Transcript BC368 Biochemistry of the Cell II Signaling Chapter 12, pp. 433-460, 471-472 February 19, 2015

BC368
Biochemistry of the Cell II
Signaling
Chapter 12, pp. 433-460, 471-472
February 19, 2015
Signal transduction
Binding of acetylcholine to its receptor opens an ion
channel.
Signal = acetylcholine
Response= ions flow
Signal transduction
Binding of insulin to its receptor results in recruitment
of GLUT4 in certain cell types.
Signal = insulin
Response= increased
glucose transport
Signal transduction
Binding of insulin to its receptor results in recruitment
of GLUT4 in certain cell types.
Signal = insulin
Response= increased
glucose transport
Here’s why you
shouldn’t trust
everything on
the web!
Signal transduction
Feedback
Regulation
Amplification
Types of signals
Intercellular signaling
Intercellular signaling can occur over long distances
(endocrine) or short distances.
some cytokines
hormone
neurotransmitter
some growth factors
Fig. 12-1
Properties of signal
transduction
Fig. 12-1
Properties of signal
transduction
Fig. 12-1
Properties of signal
transduction
Fig. 12-1
Properties of signal
transduction
Fig. 12-1
Properties of signal
transduction
In other words, signaling
proteins can interact with
more than one target,
forming complexes with
different properties.
Types of chemical signals
Cells communicate with
each other by sending
out signaling molecules.
In order to respond to the
signal, target cell must
have a receptor.
Mechanism of signal
transduction depends on
the chemistry of the
transmitter.
Interfering with chemical signals
Some molecules interfere with the normal signaling
pathway.
Interfering with chemical signals
Some molecules interfere with the normal signaling
pathway.
Agonists bind to the
receptor and mimic the
effects of the normal
signal.
Interfering with chemical signals
Some molecules interfere with the normal signaling
pathway.
Agonists bind to the
receptor and mimic the
effects of the normal
signal.
Antagonists act as
competitive inhibitors of
the normal signal.
Types of signal transducers
✓
Nuclear receptors
Nonpolar signal
molecules can pass
through the plasma
membrane.
Steroid
hormones
Thyroid
hormones
Nuclear receptors
Nonpolar signal
molecules can pass
through the plasma
membrane.
Receptor can be in
cytosol or nucleus.
Hormone-receptor
complex acts in the
nucleus to affect
gene expression.
Nuclear receptors
Problems with steroid signaling
Females who produce
excess testosterone can
have hyperandrogenism.
Caster Semenya
2009 World 800-Meter Champion
Jazz singer
Eden Atwood
X,Y Genotype
Individuals who have a
defective testosterone
receptor have androgen
insensitivity.
Anabolic Steroids
 Anabolic steroids are
testosterone agonists
used to build skeletal
muscle and stimulate
bone growth.
 Their use in sports
was pioneered by
East Germany, which
had a systematic
governmental doping
program from 1965
to 1990.
Anabolic Steroids
At the first world swimming
championships, in 1973, East
German women won 10 of the 14
gold medals available, setting eight
world records.
Three years later at the Montreal
Summer Olympics, the East
German women won 11 of 13
events.
llona Slupianek
Shot Put World
Record Holder
1980 –1984
Types of signal transducers
✓
✓
G-protein coupled
receptors
GS-protein pathway:
β-adrenergic receptor
Animation
GS-protein activation
A “GTP Switch” protein
is active when GTP is
bound; inactive when
GDP is bound.
When α subunit binds
GTP, it separates from
β and γ.
Activated α subunit
finds AC and turns it on.
Intrinsic GTPase activity
turns α subunit off- it
finds β and γ.
Activation of AC
Activated AC makes
cAMP.
One target of cAMP
is protein kinase A
(PKA).
Activation of Protein Kinase A
PKA is active
only when 4
cAMP are bound,
freeing the two
catalytic
subunits.
Active PKA has
many effects,
depending on
cell type.
Activation of Protein Kinase A
cAMP: second messenger
G-protein summary
Overview
Inhibitory G proteins
Case Study
Peter T., a teacher aged 42, had just returned from Calcutta. Within 24 h of his
arrival, he had suddenly developed diarrhea. It was not associated with pain, and as
it was watery and voluminous, P.T. took plenty of tea to make up the body fluid. As
his condition worsened, with fluid losses up to 1 L/h and with vomiting and muscle
cramps, he called his doctor.
On examination, P.T. was apathetic, his cheeks were hollow, and his eyes sunken; no
peripheral pulse was palpable, he was cyanotic and in a state of profound shock. He
had tachycardia and his respiration was rapid and shallow. The diarrheal fluid was
colorless and turbid, like “rice water.”
