General themes of transmembrane signaling

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• General themes of transmembrane signaling
• - clustering and phosphorylation
• -?receptor protein tyrosine kinases? and
  ?receptor-assoicated
• protein tyrosine kinases?
• Signaling molecules
• adaptor proteins, GEF and small G proteins,
  PLC-g,
• TCR and BCR structure
• One step before activation
• BCR signaling
• TCR signaling

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General Principles of Transmembrane Signaling

• Binding of antigen leads to clustering of antigen
  receptors on lymphocytes
• Clustering of antigen receptors leads to
  activation of intracellular signal molecules
• Phosphorylation of receptor cytoplasmic tails by
  tyrosine kinases concentrates
• intracellular signaling molecules around the
  receptors
• intracellular signaling components recruited to
  activated receptors transmit
• the signal onward from the membrane and amplify
  it

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Cross-Linking of antigen receptors is the first
step in lymphocyte activation
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Receptor Protein-Tyrosine Kinases Receptor with
Intrinsic Tyrosine Kinase Activity
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.
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Receptor Protein-Tyrosine Kinases
insulin receptor
EGF receptor
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Dimerization and Autophosphorylation of Receptor
Protein-Tyrosine Kinases
autophosphorylation by cross-phosphorylation
ligand induced dimerization
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Association of Downstream Signaling Molecules
with Receptor Protein-Tyrosine
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Adaptor Proteins
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Adaptor Proteins
Adaptor proteins are specialized signaling
molecules that usually have no enzymatic activity
themselves.
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Adaptor Proteins
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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
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Small G Proteins Are Different from GTP-binding
Stimulatory G proteins
epinephrine adrenaline
b-adrenergic receptor
Gs GTP-binding stimulatory G protein
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phospholipase C-g
PLC-g has two SH2 domains and phosphorylation of
a tyrosine residue in PLC-g activates it.
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Ca2-binding protein calmodulin ? NF-AT
(nuclear factor of activated T cells)
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Receptor without Intrinsic Tyrosine Kinase
Activity
phosphorylation of the tyrosines in ITAMs serves
as the first intracellular signal indicating that
the lymphocyte has detected its specific antigen.
BCR TCR
YXXL/VX6-9YXXL/V
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Cytokine Receptors
JAK Src
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43 kDa
34 kDa
29 kDa
12 kDa
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co-stimulatory signal
TCR triggering and co-stimulatory signal must be
delivered by the same APC.
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CD4 binds MHC class II molecules at a site on the
b2 domain through a region that lies mainly on a
lateral face of the first domain (D1)
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1. Cytoplasmic domain of the TCR heterodimer is 5-12
   a.a. long
2. The signal transduction function is carried out
   by a CD3 complex
3. CD3 complex composed of CD3g, CD3?, CD3e, and CD3z

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Receptor without Intrinsic Tyrosine Kinase
Activity
TCR BCR
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
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??? Signal Transduction ????? Phosphorylation?
Annu.Rev.Cell Dev.Biol.13513.1997
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Annu.Rev.Cell Dev.Biol.13513.1997
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Regulation of Src-Family Kinase Activity
Clinica Immunology and Immunopathology 83(3)205.
1997
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Ig? Ig?
SH2
SH2
Blk, Fyn, or Lyn
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Full phosphorylation of the ITAMs on clustered
Ig? or Ig? chains creates binding sites for Syk
Ig? Ig?
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B-cell co-receptor is a complex of three proteins
CD19, CD21, and CD81.

