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

Chloride binding properties of a macrocyclic receptor equipped with an acetylide gold(I) complex: synthesis, characterization, reactivity, and cytotoxicity studies

In this work, we report the synthesis and characterization of a mono-nuclear “two wall” aryl-extended calix4]pyrrole receptor (2Au) decorated with an acetylide-gold(I)-PTA complex at its upper rim. We describe the1 H NMR titration experiments of 2Au and its “two wall” aryl-extended calix4]pyrrole synthetic precursors: the non-symmetric mono-iodo-mono-ethynyl 2 and the symmetric bis-iodo 3 with TBACl in dichloromethane and acetone solution. In acetone solution, we use isothermal titration calorimetry (ITC) experiments to thermodynamically characterize the formed 1:1 chloride complexes and perform pair-wise competitive binding experiments. In both solvents, we measured a decrease in the binding constant of the mono-nuclear 2Au complex for chloride compared to the parent mono-iodo-mono-ethynyl 2. In turn, receptor 2 also shows a reduction in binding affinity for chloride compared to its precursor bis-iodo calix4]pyrrole 3. The free energy differences (¿G) of the 1:1 chloride complexes cannot be exclusively attributed to their dissimilar electrostatic surface potential values either at the center of the meso-phenyl wall or its para-substituent. We conclude that solvation/desolvation processes play an important role in the stabilization of the chloride complexes. In acetone solution and in the presence of TBACl, 6Au, a reference compound for the acetylide Au(I)•PTA unit, produces a bis(alkynyl)gold(I) anionic complex 7Au]-. Thus, the observation of two separate sets of signals for the bound aromatic calix4]pyrrole protons, when more than 1 equiv. of the salt is added, is assigned to the formation of the chloride complexes of 2Au and of the “in situ” formed calix4]pyrrole anionic dimer 8Au]-. Finally, preliminary data obtained in cell viability assays of 2Au and 6Au with human cancer cells lines assign them with moderate activities showing that the calix4]pyrrole unit is not relevant. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

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

Palladium-Mediated Catalysis Leads to Intramolecular Narcissistic Self-Sorting on a Cavitand Platform

Palladium-catalyzed aminocarbonylation reactions have been used to directly convert a tetraiodocavitand intermediate into the corresponding carboxamides and 2-ketocarboxamides. When complex mixtures of the amine reactants are employed in competition experiments, no ‘mixed’ products possessing structurally different amide fragments are detected either by 1H or 13C NMR. Only highly symmetrical cavitands are sorted out of a large number of potentially feasible products, which represents a rare example of intramolecular, narcissistic self-sorting. The reactivity order of the amine reactants and the changes in the Gibbs energies calculated using the semiempirical PM6 model suggest that this self-sorting process is kinetically controlled

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

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

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

Review: Nutritional ecology of heavy metals

The aim of this review is to focus the attention on the nutrition ecology of the heavy metals and on the major criticisms related to the heavy metals content in animal feeds, manure, soil and animal-origin products. Heavy metals are metallic elements that have a high density that have progressively accumulated in the food chain with negative effects for human health. Some metals are essential (Fe, I, Co, Zn, Cu, Mn, Mo, Se) to maintain various physiological functions and are usually added as nutritional additives in animal feed. Other metals (As, Cd, F, Pb, Hg) have no established biological functions and are considered as contaminants/undesirable substances. The European Union adopted several measures in order to control their presence in the environment, as a result of human activities such as: farming, industry or food processing and storage contamination. The control of the animal input could be an effective strategy to reduce human health risks related to the consumption of animal-origin products and the environmental pollution by manure. Different management of raw materials and feed, animal species as well as different legal limits can influence the spread of heavy metals. To set up effective strategies against heavy metals the complex interrelationships in rural processes, the widely variability of farming practices, the soil and climatic conditions must be considered. Innovative and sustainable approaches have discussed for the heavy metal nutrition ecology to control the environmental pollution from livestock-related activities

Role of thrombin receptor in breast cancer invasiveness

Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism. © 1999 Cancer Research Campaig

An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown., applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out. outcome and demonstrates the importance of the innate immune system in the development of tumors