Gerald Karp
Cell and Molecular Biology
Fifth Edition
Chapter 15:
Signal Transduction
Copyright © 2005 by John Wiley & Sons, Inc.
• No man is an island
• The same can be said of the cells that
make up a complex multicellular organism
• Cell signaling
• Regulation of cell growth and division
15.1 The basic elements of cell
signaling systems
• Extracellular messenger molecules
(primary messenger)
• Receptors
• Second messenger
• Effector
• Target proteins
• Transcription, survival, protein synthesis,
movement, cell death, metabolic change
15.2 A survey of extracellular
messengers and their receptors
• Messengers:
1. Small molecules:amino acids and
derivatives (glutamate, glycine,
acetylcholine, epinephrine, dopamin,
thyroid hormone)
2. Steroids, derived from cholsterol
3. Eicosanoids: derivatives from arachidonic
acid (fatty acid, 花生酸)
4. Polypeptides and proteins
Extracellular signals receptors
• 1. G protein-coupled receptors (GPCRs): a huge
family containing 7 transmembrane αhelices →
activation of G protein (GTP-binding proteins) →
vesicle fusion, microtubule dynamics, protein
synthesis etc
• 2. receptor protein-tyrosine kinases (RTKs)
→receptor dimerization → activation of the
receptor’s protein-kinase domain →serine or
threonine phosphorylation → adaptor protein →
cell division and differentiation
• 3. ligand-gated channels → change
membrane potential → ion influx →
change cytoplasmic enzyme activity
• 4. steroid hormone receptors function
as ligand-regulated transcription
factors
• 5. unique mechanisms: B- and T- cell
receptors
15.3 G protein-coupled
receptors and their second
messengers
• GPCRs or 7TM receptors
• Hundreds of different GPCRs have been
identified from yeast to flowering plant
and mammals (C. elegans, 19,000 genes
encoded 1000+ different GPCRs)
Heterotrimeric G proteins:
•
•
•
•
•
Gs, Gq, Gi, G12/13
Gs : adenyl cyclase
Gq : PLCβ
Gi : inhibit adenyl cyclase
G12/13 : GTPase-activating protein
for Ras, BTK (protein tyrosin
kinase), Src, Phospholipase D, PKC
Bacterial toxins
• Cholera toxin exerts its effect by modifying
Gαsubunits and inhibit GTPase activity in
the cells
• Adenylyl cyclase molecules remain in an
activated mode
• Increase cAMP
• Secrete large volumes of fluid
• B. pertussis (whooping cough)
• Inactivating Gα subunits
• Interring with the signaling pathway that
leads the lost of the immune response
Second messengers
• Cyclic AMP
• Ca2+,
• Inositol 1,4,5-triphosphate (IP3),
diacylglycerol (DAG),
• cGMP
• NO
The discovery of a second messenger:
cyclic AMP
• In mid-1950s, Earl Sutherland and his colleagues at
Case Western Reserve and E. Krebs and E. Fisher at
UW
• Develop a vitro system to study the physiological
responses to hormones
• They found glucagon or epinephrin can induce the
activation of glycogen phosphorylase ( in
suspension) in broken cells.
• The particulate material was required to obtain the
hormone response.
• Particulate material on the membrane is cAMP
Lipid-derived second messengers
• Phospholipases C (PLC, lipid-splitting
enzymes):
hydrolyze phosphatidylinositol - derived
second messengers (best studied)
Signals by G protein-coupled receptors and RTKs
2. Phospholipid kinases (lipid-phosphorylating
enzymes)
Phosphatidylinositol-derived
second messengers
• Effects of acetycholine on RNA synthesis in the
pancreas
• P32 orthophosphate → nucleoside triphosphates
→ RNA synthesis
• Surprise! They found radioactivity into the
phospholipid fraction
• P32 incorporated into PI (phosphotidyl inosital)
and then to phosphoinositides
• Inosital-containing lipids can be phosphorylated
by specific lipid kinases after stimulation.
DAG→PKC (activator)
Phobol esters, that resemble DAG
• Analogs can activate PKC in culture and
cause cell to divide
• Genetic engineering to continue express
PKC, exhibit the tumor cells properties
The specificity of G protein-coupled
responses
• Hormones, neurotransmitters, sensory stimuli →
cellular responses
• 9 different isoforms of adrenergic receptor
15 different isoforms of the receptors for
serotonin
• Different receptor isoforms can have different
affinities for the ligand or may interact with
different types of G proteins or the effectors
• At least 20 different Gαsubunits, 5 different Gβ
subunits, 11 Gγsubunits, 9 isoforms of the
adenylyl cyclase
• Some Gαs, some Gαi
For examples:
• When epinephrine binds to a β-adrenergic receptor
on a cardiac muscle cell, a G protein with a Gαs
subunit is activated → contraction
• When epinephrine binds to an α-adrenergic
receptor on a smooth muscle cell in the intestine,
Gαi is activated → muscle relaxation
• Clearly, the same extracellular messenger can
activate a variety of pathways in different cells.
