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When Good Signals go Bad
Lecture 18 BCHM2072 2006 Vanessa Gysbers
www.toothpastefordinner.com
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problems with signals

• ? activity
• excess ON signal
• accelerator stuck down
• lack of OFF signal
• brakes fail
• ? activity/absence
• affect ligands, receptors, downstream
  transducers, or targets

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excess deficient
G-protein coupled receptors Toxin Cholera Pertussis Night blindness (not covered in this lecture)
Intrinsic tyrosine kinase receptors Her ? breast cancer FGFR? Achondroplasia
Non-receptor TKs Bcr-abl ? CM Leukaemia BTK ? Agglobulinemia
Cytokine receptors TNFa? Rheumatoid arthritis? gc, JAK, ILxR? Severe Combined ImmunoDeficiency
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excess signal lack of OFF
G-protein Coupled Receptors

• cholera
• pertussis (whooping cough)

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InactiveG-protein bound to GDP

• Revision GPCRs normally.

N
GPCR
C
7
Ligand binds
N
GPCR
C

• adrenaline
• serotonin
• glucagon
• vision

8
Changes shape of loop
N
GPCR
C
9
G-protein can bind
N
GPCR
C
10
Exchanges GDP for GTP
N
GPCR
C
11
Exchanges GDP for GTP
N
GPCR
C
12
And becomes activated
N
GPCR
C
13
a-subunit dissociates from bg
N
GPCR
C
G-protein
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N
GPCR
C
bg
stimulatory
a-subunit (Gsa) ? activates adenylate cyclase
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N
GPCR
C
a-subunit (Gsa) ? activates adenylate cyclase
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N
GPCR
C
a-subunit (Gsa) ? activates adenylate cyclase
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cAMP 2nd messenger
N
AC
GPCR
C
cAMP
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activates Protein Kinase Aamong many others
N
GPCR
C
cAMP
cAMP
cAMP
ACTIVE
PKA
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PKAphosphorylates targets
N
GPCR
C
cAMP
cAMP
cAMP
ACTIVE
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2 ways to terminate the signal

1. hydrolysis of GTP ? GDP
2. activation of inhibitory G proteins

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Active G-protein GTP?cAMP
1. termination ? OFF
AC
Gsa is a GTPasehydrolyses GTP?GDP
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Active G-protein GTP?cAMP
1. termination ? OFF
AC
hydrolysis
GTP
GDP
Gsa is a GTPasehydrolyses GTP?GDP
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Active G-protein GTP?cAMP
1. termination ? OFF
AC
hydrolysis
GDP
GTP
GDP
Gsa is a GTPasehydrolyses GTP?GDP
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Active G-protein GTP?cAMP
1. termination ? OFF
AC
hydrolysis
GDP
GTP
GDP
inactive G-protein GDPstops activating AC ? ?
cAMP
Gsa is a GTPasehydrolyses GTP?GDP
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2. signal termination ? OFF
N
Inhibitory signal
GPCR
C
Gia
bg
GDP
different a subunit
inhibitory signal activates Ginhibitory
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2. signal termination ? OFF
N
Inhibitory signal
GPCR
C
Gia
bg
exchange GDP?GTP
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2. signal termination ? OFF
Inhibitory signal
N
GPCR
Gia
bg
inhibitory G-proteininhibits AC ? ? cAMP
Gia inhibits adenylate cyclase
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2. signal termination ? OFF
Inhibitory signal
N
GPCR
Gia
bg
inhibitory G-proteininhibits AC ? ? cAMP
Gia inhibits adenylate cyclase
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Cholera toxin interferes with termination of GCPR
signal

