1. General themes of transmembrane signaling- clustering and phosphorylation-receptor protein tyrosine kinases and receptor-assoicated protein tyrosine kinases

2. Signaling moleculesadaptor proteins, GEF and small G proteins, PLC-,

3. TCR and BCR structure

4. One step before activation

5. BCR signaling

6. TCR signaling

General Principles of Transmembrane Signaling

1. Binding of antigen leads to clustering of antigen receptors on lymphocytes

2. Clustering of antigen receptors leads to activation of intracellular signal molecules

3. Phosphorylation of receptor cytoplasmic tails by tyrosine kinases concentratesintracellular signaling molecules around the receptors

4. intracellular signaling components recruited to activated receptors transmitthe signal onward from the membrane and amplify it

Cross-Linking of antigen receptors is the first step in lymphocyte

activation

These enzyme domains are normally inactive, but when brought together by receptor clustering they are able to activate each other by trans-phosphorylation. Once activated, these tyrosine kinases can phosphorylate and activate other cytoplasmic signaling molecules.

Receptor Protein-Tyrosine Kinases Receptor with Intrinsic Tyros

ine Kinase Activity

insulin receptorEGF receptor

Receptor Protein-Tyrosine Kinases

Dimerization and Autophosphorylation of Receptor Protein-Tyrosine Kinases

ligand induced dimerizationautophosphorylation by cross-phosphorylation

Association of Downstream Signaling Molecules with Receptor Protein-

Tyrosine

Adaptor Proteins

Adaptor proteins are specialized signaling molecules that usually have no enzymatic activity themselves.

Adaptor Proteins

Adaptor Proteins

Small G proteins are switched from inactive to active states by Guanine-nucleotide Exchange Factors (SOS, Vav).

Most of the time, Ras is in the inactive state owing to its intrinsic GTPase activity

epinephrine = adrenaline

-adrenergic receptor

Gs: GTP-binding stimulatory G protein

Small G Proteins Are Different from GTP-binding StimulatoryG proteins

phospholipase C-

PLC- has two SH2 domains and phosphorylation of a tyrosine residuein PLC- activates it.

Ca2+-binding protein calmodulinNF-AT

(nuclear factor of activated T cells)

phosphorylation of the tyrosines in ITAMs serves as the first intracellular signal indicating that the lymphocyte has detected its specific antigen.

YXX[L/V]X6-9YXX[L/V]

Receptor without Intrinsic Tyrosine Kinase ActivityBCR TCR

JAKSrc

Cytokine Receptors

43 kDa

12 kDa

34 kDa29 kDa

co-stimulatory signal

TCR triggering and co-stimulatory signal must be delivered by the same APC.

CD4 binds MHC class II molecules at a site on the 2 domain through a region that lies mainly on a lateral face of the first domain (D1)

1. Cytoplasmic domain of the TCR heterodimer is 5-12 a.a. long2. The signal transduction function is carried out by a CD3 complex3. CD3 complex composed of CD3, CD3, CD3, and CD3

Receptor without Intrinsic Tyrosine Kinase Activity

receptor-associated tyrosine kinase:The antigen receptor of lymphocytes are associated with receptor-associated tyrosine kinase, mainly of the Src family, which bind to receptor tails via their SH2 domain

TCR BCR

Annu.Rev.Cell Dev.Biol.13:513.1997

為什麼 Signal Transduction 總是圍繞在 Phosphorylation?

Annu.Rev.Cell Dev.Biol.13:513.1997

Clinica Immunology and Immunopathology 83(3):205. 1997

Regulation of Src-Family Kinase Activity

SH2 SH2

Ig Ig

Blk, Fyn, or Lyn

Full phosphorylation of the ITAMs on clustered Ig or Ig chains

creates binding sites for Syk

Ig Ig

B-cell co-receptor is a complex of three proteins CD19, CD21, and CD81.

1. CD19 is expressed on all B cells from an early stage in their development,before CD21 and CD81 are expressed, and it appears to contribute to signaling through the B-cell receptor even in the absence of co-ligation through CD21.

2. CD19-/- mice

B cells from mice that lack CD19 fail to proliferate in response to B-cell receptor cross-linking and do not fully activate the intracellular signaling pathways normally generated when the B-cell receptor is cross-linked.

3. These experiments suggest that CD19 can associate with the B-cell receptor, either constitutively or after receptor activation, and contribute to signaling even when the co-receptor has not been engaged through CD21 binding to complement.

Vav

guanine-nucleotide exchange factor

Receptor crosslinking activates Blk, Fyn, and Lyn

activated Blk, Fyn, and Lyn phosphorylate ITAMs

Activated Syk phosphorylates CD19, BLNK, PLC-, and GEFs (Vav)

PLC- cleavages PIP2 to yield DAG and IP3

GEF activates small G proteins- Rac and Ras

Syk binds to phosphorylated ITAM and becomes activated

Small G proteins, Ras and Rac, activate MAP kinase cascades

The Ras-induced kinase cascade induces and activates Fos

DAG and Ca2+ activate PKC

IP3 increases intracelluloar Ca2+ concentration activating a phosphotase calcinerrin

PKC activates NF-B

The NF-B, NFAT, and AP-1 act to induce specific gene transcription

Calcineurin activates NFAT (nuclear factor activated T cells)

Antigen receptor signaling is enhanced by co-receptors that bind the same ligand

Clinica Immunology and Immunopathology 83(3):205. 1997

Binding of SH2 (Src homology 2 domain) to phosphotyrosines is a crucial mechanism for recruiting intracellular signaling molecules to an activated receptor

SH3: binds to proline rich region

Annu.Rev.Immunol. 17:89. 1999

A Second Method Controlling the Activity of Src-Family Kinases

Annu. Rev. Immunol. 17:89.1999

Adapter Proteins Grb2, Linker of Activation in T Cells (LAT), and SH2-Domain

Leukocyte Protein of 76 kDa (SLP-76) in

Mediating Positive T Cell Receptor Signals

Phosphorylated SLP-76 recruits Vav

Phosphorylated LAT recruits Grb2 and PLC to membrance

Overexpression of SLP-76 augment ERK activation and AP-1 promoter activity, suggesting that SLP-76 impacts the Ras/ERK signaling pathway

Following TCR ligation, ZAP-70 is activated and phosphorylates LAT and SLP-76.