An isotonic solution containing Na+, K+, Cl-, and HCO3- was infused intravenously at a
rate of 100 mL/min until a strong pulse was restored and thereafter in quantities
sufficient to maintain normal pulse, blood pressure, and skin turgor. When the
patient stopped vomiting, a solution of similar composition but containing 2%
glucose was given by mouth.
Microscopic examination of the diarrheal fluid established the presence of masses of
typical short, comma-shaped rods of Vibrio cholerae. A course of antibiotic therapy
was started.
After 24 h the fluid loss began to decline and the patient made a rapid and complete
recovery. He was estimated to have lost about 20 L of fluid containing 2.8 mol NaCl.
Box 12-2
Toxins that Target G proteins
•Vibrio cholerae produces the cholera
toxin, which ADP-ribosylates GSα,
inhibiting GTPase activity.
Net result:
cAMP is
high
Toxins that Target G proteins
•Bordetella pertussis produces the
pertussis toxin, which ADP-ribosylates GIα,
inhibiting nucleotide exchange.
GI
Pertussis
toxin
GI
GI is kept off, so
AC is on and
cAMP levels
increase
Net result:
cAMP is
high
Drugs that Enhance cAMP
•Caffeine and theophylline inhibit
cAMP phosphodiesterase.
cAMP
phosphodiesterase
Net result:
cAMP is
high
Termination of Response
Pathway can be terminated at any step!
Removal of signal
Desensitization of receptor
Hydrolysis of GTP (promoted
by GTPase activator proteins
[GAPS])
Degradation of 2nd messenger
Hydrolysis of phosphates
Fig. 12-8
Mechanisms of Desensitization
Strategy #1: Receptor Level
Mechanisms of Desensitization
Strategy #2: Downstream Effects
Morphine
receptor works
through GI.
Body responds
to morphine by
increasing AC
and PKA
expression.
Gq activates
phospholipase C
DAG and IP3
IP3
DAG
DAG and IP3
DAG and IP3
~Fig. 12-10
Gq acts through
DAG and IP3
DAG/IP3 as Second Messengers
http://www.youtube.com/watch?v=2bbBrpgeheY
Ca2+ as Second Messenger
Variable Ca 2+
increases
depending on the
amount of IP3
Ca2+ as Second Messenger
“Because of the calcium ion’s diverse roles in cell function, it has
plagued investigators from many biological disciplines for the last
30 years. Although its steady state concentration in the cytosol
ranges from only 0.01 – 0.1 μM, the calcium ion triggers such
diverse phenomena as lipid and glycogen degradation, the
release of neurotransmitters, muscle contraction, and cell division.
How does calcium perform its varied cellular functions when it
exists only in such minute quantities? It was first suggested in
1964 that the calcium ion itself is inactive; it must form a complex
with one of a homologous class of calcium-binding proteins.”
-Julie T. Millard
Chemistry Honors Thesis
Amherst College, 1984
~Fig. 12-11
Calmodulin Animation
Ca2+ as Second Messenger
Types of signal transducers
Receptor Tyrosine kinases
 Receptors for many growth factors, cytokines, and
hormones.
 Binding of signal initiates a kinase cascade, beginning
with autophosphorylation.
Receptor Tyrosine kinases
 Activated receptor is a dimer.
Monomer that dimerizes (e.g.,
epidermal growth factor receptor)
Starts out as dimer (e.g.,
insulin receptor)
Receptor Tyrosine kinases
 Ligand binding causes activation of the dimer (turns on
kinase activity).
 Autophosphorylation
results
Receptor Tyrosine Kinases
#1: Growth Factor Receptor
Epidermal growth factor stimulates cell
growth, proliferation, and differentiation
by binding to its receptor, which is an
RTK.
Receptor Tyrosine Kinases
#1: Growth Factor Receptor
 This pathway is an excellent example of signal
amplication.
Growth Factor Signaling
Receptor Tyrosine Kinases
#1: Growth Factor Receptor
Epidermal growth factor stimulates cell
growth, proliferation, and differentiation
by binding to its receptor.
Signal binding to the receptor leads to
a kinase cascade.
http://www.youtube.com/watch?v=OvvXgzf58MQ
Receptor Tyrosine Kinase #1:
Epidermal Growth Factor Receptor
 Ligand binding leads to
receptor dimerization,
autophosphorylation, and
recruitment of adapter
molecules:
GRB2
Sos
 Binding of adapter molecules
to receptor recruits and
activates Ras (GTP switch).
Receptor Tyrosine Kinase #1:
Epidermal Growth Factor Receptor
•Activated Ras recruits Raf.
•Binding to Ras activates Raf.
•Raf phosphorylates MEK.
•MEK phosphorylates MAP kinase (aka
ERK).
•MAP kinase/ERK phosphorylates its
targets.