• toxin binds to a receptor on enterocyte surface
• enters cell by endocytosis

Vibrio cholera
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cholera toxin cleaves inside cell active enzyme
Gsa
C
cAMP
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cholera toxin cleaves inside cell active enzyme
Gsa
GPCR
C
cAMP
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toxin catalyses ADP-ribosylation of active Gsa
Gsa
cAMP
C
NAD
nicotinamide
ADP ribose
NAD in active site of cholera toxin
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ribosylation of Gsa prevents GTPase activity of
Gsa
Gsa
cAMP
C
ADP ribose
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ribosylation of Gsa prevents GTPase activity of
Gsa
Gsa
cAMP
C
ADP ribose

• cant hydrolyse GTP?GDP
• continued activation of AC
• ? cAMP

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AC
Gsa
cAMP
ADP ribose
Cholera toxin
Gsa
GDP
cant hydrolyse GTP?GDP cannot stop activating
AC ? ? ? cAMP
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CFTR
AC
Gsa
cAMP
cAMP
ADP ribose
ions and water
Cholera toxin
Gsa
GDP

• ? cAMP ?activate CFTR
• efflux ions and water
• diarrhoea

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pertussis also interferes with termination

• inhibits an inhibitor!

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normally...
N
Inhibitory signal
GPCR
Gia
GDP
Gia activated by inhibitory ligandsexchanges
GDP? GTPinhibits AC ??cAMP
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normally...
N
Inhibitory signal
GPCR
Gia
GDP
Gia also a GTPase to terminate termination!
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pertussis toxin...
N
Inhibitory signal
GPCR
Gia
pertussis toxin
GDP
ribosylates inactive Gia prevents exchange of
GDP?GTP
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pertussis toxin
N
Inhibitory signal
GPCR
Gia
cAMP
pertussis toxin
GDP
prevents the ACTIVATION of INHIBITOR
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compare.
AC
Gsa
Gia
Cholera toxin
pertussis toxin
Gsa
GDP
GDP
cannot inhibit AC brake failure
cannot stop activating AC accelerator stuck on
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excess signaloverexpression of receptors
Receptor Tyrosone Kinases (RTKs)

• Her 2 breast cancer

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cancer

• Receptor tyrosine kinases are often receptors for
  growth factors
• dysregulated signalling through RTKs
• ? dysregulated cell proliferation ? cancer

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homodimers or heterodimers
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GF
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Ligand binds
GF
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Ligand binds
EGF
Her-2 chain

• Growth factors
• Epidermal GF
• Platelet derived GF
• Nerve GF
• Insulin

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dimerisation
GF
50
dimerisation
GF
activatesIntrinsic tyrosine-kinase domains
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Auto-phosphorylation of tyrosine residue
GF
P
Tyr
Tyr
P
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docking of partners
GF
P
Tyr
Tyr
P
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docking
GF
ENZYMES eg PLC-g PIP3 ADAPTORS eg Grb
(recruits Ras)
P
Tyr
Tyr
P
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phosphorylation of tyrosines by receptor TK
GF
ENZYMES eg PLC-g PIP3 ADAPTORS eg Grb
(recruits Ras)
P
Tyr
Tyr
P
Tyr
Tyr
P
P
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activation of downstream targets
GF
Ras
eg Ras
P
Tyr
Tyr
P
Tyr
P
mutated in 15 cancers (60 of melanomas)
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Ras
Ras activates downstream kinases
P
Tyr
Tyr
P
Tyr
P
eg. mitogen activated protein kinases
MAPKs
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Ras
P
Tyr
Tyr
P
Tyr
P
MAPKs enter nucleus and activate transcription
factors by phosphorylation
MAPKs
P
Jun
Jun
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Her-2 (erbB-2)

• one type of chain in EGF receptors (erbB)
• overexpressed in 30 breast Ca
• ? spontaneous dimerization of receptor without
  ligand
• constitutive receptor activation
• pro-proliferative (poor prognosis)

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Her-2 overexpression
Tyr
P
P
P
Tyr
P
P
P
spontaneous dimerisation
WITHOUT LIGAND!!!
eventual activation of MAPK
MAPKs enter nucleus and activate transcription
factors by phosphorylation
MAPKs
P
Jun
Jun
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ALSOanti-anti proliferative!