LAT LAT

SLP-76

Grb2

LAT may link TCR-stimulated PTKs with the phosphatidylinositol second messenger and/or Ras pathways

Shc itself is tyrosine phosphorylated and may interact with the SH2 domain of Grb2 to allow its translocation to the cell surface

recruitment of Grb2 along with Sos, results in activation of Ras

In T cells, the interaction of Shc with the tyrosine phosphorylated ITAMs of the TCR has been suggested to mediate the translocation of Grb2 to the plasma membrane

Clinica Immunology and Immunopathology 83(3):205. 1997

Shc

Clinica Immunology and Immunopathology 83(3):205. 1997

LAT

Overexpression of SLP-76 augment ERK activation and AP-1 promoter activity, suggesting that SLP-76 impacts the Ras/ERK signaling pathway

Immuol.Today 20:431.1999

cysteine residues that are palmitoylated and thus become associated with membrane lipid rafts

SLP-65 or BLNK in B-cell

Syk phosphorylates BLNK BLNKp recruits Tec Tec was phosphorylated by Src Tec phosphorylates PLC-

In B: shc-Grb2-SOS-Ras-Raf

In T: LAT-GADS-SOS-Ras-Raf

CD19 Vav-Rac

The human immunodeficiency disease X-linked agammaglobulinemia (XLA), in which B cells fail to mature, results from a mutation of Btk, while the same gene mutated in mice leads to a similar immunodeficiency called xid.

Fyn or Lck phosphorylate tyrosine residues on the CD3and , ITAMS, allowing ZAP-70 to bind

Fyn or Lck phosphorylate tyrosine residues onThe CD3and , ITAMS, allowing ZAP-70 to bind

Fyn or Lck protein tyrosine kiniase clustering activates kinase activity

Lck activated ZAP-70, which in turn phosphorylates LAT and SLP-76. SLP-76 binds and activates PLC-, GEFs, and Tec kinases

PLC- cleaves PIP2 to yield DAG and IP3

GEFs activate Ras which in turn activates a MAP kinase cascade

The Ras-induced kinase cascade induces and activates Fos, a component of the AP-1 transcription factor

IP3 increases intracellular Ca2+ concentration, activating a phosphotase, calcineurin

Calcineurin activates a transcription factor NFAT

DAG and Ca2+ activate PKC

PKC activates a transcription factor NFB

The transcription factors, NF-kB, NFAT, AP-1 act to induce specific gene transcription, leading to cell proliferation and differentiation.

Src Was Associated with Focal Adhesion

MAPK: mitogen-activated protein kinase

Raf (a serine/threonine kinase)

MEK Erk (extracellular-signal regulated kinase(phosphorylated at TEY)

MAP kinase cascades activate transcription factors

Initiation of MAP Kinase Cascadesin both Antigen Receptor and Co-stimulatory Signaling

Jnk: Jun N-terminal kinase

J. Immunol. 160:4182. 1998

Elkc-Jun

a serine/threonine protein phosphatase

cyclosporin A and FK506 Jun N-termianl kinase

Other receptors that pair with ITAM-containing chains can deliver activating

signals

KAR: killer activatory receptor

these receptors can activate NK cells to kill infected or abnormal target cells. The KARsignal through their associated ITAM-containing homodimer for the release of the cytotoxic granules by which NK cells kill their targets.

one ITAMYxx[L/V]xx6-9Yxx[L/V]

Some lymphocyte cell-surface receptors contain motifs

involved in downregulating activation.

ITIM: immunoreceptor tyrosine based inhibitory motif [I/V]xYxxL

1. It functions by recruiting one or other of the inhibitory phosphatases SHP-1, SHP-2 and SHIP.

2. SHIP is an inositol phosphatase and removes the 5’ phsophate from PI-3,4,5-P. it is thought to inhibit the activation of PLC- by inhibiting the recruitment of the Tec family of kinases, including Btk and Itk, and thus the production of DAG and IP3 and the associated mobilization of calcium

SHP-2

= IK+IK

SIIK (serine/threonine innate immunity kinase)= IRAK (IL-1R associated kinase)

The pahway that NF-B is activated by signals from TLR

Also activated is the gene for IB itself, which is rapidly synthesized and inactivates the NFB signal

1. fMLP receptor2. photoreceptor bacteriorhodopsin (the only solved structure of seven-transmembrance protein)3. receptor for anaphylotoxins4. chemokine receptors

large G protein = heterotrimeric G proteinsmall G protein

Seven-Transmembrane Receptor

Important targets for the activated G protein subunits are adenylate cyclase and phospholipase C, whose activation gives rise to the second messengers cyclic AMP, IP3 and Ca2+.

Subfamilies of Class I Cytokine Receptors Have Signaling Subunits in Common

Cytokine Receptors

JAK = Janus kinaseSTAT = signal transducers and activators of transcription

Signalling by the JAK/STAT pathway for a typical type I cytokine

Pathways for the induction of expression of IFN-  

SOCS proteins are negative-feedback inhibitors of cytokine signal transduction

The cross-talk between cytokine-signalling pathways

Schematic representation of signalling pathways activated by class II cytokine receptor

s

Early death-inducing events after trophic-factor withdrawal