Biomedical subjects

Guy C Le Breton

Publications and source records attributed to Guy C Le Breton.

8 recordsLinked to original sources

PubMed2006

Characterization of thromboxane A2 receptor signaling in developing rat oligodendrocytes: nuclear receptor localization and stimulation of myelin basic protein expression.

The present work investigates the role of thromboxane A(2) (TXA(2)) receptors in the development of oligodendrocytes (OLGs). The results demonstrate that the proteins of the TXA(2) signaling pathway, i.e., cyclooxygenase (COX-1), TXA(2) synthase (TS), and TXA(2) receptor (TPR) are expressed in the developing rat brain during myelination. Furthermore, culture of OLG progenitor cells (OPCs) revealed that the expression levels of these proteins as well as TXA(2) synthesis increase during OLG maturation. Separate studies established that activation of TPRs by the agonist U46619 increases intracellular calcium in both OPCs and OLGs as visualized by digital fluorescence imaging. Immunocytochemical staining demonstrated that TPRs are localized in the plasma membrane and perinuclear compartments in OPCs. However, during OLG differentiation, TPRs shift their localization pattern and also become associated with the nuclear compartment. This shift to nuclear localization was confirmed by biochemical analysis in cultured cells and by immunocytochemical analysis in developing rat brain. Finally, it was found that U46619 activation of TPRs in maturing OLGs resulted in enhanced myelin basic protein (MBP) expression. Alternatively, inhibition of endogenous TPR signaling led to reduced MBP expression. Furthermore, TPR-mediated MBP expression was found to be associated with increased transcription from the MBP promoter using a MBP-luciferase reporter. Collectively, these findings suggest a novel TPR signaling pathway in OLGs and a potential role for this signaling during OLG maturation and myelin production.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2006

Differential mapping of the amino acids mediating agonist and antagonist coordination with the human thromboxane A2 receptor protein.

Despite the well documented involvement of thromboxane A(2) receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent, this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein that regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding region (i.e. the C terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain). Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A, D193Q, and D193R mutants lost SQ29,548 (antagonist) binding and exhibited a dramatically reduced calcium response, which could not be restored by elevated U46619 (agonist) doses. The F184Y mutant lost SQ29,548 binding and exhibited a reduced calcium response (which could be restored by elevated U46619); and the T186A and S191T mutants lost SQ29,548 binding and retained a normal U46619-induced calcium response. Furthermore, these last three mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Two separate mutants that exhibited SQ29,548 binding yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above (e.g. E190A and F200A) produced no detectable effects on either SQ29,548 binding or the U46619-induced response. In summary, these results identify key amino acids (in particular Asp(193)) involved in TPR ligand coordination. These findings also demonstrate that TPR-specific ligands interact with different residues in the ligand-binding pocket.

Amino Acid Sequence↗

PubMed2005

Mass-dependent signaling between G protein coupled receptors.

The present study provides evidence that G protein coupled receptor (GPCR) signaling pathways participate in an interactive signaling network governed by the principles of mass action. Using an inducible thromboxane A2 receptor (TPR)/platelet activating factor receptor (PAFR) co-expressing cell model, TPR or PAFR expression was independently up-regulated. Immunostaining and radioligand binding experiments demonstrated that this receptor up-regulation resulted in increased GPCR:G protein mass ratios. This increase in mass ratio impacted both TPR and PAFR ligand affinity. Specifically, up-regulating TPR expression not only decreased TPR ligand affinity, but also decreased the ligand affinity of PAFRs. A similar effect on ligand affinities was observed when PAFRs were up-regulated. In addition, increasing the GPCR:G protein mass ratio for TPRs led to desensitization of the calcium mobilization response to PAFR activation, and increasing PAFR mass desensitized the TPR-mediated calcium response. Finally, it was observed that an increased TPR:G protein mass ratio was associated with a shift in the TPR signaling response, and revealed an additional TPR signaling pathway through G(S). Collectively, these results describe a novel mechanism, i.e., mass-dependent GPCR signaling, by which cells can modulate their GPCR signaling pathways and signaling priorities.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2005

Thromboxane A receptor-mediated cell proliferation, survival and gene expression in oligodendrocytes.

