The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C- terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha- subunits, G alpha q and/or G alpha 11

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist- induced, Ca(2+)-activated Cl- currents that were measured using a two- electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX- sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor- activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase

An overview on G protein-coupled receptor-induced signal transduction in acute myeloid leukemia

Background: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies. Methods: We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML. Results and Conclusion: Many scientific reports were found with compelling evidence for the involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometrybased protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML.acceptedVersio

Random Mutagenesis of G protein ɑ Subunit G_oɑ. Mutations altering nucleotide binding

Nucleotide binding properties of the G protein ɑ subunit G_oɑ were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [^(35)S]GTPyS (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G_oɑ cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G_oɑ for GTPyS or Mg^(2+) were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21^(ras) proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTPyS binding but retained the capacity to interact with the βy subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G_oɑ increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl^- response, suggesting that this mutation resulted in a dominant negative phenotype

Nuevo diseño de la Encuesta de Salud de Cataluña (2010-2014): un paso adelante en planificación y evaluación sanitaria

Se presenta la génesis de la Encuesta de Salud de Cataluña (2010-2014) con sus submuestras semestrales. Se detallan las características básicas de su diseño muestral polietápico. Entre las ventajas organizativas de esta nueva operación, en comparación con las anteriores, destacan la agilidad en la disponibilidad de datos y la capacidad de monitorización continuada de la población. Se señalan como beneficios la puntualidad en la obtención de indicadores y la posibilidad de introducir nuevos tópicos a través del cuestionario complementario, según las necesidades de información. Como limitación se apunta la mayor complejidad del diseño muestral y la falta de seguimiento longitudinal de la muestra. Se hace hincapié en la necesidad de utilizar ponderaciones adaptadas a las submuestras para el análisis estadístico que emplee microdatos, así como de acumular oleadas si se desea elevar el grado de desagregación del análisis, ya sea en el territorio o por subgrupos de población

Desensitization of Inositol 1,4,5-Trisphosphate/Ca2+-induced Cl- Currents by Prolonged Activation of G Proteins in Xenopus Oocytes

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4,5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha 16 subunit and the murine homologue Galpha 15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha 16 subunit cRNA cause modest (~2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha 16 and Galpha 15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbeta gamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha 16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbeta gamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha 16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha 16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha 16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein

Gαq activation modulates autophagy by promoting mTORC1 signaling.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.We thank Paula Ramos, Susana Rojo-Berciano, and Laura López for helpful technicalassistance. Dr. Marta Cruces (Universidad Autónoma de Madrid, Spain) for herinvaluable help regarding the liver explants experiments, Dr. Badford Berk (University ofRochester, NY, USA) for providing the GFP-Flag-PB1-p62 plasmid, Drs. Stefan Offer-manns and Nina Wettschureck (Max-Planck-Institute for Heart and Lung Research,Germany) for providing Tie2-CreERT2; Gnaq f/f; Gna11−/−[EC-q/11-KO) mice, andDr. Guzmán Sánchez for scientific advice. We thank also Ricardo Ramos from theGenomic facility of Fundación Parque Científico de Madrid (Universidad Autónoma deMadrid, Spain) and Gemma Rodríguez-Tarduchy from the Genomic facility of theInstituto de Investigaciones Biomédicas“Alberto Sols”for their help with cell linesauthentication. The help from CBMSO Animal Care, Flow Cytometry, Electron andOptical and Confocal Microscopy facilities is also acknowledged. This work was sup-ported by Ministerio de Economía; Industria y Competitividad (MINECO) of Spain(grant SAF2017-84125-R to F.M.), (grant BFU2017-83379-R to A.M.A.), Instituto deSalud Carlos III (PI18/01662 to CR, co-funded with European FEDER contribution),CIBERCV-Instituto de Salud Carlos III, Spain (grant CB16/11/00278 to F.M., co-fundedwith European FEDER contribution), Fundación Ramón Areces (to C.R. and F.M.) andPrograma de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to F.M. and NIH grants AG021904 and AG038072 to A.M.C. Wealso acknowledge the support of a Contrato para la Formación del Profesorado Uni-versitario (FPU13/04341) and (FPU14/06670), an EMBO short-term fellowship (ASTF600-2016). We also acknowledge institutional support to the CBMSO from FundaciónRamón Areces.S

G12 Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of Gα12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Gα12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of Gα(o) did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12 in the mitogenic response to thrombin in human astroglial cells

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures

G proteins at the mitocondria

Trabajo presentado en la Molecular Pharmacology. Gordon Conference, celebrada en Ventura, CA, US, del 1 al 6 de febrero de 2015Peer Reviewe