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Antigen recognition. out. + -. Lipid bilayer of plasma membrane. in. Signal transduction (contains one or more ITAMs). Src-family tyrosine kinase reversibly associated with receptor. Consensus ITAM (amino acid)

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  1. Antigen recognition out + - Lipid bilayer of plasma membrane in Signal transduction (contains one or more ITAMs) Src-family tyrosine kinase reversibly associated with receptor

  2. Consensus ITAM (amino acid) ….---.asp.---.---.tyr.---.---.leu.---.---.---.---.---.---.---.tyr.---.---.leu.---…. glu ile ile The one letter code is usually used: xx(D/E)xxYxx(L/I)xxxxxxxYxx(L/I) These tyrosines are phosphorylated by various src family kinases upon recognition receptor ligation

  3. Src-family tyrosine kinases Myristylation/palmitylation sites present in many family members, promotes association with membranes and membrane “rafts”. (e.g. CH3(CH)12CONH-Glycine..) Unique region reversibly associates with certain membrane receptors. Differs among family members. SH3 motif involved in binding to many proteins that carry a proline rich motif (e.g. P-X-X-P). SH2 motif involved in binding to phospho-tyrosines in certain sequence contexts, including phosphorylated ITAMs. Kinase domain motif catalyzes tyrosine phosphorylation.

  4. CD45 (plasma membrane tyrosine phosphatase) csk (cytoplasmic tyrosine kinase)

  5. Adapted from Molecular Cell Biology 3rd Ed., p894

  6. Table 1. Expression of Src family kinases Src Ubiquitous; two neuron-specific isoforms Fyn Ubiquitous; T cell-specific isoform (Fyn T) Yes Ubiquitous Lyn Brain, B-cells, myeloid cells; two alternatively spliced forms Hck Myeloid cells (two different translational starts) Fgr Myeloid cells, B-cells Blk B-cells Lck T-cells, NK cells, brain Frk subfamily Primarily epithelial cells

  7. T Cell Receptor a b CD3 functions both as signal transducers and in transport of antigen receptors to the plasma membrane CD3 d e Antigen recognition g e out + - - - + CD3 z in Signal transduction (with ITAMs)

  8. + + - - + + - - Lyn Blk Hck Fyn Lck syk ACTIVATION! B Cell Receptor Ig a b Antigen recognition out Signal transduction (with ITAMs) in

  9. Antigen receptor signaling may be similar to that of other surface receptors such as receptor tyrosine kinases like epidermal growth factor in which receptor crosslinking leads to initial trans-phosphorylation.

  10. CD4 CD3 d e g e Cholesterol and sphingolipid rich microdomain - Lck CD3 z T Cell Receptor And coreceptor a b out + - - + in Signal transduction (with ITAMs) Fyn

  11. CD4 CD3 d e g e - ZAP70 ACTIVATION! Lck CD3 z T Cell Receptor a b + - - +

  12. TABLE 2. Protein with Src homology-2 domains that may be associated with T cell-activation (partial list) Protein tyrosine kinases Fyn Lck Syk ZAP-70 Csk ltk Protein tyrosine phosphatases SHP-1 SHP-2 Proteins with enzymatic functions Phospholipase C 1 (PLC 1) Phosphatidylinositol 3”-kinase (P1 3-kinase), p85 subunit Adaptors and Regulators GTPase acttivating protein (GAP) SOS Vav Slp-76 Shc Nck Grb2 Crk From Fundamental Immunology 4th Ed, Paul WE Ed., pp 425

  13. TABLE 3. Some of the proteins that are tyrosine phosphorylated following TCR stimulation. TCR subunits CD3 , ,  ,  Protein tyrosine kinases ltk Lyn Lck MAPKs Pyk2 Syk ZAP-70 Proteins with enzymatic function Phospholipase C 1 (PLC 1) Others Cbl CD5 CD6 Ezrin LAT Shc Slap-130 Slp-76 Shc  Tubulin Valosin containing protein Vav From Fundamental Immunology 4th Ed, Paul WE Ed., pp 427

