scholarly journals c-Cbl-Mediated Regulation of LAT-Nucleated Signaling Complexes

2007 ◽  
Vol 27 (24) ◽  
pp. 8622-8636 ◽  
Author(s):  
Lakshmi Balagopalan ◽  
Valarie A. Barr ◽  
Connie L. Sommers ◽  
Mira Barda-Saad ◽  
Amrita Goyal ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Signaling Molecules ◽  
Endocytic Machinery ◽  
Intracellular Compartments ◽  
Tcr Activation ◽  
Mutant Forms ◽  
Multiple Signaling

ABSTRACT The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.

Participation of RGS8 in the ternary complex of agonist, receptor and G-protein

2004 ◽  
Vol 32 (6) ◽  
pp. 1045-1047 ◽  
Author(s):  
A. Benians ◽  
M. Nobles ◽  
A. Tinker
Keyword(s):  
G Protein ◽  
Ternary Complex ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Receptor Activation ◽  
K Channel ◽  
Physical Interaction ◽  
Rgs Proteins ◽  
Α Subunit ◽  
G Protein Α Subunit

The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein α subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K+ channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and Go αA-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gβγ overexpression. FRET was also seen between RGS8 and Gγ, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein α subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the ‘kinetic efficacy’ of G-protein activation.

scholarly journals T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

2002 ◽  
Vol 158 (7) ◽  
pp. 1263-1275 ◽  
Author(s):  
Stephen C. Bunnell ◽  
David I. Hong ◽  
Julia R. Kardon ◽  
Tetsuo Yamazaki ◽  
C. Jane McGlade ◽  
...  
Keyword(s):  
T Cells ◽  
Lipid Raft ◽  
Synapse Formation ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Receptor ◽  
Rich Cluster ◽  
Enhanced Yellow Fluorescent Protein ◽  
Immune Synapse ◽  
Signaling Complex

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

scholarly journals Inhibition of T Cell Receptor Activation by Semi-Synthetic Sesquiterpene Lactone Derivatives and Molecular Modeling of Their Interaction with Glutathione and Tyrosine Kinase ZAP-70

Molecules ◽  
2019 ◽  
Vol 24 (2) ◽  
pp. 350 ◽  
Author(s):  
Andrei Khlebnikov ◽  
Igor Schepetkin ◽  
Anarkul Kishkentaeva ◽  
Zhanar Shaimerdenova ◽  
Gayane Atazhanova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
T Cell Receptor ◽  
Sesquiterpene Lactones ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Tcr Signaling ◽  
Jurkat T Cells ◽  
Tcr Activation

A variety of natural compounds have been shown to modulate T cell receptor (TCR) activation, including natural sesquiterpene lactones (SLs). In the present studies, we evaluated the biological activity of 11 novel semi-synthetic SLs to determine their ability to modulate TCR activation. Of these compounds, α -epoxyarglabin, cytisinyl epoxyarglabin, 1 β ,10 α -epoxyargolide, and chloroacetate grosheimin inhibited anti-CD3-induced Ca2+ mobilization and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in Jurkat T cells. We also found that the active SLs depleted intracellular glutathione (GSH) in Jurkat T cells, supporting their reactivity towards thiol groups. Because the zeta-chain associated tyrosine kinase 70 kDa (ZAP-70) is essential for TCR signaling and contains a tandem SH2 region that is highly enriched with multiple cysteines, we performed molecular docking of natural SLs and their semi-synthetic derivatives into the ZAP-70 binding site. The docking showed that the distance between the carbon atom of the exocyclic methylene group and the sulfur atom in Cys39 of the ZAP-70 tandem SH2 module was 3.04–5.3 Å for active compounds. Furthermore, the natural SLs and their derivatives could be differentiated by their ability to react with the Cys39 SH-group. We suggest that natural and/or semi-synthetic SLs with an α -methylene- γ -lactone moiety can specifically target GSH and the kinase site of ZAP-70 and inhibit the initial phases of TCR activation.

scholarly journals Bcl-2 differentially regulates Ca2+ signals according to the strength of T cell receptor activation

2006 ◽  
Vol 172 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Fei Zhong ◽  
Michael C. Davis ◽  
Karen S. McColl ◽  
Clark W. Distelhorst
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Receptor Subtypes ◽  
Activated T Cells ◽  
High Concentrations ◽  
Tcr Activation ◽  
Insp3 Receptor

To investigate the effect of Bcl-2 on Ca2+ signaling in T cells, we continuously monitored Ca2+ concentration in Bcl-2–positive and –negative clones of the WEHI7.2 T cell line after T cell receptor (TCR) activation by anti-CD3 antibody. In Bcl-2–negative cells, high concentrations of anti-CD3 antibody induced a transient Ca2+ elevation, triggering apoptosis. In contrast, low concentrations of anti-CD3 antibody induced Ca2+ oscillations, activating the nuclear factor of activated T cells (NFAT), a prosurvival transcription factor. Bcl-2 blocked the transient Ca2+ elevation induced by high anti-CD3, thereby inhibiting apoptosis, but did not inhibit Ca2+ oscillations and NFAT activation induced by low anti-CD3. Reduction in the level of all three inositol 1,4,5-trisphosphate (InsP3) receptor subtypes by small interfering RNA inhibited the Ca2+ elevation induced by high but not low anti-CD3, suggesting that Ca2+ responses to high and low anti-CD3 may have different requirements for the InsP3 receptor. Therefore, Bcl-2 selectively inhibits proapoptotic Ca2+ elevation induced by strong TCR activation without hindering prosurvival Ca2+ signals induced by weak TCR activation.

T cell receptor activation status as biological context for interpreting PD1/PDL1 biomarker measurements.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 54-54
Author(s):  
Ralph E. Parchment ◽  
Tony Navas ◽  
Kristin Fino ◽  
Andy Fung ◽  
Facundo Cutuli ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Tumor Immunity ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Immunofluorescence Microscopy ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Tcr Activation

54 Background: Direct cytolysis of tumor cells by CD8+ T cells results from the net effect of at least two biochemical pathways: (1) stimulatory signaling from the activated T cell receptor (TCR) complex in response to its recognition of a tumor neoantigen presented in the context of autologous MHC class I, and (2) suppressive signaling from immune checkpoints, such as the response of PD1 to binding its ligand, PDL1. Because the PD1:PDL1 immune checkpoint is significant for therapy only when there is tumor cell-specific TCR activation and signaling, it is not surprising that simple measurements of either PD1 or PDL1 in tumor biopsies are, at best, imperfect predictive biomarkers. Instead, a more precise test that quantifies PD1 signaling due to PDL1 binding only in the subset of CD8+ T cells exhibiting activated TCR signaling should provide a more accurate assessment of the extent of immune checkpoint suppression of tumor immunity and therefore be a more predictive biomarker of response to PD1/PDL1-targeted immunotherapy. Methods: We have developed a multiplexed immunofluorescence microscopy test capable of simultaneous quantitation of TCR activation (phospho-CD3zeta), immune checkpoint signaling via PD1 (phospho-SHP1 and -SHP2), and the net stimulation or inhibition resulting from the integration of these two pathways (phospho-ZAP70). Results: Specific antibodies to these biomarkers have been qualified, including peptide inhibition studies to establish antibody specificity, and their performance established by fit-for-purpose studies of in vitro models of CD8+ T cell activation. This multiplex biomarker panel is suitable for clinical use with formalin-fixed, paraffin embedded core needle biopsies of tumor and quantitative immunofluorescence microscopy (qIFA). Conclusions: The additional biomarkers of tumor immunity are expected to add an important context for interpreting PD1/PDL1 measurements. Funded by NCI Contract No. HHSN261200800001E.

scholarly journals PPG neurons of the lower brain stem and their role in brain GLP-1 receptor activation

2015 ◽  
Vol 309 (8) ◽  
pp. R795-R804 ◽  
Author(s):  
Stefan Trapp ◽  
Simon C. Cork
Keyword(s):  
Energy Balance ◽  
Brain Stem ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Receptor Activation ◽  
Vagal Afferents ◽  
Reticular Nucleus ◽  
Autonomic Neurons ◽  
The Brain

Within the brain, glucagon-like peptide-1 (GLP-1) affects central autonomic neurons, including those controlling the cardiovascular system, thermogenesis, and energy balance. Additionally, GLP-1 influences the mesolimbic reward system to modulate the rewarding properties of palatable food. GLP-1 is produced in the gut and by hindbrain preproglucagon (PPG) neurons, located mainly in the nucleus tractus solitarii (NTS) and medullary intermediate reticular nucleus. Transgenic mice expressing glucagon promoter-driven yellow fluorescent protein revealed that PPG neurons not only project to central autonomic control regions and mesolimbic reward centers, but also strongly innervate spinal autonomic neurons. Therefore, these brain stem PPG neurons could directly modulate sympathetic outflow through their spinal inputs to sympathetic preganglionic neurons. Electrical recordings from PPG neurons in vitro have revealed that they receive synaptic inputs from vagal afferents entering via the solitary tract. Vagal afferents convey satiation to the brain from signals like postprandial gastric distention or activation of peripheral GLP-1 receptors. CCK and leptin, short- and long-term satiety peptides, respectively, increased the electrical activity of PPG neurons, while ghrelin, an orexigenic peptide, had no effect. These findings indicate that satiation is a main driver of PPG neuronal activation. They also show that PPG neurons are in a prime position to respond to both immediate and long-term indicators of energy and feeding status, enabling regulation of both energy balance and general autonomic homeostasis. This review discusses the question of whether PPG neurons, rather than gut-derived GLP-1, are providing the physiological substrate for the effects elicited by central nervous system GLP-1 receptor activation.

scholarly journals SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

2011 ◽  
Vol 441 (2) ◽  
pp. 731-741 ◽  
Author(s):  
Susan Treves ◽  
Raphael Thurnheer ◽  
Barbara Mosca ◽  
Mirko Vukcevic ◽  
Leda Bergamelli ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
C2c12 Cell ◽  
Receptor Activation ◽  
Short Chain ◽  
C2c12 Myotubes ◽  
Retinol Dehydrogenase ◽  
Short Chain Dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35–EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca2+ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.

Sign in / Sign up

Export Citation Format

Share Document