Regulation of blood glucose levels
• Glucose stored as glycogen
• Glucagon and epinephrine can stimulate
the breakdown the glycogen
• Glucagon in response to low conc. of
blood glucose is produced by α cells of the
pancreas
• Epinephrine “fight or flight” hormone, is
produced by the adrenal gland in stressful
situations
• Glucagon and epinephrin recognized by
different receptors induce the same
response in a single target.
HOW?
1.The receptor for glucagon is a G
protein-coupled receptor
•
•
•
•
Glucagon: 29 aa
Epinephrine: tyrosine derivatives
Receptors are different.
Following activating by their respective
ligands, both receptors activate the same
type of heterotrimeric G proteins that
cause an increase of cAMP
2. Glucose mobilization: an example of a
response induced by cAMP (PKA
pathway)
15.4 protein-tyrosin
phosphorylation as a mechanism
for signal transduction
• Regulation of growth, division,
differentiation, survival, attachment to the
ECM, migration
•
•
•
Src-homology 2 (SH2) domain: More than 110
SH2 domains are encoded by the human
genome.
They mediate a large number phosphorylationdependent protein-protein interaction
Phosphotyrosine-binding (PTB) domain. They
can bind to phosphorylated tyrosine as asnpro-x-tyr motif.
Activation of downstream signaling
pathways
• A variety signaling proteins with SH2, SH3 or
PTB domains are present in the cytoplasm
• Adaptor proteins: Grb2 (one SH2 and two SH3
domains)
(a). SH3 domains of Grb2 bind to Sos and Gab
(b). SH2 domains bind to phosphorylated tyrosine within
a Tyr-X-Asn motif
(c). Tyrosine –P in the motif results in translocation of
Grb2-Sos and Grb2-Gab to receptor at the plasma
membrane
• Docking proteins: IRS containing either a PTB
domain or an SH2 domain and a number of tyrosine
phosphorylation sites
• Transcription factors: belong to STAT
family-immune system
• Signaling enzymes: PK, protein
phosphatase, GTPase activating proteins--change conformations, close to substrate
Ending the response
• Internalization the receptors
• Receptor binding protein Cbl
• Cbl bind to receptor and brings along an
enzyme capable of attach a ubiquitine
molecule to the receptor
The Ras (small G protein) and MAP
kinase pathway
• Retrovirus (RNA virus) containing genes,
called oncogens (Ras), that enable them
to transform normal cells into tumor cells.
• Ras originally a retroviral oncogene, but
later found that it was derived from their
mammalian host.
• 30+ human cancers contain mutant
versions of Ras genes.
• Ras, a small GTPase, is held at the inner
surface of the plasma membrane by a lipid
group
• Ras superfamily contain numerous subfamily of
monomeric G proteins including Ras, rab, Arf,
ran, Rho.
• Ras proteins present in two different forms:GTPbound form and GDP-bound form.
• Ras mutation, GTP-bound form can not
transform to GDP-bound form → proliferation
→ tumor
G protein cycle and MAP kinase
cascade
• 1. GTPase-activating proteins (GAPs): dramatically
shorten the duration of a G protein-mediated response.
Mutation in one of the Ras-GAP genes (NF1) cause
neurofibromatosis
• 2. Guanine nucleotide-exchange factors (GEFs): bind to
GDP-protein and change it to GTP-protein
• 3. Guanine nucleotide-dissociation inhibitors (GDIs):
inhibit the release of a bound GDP from a monomeric G
protein, thus maintaining the protein in the inactive,
GDP-bound state.
Signaling by the insulin receptor
• Blood glucose: maintaining within a narrow
range, a decrease in blood glucose cause
loss of consciousness and coma.
• An increase in blood glucose cause loss of
fluid and electrolytes in urine.
• Blood glucose was monitored by pancreas
• After a meal → glucose increase → β cells
secreting insulin → cell express insulin
receptors in liver → increase glucose
uptake → increase glycogen and
triglyceridessynthesis and decreasing
gluconeogenesis
1. The insulin receptor is a proteintyrosin kinase
• Insulin receptor is composed of an α and a
βchain
• Insulin → dimer receptor → disulfide bond
→ tetramer
2. Insulin receptor substrate 1 and
2 (IRS-1, IRS-2)
• Usually, RTKs autophosphorylation → recruit SH2
domain-containing signaling proteins (Fig. 15
a,c,d).
• But insulin receptor autophosphorylation →
insulin-receptor substrates (IRSs: small docking
protein).
• After stimulation, PH domain binds to
phosphoinositides, PTB (phosphotyrosine
binding) domain) binds to SH2 domains and
other signaling prtoeins.
A diversity of signaling proteins
The role of tyrosine-phosphorylated IRS in activating
a variety of signaling pathways
Bind to
membrane
Bind to
receptor
IRS polypeptide. Y: tyrosine residue may be phosphorylated after
stimulation. These tyrosine-P can serve as binding sites for other
proteins, including a lipid kinase (PI3K), an adaptor protein
(Grb2), and a protein-tyrosine phosphotase (Shp2).