• 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.
• 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.
• 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
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guanine-nucleotide exchange factor
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Receptor crosslinking activates Blk, Fyn, and Lyn
activated Blk, Fyn, and Lyn phosphorylate ITAMs
Syk binds to phosphorylated ITAM and becomes
activated
Activated Syk phosphorylates CD19, BLNK, PLC-g,
and GEFs (Vav)
GEF activates small G proteins- Rac and Ras
PLC-g cleavages PIP2 to yield DAG and IP3
IP3 increases intracelluloar Ca2 concentration
activating a phosphotase calcinerrin
Small G proteins, Ras and Rac, activate MAP
kinase cascades
DAG and Ca2 activate PKC
The Ras-induced kinase cascade induces and
activates Fos
PKC activates NF-?B
Calcineurin activates NFAT (nuclear factor
activated T cells)
The NF-?B, NFAT, and AP-1 act to induce specific
gene transcription
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Antigen receptor signaling is enhanced by
co-receptors that bind the same ligand
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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
Clinica Immunology and Immunopathology 83(3)205.
1997
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A Second Method Controlling the Activity of
Src-Family Kinases
Annu.Rev.Immunol. 1789. 1999
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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
LAT
LAT
Grb2
?Phosphorylated LAT recruits Grb2 and PLC to
membrance
?Following TCR ligation, ZAP-70 is activated and
phosphorylates LAT and SLP-76.
Overexpression of SLP-76 augment ERK activation
and AP-1 promoter activity, suggesting that
SLP-76 impacts the Ras/ERK signaling pathway
SLP-76
?Phosphorylated SLP-76 recruits Vav
Annu. Rev. Immunol. 1789.1999
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LAT may link TCR-stimulated PTKs with the
phosphatidylinositol second messenger and/or Ras
pathways
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Shc
? 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
? 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
Clinica Immunology and Immunopathology 83(3)205.
1997
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LAT
Overexpression of SLP-76 augment ERK activation
and AP-1 promoter activity, suggesting that
SLP-76 impacts the Ras/ERK signaling pathway
Clinica Immunology and Immunopathology 83(3)205.
1997
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cysteine residues that are palmitoylated and
thus become associated with membrane lipid rafts
? SLP-65 or BLNK in B-cell
Immuol.Today 20431.1999
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Syk phosphorylates BLNK ? BLNKp recruits Tec ?
Tec was phosphorylated by Src ? Tec
phosphorylates PLC-g 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.
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Fyn or Lck phosphorylate tyrosine residues on the
CD3e and z, ITAMS, allowing ZAP-70 to bind
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Fyn or Lck phosphorylate tyrosine residues onThe
CD3e and z, 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-g, GEFs, and Tec kinases
PLC-g cleaves PIP2 to yield DAG and IP3
IP3 increases intracellular Ca2 concentration,
activating a phosphotase, calcineurin
GEFs activate Ras which in turn activates a MAP
kinase cascade
DAG and Ca2 activate PKC
The Ras-induced kinase cascade induces and
activates Fos, a component of the AP-1
transcription factor
Calcineurin activates a transcription factor NFAT

PKC activates a transcription factor NF?B
The transcription factors, NF-kB, NFAT, AP-1 act
to induce specific gene transcription, leading to
cell proliferation and differentiation.
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Src Was Associated with Focal Adhesion
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MAP kinase cascades activate transcription factors
Raf (a serine/threonine kinase)
MEK
Erk (extracellular-signal regulated
kinase (phosphorylated at TEY)
MAPK mitogen-activated protein kinase
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Initiation of MAP Kinase Cascades in both Antigen
Receptor and Co-stimulatory Signaling
Jnk Jun N-terminal kinase
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c-Jun
Elk
J. Immunol. 1604182. 1998
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Jun N-termianl kinase
cyclosporin A and FK506
a serine/threonine protein phosphatase
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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 KAR
• signal through their associated ITAM-containing
• homodimer for the release of the cytotoxic
• granules by which NK cells kill their targets.

one ITAM YxxL/Vxx6-9YxxL/V
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Some lymphocyte cell-surface receptors contain
motifs involved in downregulating activation.

• ITIM immunoreceptor tyrosine based inhibitory
  motif I/VxYxxL
• It functions by recruiting one or other of the
  inhibitory
• phosphatases SHP-1, SHP-2 and SHIP.
• 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
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The pahway that NF-?B is activated by signals
from TLR
IkKaIkKb
SIIK (serine/threonine innate immunity kinase)
IRAK (IL-1R associated kinase)
Also activated is the gene for I?B itself, which
is rapidly synthesized and inactivates the NF?B
signal
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Seven-Transmembrane Receptor

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

large G protein heterotrimeric G protein small
G protein
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.
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Subfamilies of Class I Cytokine Receptors Have
Signaling Subunits in Common
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Cytokine Receptors
JAK Janus kinase STAT signal transducers and
activators of transcription
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 Signalling by the JAK/STAT pathway for a typical
type I cytokine
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Pathways for the induction of expression of IFN-g
 
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SOCS proteins are negative-feedback inhibitors of
cytokine signal transduction
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The cross-talk between cytokine-signalling
pathways
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Schematic representation of signalling pathways
activated by class II cytokine receptors
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Early death-inducing events after trophic-factor
withdrawal
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