• ? inhibition of a cdk inhibitor

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normally
Cdk2
proliferative signal ? mitosis
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normally
cdk2 inhibitor
Cdk2
inhibit
cdk2 inhibitor
anti-proliferative signal ? arrest
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Her-2 overexpression
Cdk2
cdk2 inhibitor
inhibit
P
cdk2 inhibitor
anti-proliferative signal ? cannot arrest
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Herceptin (Trastuzumab)

• Herceptin is a monoclonal antibody
• binds extracellular bit of Her-2 Receptor.
• disrupts dimerisation ? ? signal initiation
• ? activation of MAPKs
• ? phosphorylation of cdk2-inhibitor (via PI3K)
• ?can enter the nucleus
• inhibit cdk2 activity
• ? cell cycle arrest during G1 phase (Kute et al
  2004).
• ? proliferation

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Herceptin monoclonal antibody
binds Her-2 prevents signal initiation
Tyr
MAPKs
cdk2 inhibitor
cdk2 inhibitor
Jun
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Her-2 overexpression Herceptin
Cdk2
cdk2 inhibitor
inhibit
P
cdk2 inhibitor
cdk2 inhibitor
can inhibit cdk2 ? arrest
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Herceptin

• (70K/year)
• Aug 06? Herceptin listed on the Pharmaceutical
  Benefits Scheme (PBS).
• Around 2,100 patients are expected to be treated
  with Herceptin each year.
• 46 decrease in reoccurrence

not examinable!
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deficient signalmutation of receptors
Receptor Tyrosone Kinases (RTKs)

• achondroplasia

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Achondroplasia

• deficient RTK signal
• mutation of Fibroblast Growth Factor Receptor
  (FGFR3 gene )

• ? proliferation of cartilage at growth plates
  in bone ? shorter bones, and shorter stature

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• 98 Glycine ? Arginine in TMD
• single point mutation of gene
• arises spontaneously in 80 of cases!!
• neither parent affected
• then autosomal dominant inheritance
• lethal if homozygous

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deficient signal underexpression of receptor
chains or of associated kinases
Cytokine receptors

• Severe Combine Immunodeficiency Syndrome (SCID)

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Typical cytokine receptor Receptor that
interacts with tyrosine kinases
cytokine
revision!!!
JAK
JAK
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The interacting tyrosine kinase JAK
cytokine
JAK
JAK
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JAK the two faced kinase
JAK Janus kinase
Hello my JAK twin!! Let me phosphorylate you,
STAT and the receptor
Hi JAK! Let me phosphorylate you, STAT and the
receptor too!
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Ligand binds
Interleukin 3

• Interleukins
• Interferons
• Growth hormone
• Erythropoietin

JAK
JAK
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dimerisation
Interleukin 3
JAK
JAK
77
dimerisation
Interleukin 3
JAK
JAK
78
JAKs meet phosphorylate each other
Interleukin 3
P
P
JAK
JAK
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JAKs can then phlate the RECEPTOR
Interleukin 3
P
P
JAK
JAK
P
P
Tyr
Tyr
80
JAKS phlate tyrosine residues
Interleukin 3
P
P
JAK
JAK
P
P
Tyr
Tyr
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STAT docks to P-tyr on Receptor
Interleukin 3
P
P
JAK
JAK
Signal Transducer and Activator of Transcription
P
P
Tyr
Tyr
SH2
STAT
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STAT gets phlated by JAK
Interleukin 3
P
P
JAK
JAK
P
P
Tyr
Tyr
P
P
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P-STAT then dissociates
Interleukin 3
P
P
JAK
JAK
P
P
Tyr
Tyr
P
P
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P-STAT then dissociates
Interleukin 3
P
P
JAK
JAK
P
P
Tyr
Tyr
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then dimerises
P
P
JAK
JAK
P
P
Tyr
Tyr
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then translocates
P
P
JAK
JAK
P
P
Tyr
Tyr
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then translocates to the nucleus
P
P
JAK
JAK
P
P
Tyr
Tyr
88
Where it activates transcription
promoter
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eg immune cell development,anti or pro
inflammatory genes
specific ligand ? specific JAKS and STATS?
specific genes
promoter
90
interleukins (IL)
ILxR
gc
P
JAK
P
JAK
P
Tyr
Tyr
SH2
P
SH2
STAT
STAT
P
P
gc chain shared by receptors of many different
interleukins
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interleukins (IL)
ILxR
gc
JAK
JAK
Tyr
SH2
STAT
affects immune cell development
gene mutation of g chain ?X-linked Severe
Combined ImmunoDeficiency
92
IL-xR or JAK3 mutation
less common
specific for particular interleukins
gc
JAK
Tyr
SH2
SH2
STAT
STAT
autosomal recessive (AR)SCID affects fewer
receptor types
93
SCID

• affects immune cell development
• severe infections
• g chain mutation worst shared by many
  interleukins
• most common (50)
• affects more cell types

94
treatment of SCID

• Gene therapy to replace defective gene

• gc chain
• JAK
• ILxR chain
• ADA

95
excess signal overexpression of ligand
Cytokine receptors

• Rheumatoid arthritis

96
normally negative feedback loopvia Supressors
of Cytokine Signals
TNFa
SOCS is induced by same receptor
P
P
JAK
JAK
Tyr
Tyr
P
P
P
P
inhibits JAK target proteins for ubiquitin
degradation
SOCS
97
normally negative feedback loopvia Supressors
of Cytokine Signals
TNFa
SOCS is induced by same receptor
P
P
JAK
JAK
Tyr
P
98
? excess TNFa ligand
TNFa
P
P
JAK
JAK
P
P
Tyr
Tyr
P
P
dimerisation
translocation
activates transcription eg of inflammatory genes
99
? treatments for RhA
TNFa
eg Infliximab binds TNFa
what would happen subsequently?
P
P
JAK
JAK
what about other potential targets?
P
P
Tyr
Tyr
P
P
dimerisation
translocation
SOCS
stimulate
100
Non-receptor TKs

• excess signal
• bcr-abl fusion protein
• chronic myelogenous leukaemia
• (CML)

101
chronic myelogenous leukaemia
bcr
22
tyrosine kinase
SH3
control?
abl
Philadelphia chromosome
bcr
bcr-abl fusion protein

• fusion ?change in SH3 domain of abl?
• bcr-abl constitutively active

102
SH3 domain
constitutively active TK
abl
SH3
abl
bcr
SH3
ATP
bcr
ADP
kinase domain
uncontrolled proliferation, ?apoptosis
Ras STAT myc
Ras STAT myc
103
Treatment imatinib (Glivec ) competes with
ATP at active site of abl
ATP
SH3
abl
bcr
glivec
Gleevec (purple) in active site of kinase
Ras STAT myc
Ras STAT myc
structure of Gleevec
104
deficient signal mutation in B-cell Tyrosine
Kinase (BTK)
Non-receptor tyrosine kinase
red spots show mutations that have been located
in BTK

• X-linked agammaglobulinaemia

105
normal btk signal

• B cell receptor?
• PIP3K ? ? BTK
• ?
• phospholipase Cg
• ?
• Inosityl triphosphate
• (IP3)
• ?
• ? Ca2

I certainly dont expect you to know details of
this diagram , but you should know the PIP3K,
PLCg, IP3 pathway for Dr E-S, and appreciate how
mutation of btk interrupts the pathway.
106
X-linked agammaglobulinemia

• mutation in btk
• (B-cell Tyrosine Kinase) prevents binding to
  upstream and downstream partners

usually signals via phospholipase Cg ? ? Ca2
107
X-linked agammaglobulinaemia

• btk
• (B-cell Tyrosine Kinase) needed for
• production of antibodies
• B-cell maturation
• deficiency?
• humoral immunity

• susceptible to BACTERIAL infections