2 SH2 domain
1 catalytic
domain
Liver cell
Close to PKB and
phosphorylated
PKB)
(glycogen synthase kinase-3 inactivated)
Diabetes Mellitus
•
•
•
•
Defects in insulin signaling
Type 1: 5-10% : inability to produce insulin
Type 2: 90-95%: complex (living habits)
A high-calorie diet cause a chronic increase in
insulin secretion → over-stimulate target cells in
liver and elsewhere in the body → insulin
resistance (these target cells stop responding to
the presence of the hormone)
• Elevated blood sugars stimulates additional
secretion of insulin from the pancreas →
destruction of the β cells of pancreas
Drugs: make cells more insulin
sensitive
• PTP-1B is a phosphatase cause insulin
receptor inactive
• If lacking PTP-1B enhance insulin
sensitivity
• Drugs development on insulin receptor
sensitivity
Signaling pathway in plants
• Plants contain a type of PK that is absent
from animal cells.
• Histidine phosphorylation found in bacteria
and then in plant and yeast.
• Etr1 gene → receptor for C2H4 → MAP
kinase → cascade a diverse development
processes i.e. seed germination, flowering,
and fruit ripening
The role of calcium as an
intracellular messenger
•
•
•
•
•
•
Muscle contraction
Cell division
Secretion
Fertilization
Synaptic transmission
Metabolism, transcription, and cell
movement
• Ca2+ in cytosol (10-7 M)
• GPCR → PLCβ → PIP2 → IP3 → open Ca2+
channel in ER membrane → raise cytosolic
Ca2+ concentration
• RTKs → PLCγ (posses an SH2 domain)
→IP3
• A nerve impulse → depolymerization of the
PM →Voltage-gated channel open → influx
of Ca2+
Calcium can affect a number of different
cellular effectors
• 1. activate and inhibit various enzymes,
membrane fusion, alter cytoskeleton,
structure and function.
• 2. need calcium binding proteins (best
known: calmodulin, others: synaptotagminfusion in nerve cell, troponin C)
15.6 convergence, divergence, and
crosstalk among different
signaling pathways
• Signals from unrelated receptors, binding to its
own ligands, can converge to activate a common
effector, Ras or Raf.
• Signals from the same ligand, such as EGF or
insulin can diverge to activate different effectors,
leading to diverse cellular response.
• Signals can be passed back and forth between
different pathways, a phenomenon known as
“crosstalk”.
15.7 The role of NO as an
intercellular messenger
– No is formed from L-arginine catalyzed by
nitric oxide synthase (NOs).
– It has been known that acetylcholine acts
to relax the smooth muscle cells of blood
vessel, but the response could not
duplicated in vitro.
– Shift from aortic strips to rings and
realized the relaxation response---EC
Nitroglycerine :treat the pain of angina that
results from a inadequate flow of blood to
the heart (1860s)
• Nitroglycerine → NO → relaxation of the
smooth muscle cells → increasing blood
flow to the organ
• Viagra: inhibitor of cGMP
phosphodiesterase → maintain elevated
levels of cGMP →relaxation.
Apoptosis (programmed cell death)
•
•
•
•
•
A neat orderly process
Shrinkage in volume of the cell and its nucleus
The loss of adhesion to neighboring cells
The formation of blebs at the cell surface
The dissection of the chromatin into small
fragments
• The rapid engulfment of the “corpse” by
phagocytosis
• During development, those nerves that fail to
find their way to the target tissue do not receive
the survival signal and ultimately eliminated by
apoptosis.
• T-lymphocytes apoptosis during development.
• Irreparable genomic damages
• Neurodegenerative diseases: elimination of
neurons during disease progression → loss of
memory or decrease in motor coordination.
• 1972, C. elegans: during development,
131 cells out of 1090 cells are destined to
die by apoptosis
• 2002, Robert Horvitz et al. CED-3 gene
mutation → without losing any of their cells
to apoptosis
• Homologous family of proteins in
mammals→ caspases
• Caspases: cysteine protease
Target of caspases
• 1. more than a dozen of PKs: FAK, PKB, PKC,
and Raf1
• 2. lamins
• 3. Proteins of the cytoskeleton → change in cell
shape
• 4. an endonuclease called caspase activated
DNase (CAD): once activated, CAD translocate
from the cytoplasm to the nucleus where it
attachs DNA, severing it into fragments
1. Extrinsic (receptor-mediated)
pathway of apoptosis
• External stimuli: removal of growth factor
in the medium
• Deprived of the male sex hormone
testosterone → epithelial cells of the
prostate become apoptotic.
• Prostate cancer is often treated with drugs
that interfere with testosterone production.
2. The intrinsic pathway of
apoptosis (mitochondria-induced)
• Genetic damage, extremely high conc. of
Ca2+, severe oxidative stress, lack of
survival signals.