  14. LAT CRAC DAG RAS CD4 PKC CD3 d e g e GTP GDP IP3 Ca++ ACTIVATION! Ca++ Ca++ Ca++ Raf-1 Ca++ - [Ca++]i calcineurin IP3 receptor MEK Ca++ Endoplasmic reticulum ZAP70 NFAT NFAT PO4 Lck MAPK NFAT Fos/Jun CD3 z Immediate early genes Jun/Fos T Cell Receptor activation (cont) a b Ca++ + - - + RasGRP PIP2 Plcg nucleus IL-2 gene

  15. From Fundamental Immunology 4th Ed, Paul WE Ed., pp 428

  16. LAT Ras GTP Ras GDP Grb2 GAPs CD4 CD3 d e sos g e Gads Ras effectors vav Slp-76 ACTIVATION! Nck rho/rac SLAP 130 Pak - WASP Actin/cytoskeletal reorganization ZAP70 Adhesion migration Lck CD3 z T Cell Receptor activation (cont) a b + - - + nucleus

  17. Antigen presenting cell “Costimulatory” B7 molecules on antigen presenting cells B7.1 or B7.2 LAT CD28 CD3 d e PIP2 PIP3 ? g e p85 PI3K PI3K ACTIVATION! ACTIVATION! - PKB Itk CD3 z T Cell Receptor activation (cont) a b CD4 + - - + Lck nucleus

  18. PI3K Removed by SHIP--1 P

  19. Where does cell type specificity come from? • Preexisting substrate differences. • Specificity of src and ZAP70/syk kinases • PKC isoform expression • PLCg isoform expression • NFAT isoform expression • Transcription factor isoform expression • Inhibitory receptor expression • etc….

  20. Activating Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells) 2-4 Ig-like extracellular domains FcgRI FcgRIIa* FcgRIII FcaRI ILT1 ILT7 ILT8 LIR6a 1-3 Ig-like extracellular domains NK activatory receptors FcaRI 1,2 Ig-like extracellular domains NKp46 NKp30 FcRIII 1,2-? Ig-like extracellular domains FcgRIII FceRI Type 2 membrane protein NKp80 “Antigen” recognition out + - + - CD3 z in Fc common g chain DAP12 Fc g chain Signal transduction (with ITAMs) b

  21. Activating ligand Inhibitory ligand Activating receptor Motif: I/VxYxxL/V out + - in ITAMs ITIMs SHIP lipid phosphatase Tyrosine phosphatase e.g., SHP-1 Src-family tyrosine kinase reversibly associated with receptor Tyrosine kinase e.g., ZAP-70, syk

  22. MHC class I - + Normal cell Defective cell “Missing self” “Natural killer” cell recognition + Weak signal no response Initiation of killing program nucleus nucleus

  23. APC Late in the response... T cell down-regulation late in response B7 B7 CD28 + - + CTLA4 on surface + Ag signal + costimulation T cell activation APC MHC/peptide CD28 TCR CTLA4 signaling turns response off CTLA4 sequestered nucleus nucleus

  24. Type 2 membrane proteins Ly49 CD94/ NKG2A CD72 Inhibitory Receptors (on Monocytes, Macrophages, Neutrophils, Mast cells, and NK cells) 1-6 Ig-like extracellular domains ILT5 LIR8 ILT4 Siglec 6 CD33 CD22 FcgRIIb PIRB SIRPs LAIR1 ILT3 LIR5 PD-1 KIR2DL KIR3DL CTLA4 “Antigen” recognition out in Signal transduction (with ITIMs)

  25. Immunoprecipitation Detergent Anti-Y-PO4 precipitate antibodies with S. aureus protein A-coupled resin, wash, elute sample control markers - Fractionate by electrophoresis on denaturing polyacrilamide gel Mobility inversely proportional to the Log of molecular weight +

  26. Generation of gene knockouts in mice: an important technique in establishing the role of signaling molecules in normal cells. Adapted from Molecular Cell Biology 3rd Ed., p294-5

  27. Adapted from Molecular Cell Biology 3rd Ed., p296

  28. Yeast two hybrid assay is one way that protein: protein interactions can be measure and is an important way that new molecules are discovered. Adapted from Molecular Cell Biology 3rd Ed., p896

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