NEUE IMMUNOLOGISCHE SIGNALE AUS MAGDEBURG

Das Fach „Immunologie“ hat sich in den vergangenen zwanzig Jahren zu einer interdisziplinären Forschungsrichtung entwickelt, welche in nahezu allen Bereichen der Medizin eine wichtige Rolle spielt. Es gibt kaum ein medizinisches Fachgebiet, in der immunologische Fragestellungen im klinischen Alltag keine Rolle spielen. In diesem Artikel diskutieren wir im ersten Teil einige immunologische Probleme, die es in der Zukunft zu lösen gilt. Dieser Teil kann das komplexe Gebiet der klinischen Immunologie nur anreißen und entbehrt daher jeder Vollständigkeit (wofür wir um Verständnis bitten). Im zweiten Teil haben wir für den interessierten Leser in einem Überblick das derzeitige Wissen der membrannahen Signaltransduktionsmechanismen in T-Lymphozyten komprimiert dargestellt. Der dritte Teil befasst sich dann mit den speziellen Forschungsthemen des Instituts für Immunologie an der Medizinischen Fakultät der Otto-von-GuerickeUniversität Magdeburg sowie einigen Perspektiven, die wir für die biomedizinische Forschung am Standort Magdeburg sehen

The Csk-binding protein PAG regulates PDGF-induced Src mitogenic signaling via GM1

Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk

T-Cell Receptor/CD28-Mediated Activation of Human T Lymphocytes Induces Expression of Functional -Opioid Receptors

ABSTRACT Opiates function as immunomodulators, partly by their effects on T cells. Opioids act via -, ␦-, and -opioid receptors, among which the -type is of particular interest, because morphine-like opioids preferentially bind to it. Here we report that -opioid receptor mRNA was induced after CD3/28-mediated activation of primary human T lymphocytes and Jurkat T cells, neither of which expresses the gene constitutively. Moreover, a reporter gene construct containing 2624 base pairs of the -opioid receptor promoter was transactivated by CD3/28 stimulation. Transcriptional induction of the -opioid receptor gene was mediated by activator protein-1 (AP-1), nuclear factor-B, and nuclear factor of activated T cells (NFAT). NFAT was found to bind to three sequences of the -opioid receptor promoter, located at nucleotides Ϫ1064, Ϫ785, and Ϫ486. Although the Ϫ486 element is in close proximity to a putative AP-1 site, there was no evidence for a combined AP-1/NFAT site. Furthermore, we demonstrated that the induction of interleukin-2 mRNA and protein in activated T cells was inhibited by morphine in cells, in which -opioid receptors had been induced by CD3/28 monoclonal antibodies (mAbs), and that this effect was blocked by the -opioid receptor-specific antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 . CD3/28 mAb-induced interleukin-2 transcription was also inhibited by the opioids fentanyl and loperamide. This indicates that the induced -opioid receptor mRNA is translated into functional receptor protein. Furthermore, a -opioid receptor-enhanced green fluorescent protein-fusion protein was localized in membranes of Jurkat cells and internalized in response to [D-Ala 2 ,NMe-Phe 4 ,Gly 5 -ol]-enkephalin but not morphine. In conclusion, these data emphasize the role of opioids in the modulation of T lymphocyte signaling

Analysis of TCR activation kinetics in primary human T cells upon focal or soluble stimulation.?

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Stoichiometry and intracellular fate of TRIM-containing TCR complexes

<p>Abstract</p> <p>Background</p> <p>Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The αβTCR includes the antigen-binding TCRαβ heterodimer as well as the signal transducing CD3εγ, CD3εδ and ζ₂subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.</p> <p>Results</p> <p>We used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing αβTCR is TCRαβCD3ε₂γδζ₂TRIM₂. Smaller αβTCR complexes possess a TCRαβ CD3ε₂γδζ₂stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the αβTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3ε colocalizes with ζ and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with ζ. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the γδTCR.</p> <p>Conclusions</p> <p>Small αβTCR complexes have a TCRαβ CD3ε₂γδζ₂stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε₂γδζ₂TRIM₂. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.</p

The Transmembrane Adaptor Protein Trim Regulates T Cell Receptor (Tcr) Expression and Tcr-Mediated Signaling via an Association with the Tcr ζ Chain

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-ζ chains and to a lesser extent via the CD3-ε/γ heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca2+ mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface

Adhesion and Degranulation Promoting Adapter Protein (ADAP) Is a Central Hub for Phosphotyrosine-Mediated Interactions in T Cells

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486–783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCγ, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76

LIME: A New Membrane Raft-associated Adaptor Protein Involved in CD4 and CD8 Coreceptor Signaling

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors