Chapter 14 Signal-transduction pathways
[proteins in cell membrane]
Delivery
[2 messenger]
Hormones, Aromas (volatiles) sense1 messenger[threshold conc.][ligand]
changes in enzyme activity, gene expression, or ion-channel activity
metabolic pathways
Branch / feedback / terminate
Common second messengers
(1) Free to diffuse to other compartments of the cell
(2) Significantly amplified signals
(3) Common 2º messengers utilized
— cross talk: opportunities and potential problems
[NO]
H2O2
cADP ribose
Hormones:
Signal transduction
Signal molecules
nonpolar large and polar
through membrane bind to receptor
bind to protein alter receptor structure
interact with DNA [2° messengers]
modulate gene expression protein P / de-P
biochemical reaction
intracellular
steroidsligand,
primary messenger
Type 1: Seven-transmembrane-helix (7TM) receptor
–– serpentine receptor
ca. 50 % therapeutic drugs
rhodopsin
C-terminal and cytoplsmic loop conformation changes
Guanyl nucleotide-binding protein (G protein)
7TM
G-protein-coupled receptors
(GPCRs)
ATP cAMP
an intermediary in signal transduction from 7TM
a heterotrimeric G proteins
G protein: a heterotrimer
: bind the nucleotide (activated and unactivated state): a seven-bladed propeller: a pair of -helices
p. 381
G-protein-coupled receptors (GPCRs)(02)
Activated G proteins transmit signals by binding adenylate cyclaseATP cAMP
The epinephrine receptor signal-transduction
pathway [cAMP] stimulate ATP production for muscle contrac
tion
enhance the degradation of storage fuels
increase the secretion of acid by gastric mucosa
lead to the dispersion of melanin pigment granules
diminish the aggregation of blood platelets
induce the opening chloride channel
mediated by protein kinase A(PKA)
target protein—ser/thr-P
cAMP- response element binding (CREB) protein, in nucleus
- a transcriptional activator
Serotonin
Close K+ channel
cAMP- response element binding protein
1. Resetting G subunit: a intrinsic GTPase activity, spontaneo
us
seconds ~minutes
A build-in clock
Mechanism?
How to terminate the signal transduction?
Signal termination
cytosol
dep. hormone conc.
Specific
(for C-terminal
and occupied)
diminishes its ability to activate G proteins
Phosphatidyl inositol bisphosphate hydrolysis two messengers
Vasopressin
7TM
Gq
Phospholipase C
Cleavage PIP2
IP3 (soluble form)
DAG (insoluble form)
A1
A2
C
D
1,4,5
DAG and IP3
work in tandem
IP3 open channels to release Ca2+
3IP3 + IP3 -gate channel
Ca2+ release (from ER, SR)
Smooth muscle contraction
Glycogen breakdown
Vesicle release
Early fertilization
a short-lived messenger
(nM)
(02)
Diacylglycerol metabolism
Arachidonate
Prostaglandin H2
phosphorylated
hydrolyzed
(02)
Ca2+ — a ubiquitous cytosolic messenger
The reasons for Ca2+ mediate many signaling processes1. Fleeting changes in [Ca2+] are readily detected
The low level of [Ca2+]cyto can be easily and abruptly
raised for signaling purposes 100 nM
avoid insoluble compound formation
(an apparent drawback is in fact an advantage)
is via Ca2+-ATPase and Na+- Ca2+ exchanger (02)
2. Ca2+ can bind tightly to proteins and result in
significant conformational changes coordinated with six to eight oxygen atoms
from proteins or water
6 O from aa/protein
1 O from water
Plagiarism
How to detect or monitor the variation of [Ca2+] in real time
- The fluorescent calcium-binding dye
Red: high
Blue: low
Calcium-specific reagents (02)
ionophores
raise the cytosolic Ca2+ level reduce the unbound Ca2+ level
EDTA: ethylenediamine tetraacetic acid
Calmodium – a calcium sensor
¤ a 17-kd with 4 calcium-binding sites
¤ is activated when [Ca2+]cyto 500 nm
¤ a member of EF-hand protein family, a calcium binding motif
- a helix-loop-helix unit
¤ Parvalbumin: vitamin D3-dependent Ca2+ binding proteins
thumb
forefinger
Calmoduline-dependent protein kinase (CaM kinase)– recognize positively charged,
amphipatic helix
Calcium bind
calmodium conformational changes
A pair of EF-hand motifs
a flexible helix
expose hydrophobic surfaces that can be used to bind other proteins
calcium + calmodulin
CaM kinase Ca2+-ATPase pump
active target protein [Ca2+]cyto level decrease
signal propagation signal termination (the memory of a
previous calcium pulse)
(02)
Type 2: Insulin signaling
receptors that include protein kinase as part of their structures
receptor is a dimer of two identical units
each unit: - and -chain linked by a disulfide bond
-chain-chain
2 inter- and 1-intra-chain disulfide bond
One insulin binding on the outside of the cell
A membrane-associated kinase within the cell is activated;
cross-phosphorylation
Protein kinase A: Ser/Thr P
in subunit
3 tyr residues in activation loop of subunit
IRS : insulin-receptor substrate
a series of membrane-anchored molecules
IRS : insulin-receptor substrate
IRS1/IRS2, act as adaptor proteins
N-terminal: Pleckstrin homology domain, binds phosphoinositide lipids
phosphotyrosine-binding domain:
Tyr-X-X-M sequence: are phosphorylated by the receptor tyrosine kinase
Met
IRS phosphoinositide 3-kinase:
a lipid kinase, 110 kd catalytic subunit and 85 kd regulatory subunit
containing a SH2 domain: Src homology 2, recognize the
phosphotyrosine residues in the IRS, via two Arg residues that are
conserved in all SH2 domain
phosphoinositide 3-kinase PIP3
PIP3-dependent protein kinase
Akt: a kind of protein kinase, is not membrane anchored
Glucose transporters (GLUT4)
Stimulate glycogen synthesis
Membrane-anchor molecules
Amplicification/ termination
phosphatase phosphatase
phosphatase
Type 3: EGF (epidermal growth factor) signaling
stimulate the growth of epidermal and epithelial cells
a receptor tyrosine kinase, a 6 kd polypeptide
3 intrachain disulfide bonds
EGF receptor structure
EGF receptor:
is a dimer of two identical units, but exist as monomers until EGF
ligands bind to them
each monomer binds a EGF molecule in its extracellular domain
each EGF molecule lies far away from the dimer interface
a dimerization arm from each monomer that reaches out and inserts
into a binding pocket on the other monomer
If EGF is absent?
binds to a part of within the same monomer
Once EGF present,
Change into a active conformation
A constitutive active form ?
Her 2 receptor, 50 % identical in aa sequence
with the EGF receptor and has the same domain
structure
Her 2 is overexpressed in some cancers
EGF phosphorylation:
also like insulin receptor, cross-phosphorylation of one unit by another unit
within a dimer, but
its carboxyl - terminal tail containing tyrosine rich (5 residues)
the kinase itself is an active conformation without phosphorylation
Dimerization C-terminal region on one receptor into the active site
of its partner’s kinase
Grb-2: an adaptor protein
SH2 domain phosphotyrosine residues of receptor
SH3 domain proline-rich region of Sos
Sos: a guanine-nucleotide-exchange factor (GEF)
Ras: small G proteins, small GTPase
localized to the inner surface of plasmamembrane
two
phosphatase
GTPase-activating proteins (GAPs)
G proteins vs. small G proteins(divergent evolution)
G proteins small G proteins
30-35 kd 20-25 kd
heterotrimer monomer (similar to G)
7TM dimerization
GTPase act. GTPase act. (low)
GTPase-activating proteins (GAPs):
facilitate GTP hydrolysis Sos + GAPs adjust small G cycle
ras mutation cancer
14.4 Many elements recur with variation
in different signal transduction pathways
Protein kinases are central
Second messengers
Specialized domains
pleckstrin homology domains: interact with lipids PIP3
SH2 domains: interact with the phosphorylated tyrosine residues
Some virus induced cancer– to understand the signal-transduction proteins and
pathwaysRous sarcoma virus: a retrovirus, a oncogenic RNA virusviral sarcoma (v-src): oncogene [A cancer-causing gene; any of several m
utant genes that cause cells to exhibit rapid, uncontrol proliferation.] cellular sarcoma (c-src): proto-oncogene, does not induce cell transformat
ion
a. SH2 bind to tyr-P of C-terminalb. The linker between SH2 and protein kinase is bounded by SH3 c-Src inactive
v-Src: 11 aa of C-terminal, lack Y residue
always active
Biology/chemical/physical factors
c-Src
19 aa
Ras: a small G protein or GTPase– localized to the inner surface of plasmamembrane
The small G proteins
Three 21-kd Ras proteins in mammalian cells
H-Ras: Harvey rat sarcoma
K-Ras: Kirsten rat sarcoma A loss of the ability to hydrolyze GTP
N-Ras: Neuroblastoma rat sarcoma continue on
Tumor-suppressor genes (contribute to cancer development):
to develop cancer only when both copies of the genes normally present in a cell
are deleted or otherwise damaged.
e.g., genes for some of the phosphatase
Monoclonal antibodies utilization:
inhibit the signal transduction in activated tumor formation
In some human epithelial cancers, such as breast, ovarian, and
colorectal cancers, overexpressed the epidermal-growth-factor receptor (EGFR)
Monoclonal Ab offend receptor
e.g., Cetuximab, target a receptor tyrosine kinase
Trastuzumab (Herceptin): inhibit Her2 overexpressed in breast cancers
Protein kinase inhibitor– a potential anticancer drugs
Chronic myologenous leukemia (CML)
chromosome defect:
the translocation between chromosome 9 and 22
(reciprocally)
Bcr-Abl fused protein:
overexpress kinase activity and is not regulated appropriately
STI-571: a specific Bcr-Abl kinase inhibitor
encode tyrosine kinase
To understanding the signal-transduction pathways is leading to conceptually new disease treatment.
Metabolism disease
Choleragen
a cholera toxin from Vibrio cholera (G -) two functional units: subunit B: bind to GM1 gangliosides of intestinal epithelium (p. 738) subunit A: enters the cell, catalyze the covalent modification of Gs
proteinGs + subunit A Gs-Arg-ADP-ribose
stabilize Gs-GTP form (perpetually stimulation)
activate adenylate cyclase
[cAMP] activate protein kinase A
open Cl- channel / inhibit Na+-H+ exchanger
NaCl and H2O loss
Treatment consists of rehydration with a glucose-electrolyte solution.
Pertussis toxin
is secreted by Bordetella pertussis
Gi + pertussis Gi-ADP-ribose
reduced Gi-GTP affinity
inhibit adenylate cyclase
[cAMP] close Ca2+ channels and open K+ channels
Altered G-protein activity caused diseases
(02)
96T
(191)
96T (192)
STAT5(signal transducers and activators of
transcription)–– a regulator of gene expression
–– is phosphorylated by JAK2
recirpocal interaction
(02)
97T
Which of the event is important for epidermal growth factor signaling?(A) ADP-ribosylation (B) Farnesylation (C) Mono-oxygenation (D) Peroxidation (E) Glycosylation (94, 台大 ) p. 284
Nelson p. 474
96C