Thromboxane A(2) receptors (TP) were previously localized to discrete regions in the rat brain on myelinated fiber tracts and oligodendrocytes (OLGs). The present studies extended these findings and investigated the effects of TP signaling on cell proliferation, survival, and gene expression in OLG progenitor cells (OPCs) and OLGs. It was found that the TP agonist, U46619 stimulated the proliferation of OPCs and promoted the survival of mature OLGs. Examination of the early gene expression events involved in OPC proliferation, revealed that c-fos expression was substantially increased by U46619 stimulation. Treatment of OPCs or OLGs with U46619 caused activation of the mitogen-activated protein kinases (MAPK) ERK 1/2. In OPCs this activation was blocked by inhibition of src. However, in OLGs this phosphorylation was not only blocked by inhibition of src but also by inhibition of protein kinase C (PKC). Furthermore, U46619 was found to increase CREB phosphorylation in both OPCs and OLGs. Similar to ERK 1/2 activation, there was a divergence in the mechanism of the TP-mediated CREB response for each cell type. Specifically, U46619 activation was attenuated by src and protein kinase A (PKA) inhibition in OPCs, whereas in OLGs this effect was blocked by inhibition of src, PKA as well as by inhibition of PKC. Collectively, these results provide the first demonstration that TP-activated nuclear signaling events are involved in the proliferation of OPCs, the survival of mature OLGs, and the stimulation of gene expression.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2004

Cell signalling through thromboxane A2 receptors.

Thromboxane A2 receptors (TPs) are widely distributed among different organ systems and have been localized on both cell membranes and intracellular structures. Following the initial cloning of this receptor class from human placenta, the deduced amino acid sequence predicted seven-transmembrane spanning regions, four extracellular domains and four intracellular domains, making TP a member of the seven-transmembrane G-protein-coupled receptor (GPCR) super family. A single gene on chromosome 19p13.3 leads to the expression of two separate TP isoforms: TPalpha which is broadly expressed in numerous tissues, and a splice variant termed TPbeta which may have a more limited tissue distribution. Mutagenesis, photoaffinity labelling, and immunological studies have indicated that the ligand binding domains for this receptor may reside in both the transmembrane (TM) and extracellular regions of the receptor protein. In addition, separate studies have provided evidence that this receptor can couple to at least four separate G protein families. As a consequence, TP signalling has been shown to result in a broad range of cellular responses including phosphoinositide metabolism, calcium redistribution, cytoskeletal arrangement, integrin activation, kinase activation, and the subsequent nuclear signalling events involved in DNA synthesis, cell proliferation, cell survival and cell death. While activation of these different signalling cascades can all derive from TP stimulation, the relative signalling preference for a given cascade appears to be both tissue and cell specific. Finally, separate studies have indicated that TP signalling capacity can be both down-regulated by protein kinase activation and up-regulated by GPCR cross-signalling. Thus, the multitude of signalling events which derive from TP activation can themselves be modulated by endogenous cellular messengers.

Cells, Cultured↗

PubMed2003

Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases.

Thromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kgamma knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kgamma deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2002

Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation.

The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha(13) and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha(13) and the PKA catalytic subunit established that PKA directly phosphorylates Galpha(13). The location of this phosphorylation site was next investigated with a new synthetic peptide (G(13)SRI(pep)) containing the PKA consensus sequence (Arg-Arg-Pro-Thr(203)) within the switch I region of Galpha(13). G(13)SRI(pep) produced a dose-dependent inhibition of PKA-mediated Galpha(13) phosphorylation. On the other hand, the Thr-phosphorylated derivative of G(13)SRI(pep) possessed no inhibitory activity, suggesting that Galpha(13) Thr(203) may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha(13)-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Galpha(13) phosphorylation stabilized coupling of Galpha(13) with platelet thromboxane A(2) receptors but destabilized coupling of Galpha(13) to its betagamma subunits. In order to determine the functional consequences of this phosphorylation on Galpha(13) signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Galpha(13) wild type (Galpha(13)-WT) or Galpha(13)-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Galpha(13) wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Galpha(13) Thr(203).

Animals↗

PubMed2002

Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.

The human thromboxane A(2) (TP) receptor, a member of the G protein-coupled receptor superfamily, consists of seven transmembrane segments. Attempts to elucidate the specific segment(s) that define the receptor ligand-binding pocket have produced less than definitive and sometimes conflicting results. On this basis, the present work identified an amino acid sequence of the TP receptor that is directly involved in ligand binding. Mapping of this domain was confirmed by two separate approaches: photoaffinity labeling and site-specific antibodies. The newly synthesized, biotinylated photoaffinity probe, SQBAzide, was first shown to specifically label TP receptor protein. Sequential digestion of this protein with CNBr/trypsin revealed photolabeling of a 2.9-kDa peptide. Using anti-peptide antibodies directed against different regions of the receptor protein, it was established that this peptide represents the predicted cleavage product for CNBr/trypsin and corresponds to amino acids Arg(174)-Met(202) of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys(183)-Asp(193) was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly(172)-Cys(183) was not. Collectively these findings provide evidence that ligands interact with amino acids contained within the C-terminal portion of the third extracellular domain (ED3) of the receptor protein. This information should be of significant value in the study of TP receptor structure and signaling.

Amino Acid Sequence↗