kisspeptin in glioblastoma cells. MATERIALS AND METHODS: Glioblastoma cells were treated with kisspeptin and subjected to proliferation, migration, and invasion assays. KISS1R dependency was tested by KISS1R silencing with KISS1R siRNAs. RESULTS: Kisspeptin inhibited migratory and invasive abilities of U87-MG, U-251-MG and U373-MG glioblastoma cells with no effect on cell viability. KISS1R gene silencing with KISS1R siRNAs blocked kisspeptin-induced glioblastoma cell invasiveness. Moreover, chemical inhibitors against Gq, PLC or PKC blocked kisspeptin-induced glioblastoma cell invasiveness. CONCLUSION: Kisspeptin induces glioblastoma cell invasiveness via the KISS1R-Gq-PLC-PKC signaling pathway.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Kisspeptinas/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Invasividade Neoplásica , Metástase Neoplásica
16.
Proc Natl Acad Sci U S A ; 117(3): 1753-1761, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31896584

RESUMO

Carbon dioxide (CO2), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H+ receptor, and endothelial Gαq/11 proteins mediate the CO2/H+ effect on cerebrovascular reactivity in mice. CO2/H+ leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO2, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO2 dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO2 effect on anxiety. Even at atmospheric CO2 concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO2 is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.


Assuntos
Ansiedade/metabolismo , Sistema Cardiovascular/metabolismo , Endotélio/metabolismo , Transtornos Respiratórios/metabolismo , Tonsila do Cerebelo , Animais , Arteríolas/patologia , Encéfalo/fisiologia , Tronco Encefálico/metabolismo , Dióxido de Carbono/metabolismo , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Endotélio/patologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Humanos , Hipercapnia/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Respiração , Fatores de Risco , Transdução de Sinais
17.
Biochem J ; 476(24): 3721-3736, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31794025

RESUMO

We previously purified lysophospholipase D (lysoPLD), which hydrolyzes lysophosphatidylcholine (lysoPC) to lysophosphatidic acid (LPA), from rat brain and identified the heterotrimeric G protein subunits Gαq and Gß1 in the lysoPLD active fractions. Tag-affinity purified Gαq exhibits lysoPLD activity but a mutant that affected cellular localization or interaction with the Gß subunit reduced lysoPLD activity. Size exclusion chromatography revealed that active lysoPLD is a much higher molecular mass complex than is heterotrimeric G protein, suggesting the presence of other components. Liquid chromatography-tandem mass spectrometry of lysoPLD purified from rat brain identified glycerophosphodiesterase 4 (GDE4), recently reported as lysoPLD, in the same fraction as G proteins. The overexpressed and tag-purified Gαq fractions, which exhibit lysoPLD activity, contained GDE4. Exogenously expressed GDE4 was co-immunoprecipitated with endogenous Gαq and Gß and exhibited high lysoPLD activity. The results of confocal microscopy and cell fractionation experiments indicated that exogenously expressed GDE4 in cells mainly localized at the endoplasmic reticulum and partially co-localized with Gαq protein at the plasma membrane. Proteinase K protection assay results suggested that the catalytic domain of GDE4 faces the lumen/extracellular space. Mutations at the conserved amino acids in the C-terminus cytoplasmic regions amongst GDE1, 4 and 7, dramatically suppressed GDE4 enzyme activities. When both the Gαq and Gα11 genes in Neuro2A cells were disrupted using the CRISPR-Cas9 system, endogenous lysoPLD activity was partially reduced but rescued by overexpression of Gαq. These results suggest that GDE4 is a new effector of G protein signaling that produces bioactive phospholipid LPA and/or modulates membrane homeostasis.


Assuntos
Cromograninas/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular , Cromograninas/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Camundongos , Diester Fosfórico Hidrolases/genética
18.
Int J Mol Sci ; 20(22)2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703413

RESUMO

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the ß-subunit of thyrotropin (TSHB). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype.


Assuntos
Hipotireoidismo Congênito , Sistema de Sinalização das MAP Quinases , Mutação , Receptores da Tireotropina , Sistemas do Segundo Mensageiro , Tireotropina Subunidade beta , Linhagem Celular Tumoral , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Receptores da Tireotropina/química , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Tireotropina Subunidade beta/química , Tireotropina Subunidade beta/genética , Tireotropina Subunidade beta/metabolismo
19.
Int J Mol Sci ; 20(21)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31653051

RESUMO

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor's preference for the Gαq protein and the M2 receptor's preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor-G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor-G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Simulação de Dinâmica Molecular , Receptores Muscarínicos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores Muscarínicos/química , Termodinâmica
20.
Nat Commun ; 10(1): 4836, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645561

RESUMO

Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the ß2-adrenergic receptor (ß2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. ß2-AR-Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta , Regulação Alostérica , Animais , Sítios de Ligação , AMP Cíclico/metabolismo , Epinefrina/metabolismo , Fenoterol/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isoproterenol/metabolismo , Cinética , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais
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