Receptor Tyrosine Kinase (RTK)/
Ras GTPase/MAP kinase (MAPK)
signaling pathway
Raf
MEK
ERK
Animation
Receptor Tyrosine Kinase #1:
Epidermal Growth Factor Receptor
•Activated ERK has many
substrates in the cytosol [e.g.
cytoskeletal proteins,
phospholipase A2, signalling
proteins, and activation of
transcription proteins (STATs).
•ERK can also enter the nucleus to control gene expression by
phosphorylating transcription factors such as Elk-1, growth-factorreceptor-binding protein 2, and SRF, serum response factor.
Regulation of GTP-Switch Proteins
Guanine nucleotide
Exchange Factor
GTPase Activating
Protein
Regulator of Gprotein Signaling
Ras: a GTP-Switch Protein
https://www.youtube.com/watch?v=NL3ndoSzFo4
RTK Example 2: Insulin Receptor
•Insulin receptor is
already a dimer
•Insulin binding triggers
conformational change
and autophosphorylation
of Tyr residues in the
cytosolic region
•Receptor then binds and
phosphorylates target
proteins
Fig. 12-15
RTK Example 2: Insulin Receptor
•One target is IRS-1.
•Activated IRS-1 recruits Grb2.
•Grb2 binds Sos.
•Sos binds Ras, leading to
nucleotide exchange.
•Ras activates Raf-1.
•Raf-1 phosphorylates MEK.
•MEK phosphorylates ERK/
MAPK, which phosphorylates
transcription factors.
Fig. 12-16
RTK Example 2: Insulin Receptor
•Insulin signaling has many effects. Here’s another one.
RTK Example 3: EPO Receptor
Erythropoietin (EPO)
is a peptide hormone
that stimulates red
blood cell production
in the bone marrow.
An estimated 70% of
professional cyclists
in Europe used EPO
in the mid 1990s.
RTK Example 3: EPO Receptor
EPO receptor has no
intrinsic protein kinase
activity, but recruits a
tyrosine kinase (JAK).
RTK Example 3: EPO Receptor
EPO receptor has no
intrinsic protein kinase
activity, but recruits a
tyrosine kinase (JAK).
JAK phosphorylates
STAT, which dimerizes,
goes to nucleus, and
affects gene expression.
RTK Example 3: EPO Receptor
EPO receptor has no
intrinsic protein kinase
activity, but recruits a
tyrosine kinase (JAK).
JAK phosphorylates
STAT, which dimerizes,
goes to nucleus, and
affects gene expression.
JAK also binds Grb2,
initiating the MAPK
cascade.
Types of signal transducers
Guanylyl Cyclases
When activated,
these receptor
enzymes convert
GTP to the second
messenger cGMP.
Guanylyl Cyclases
When activated,
these receptor
enzymes convert
GTP to the second
messenger cGMP.
Two types:
Singletransmembrane
pass receptor
Cytosolic NO
receptor
Guanylyl Cyclases
The effects of cGMP are
often mediated by protein
kinase G (cGMP-dependent
protein kinase).
cGMP is degraded by a
specific phosphodiesterase.
Case Study
A 56-year-old man presents to the emergency department complaining of substernal
chest pain described as "something very heavy on my chest." The pain started while
carrying boxes up a flight of stairs. Originally, the pain was a 9 out of 10 but after rest
is now a 4 out of 10. The pain radiates to his left shoulder and is accompanied by
shortness of breath. The patient states that he's had similar but less severe pain in the
recent past with exertion. He wasn't going to come to the ED ("I told her it was just
indigestion”), but his wife, concerned about a heart attack, made him. He has
hypertension, hypercholesterolemia, and smokes a pack of cigarettes a day.
The ED nurse gives him nitroglycerine tablets, 0.4 mg sublingual (under his tongue),
every 5 minutes three times. She also gives him chewable aspirin (324 mg), oxygen by
nasal cannula, nitroglycerine paste on his skin, and obtains an EKG. The EKG
demonstrates changes consistent with ischemia (low oxygen). Ten minutes later when
the ED physician sees him, his pain is nearly gone at a 1 out of 10. Blood work,
including tests for cardiac enzymes, is obtained.
The results of the blood work are normal, but due to concerns of angina the patient is
admitted to the hospital. Further blood work rules out a myocardial infarction, and a
stress test reveals reversible cardiac ischemia. He is diagnosed with unstable angina
and is prescribed nitroglycerine to take as needed, a beta-blocker, daily aspirin, an
exercise regimen, and told to stop smoking. He is scheduled for a cardiac angiogram
and sent home.
Enhancing cGMP
NO
Vasodilation
Case Study
Researchers at Pfizer realized that a phosphodiesterase (PDE) inhibitor could
be a therapeutic option for treatment of angina. Screening of existing
compound collections resulted in several lead compounds that acted as
potent inhibitors of PDE-5, a cGMP-specific phosphodiesterase in coronary
smooth muscle. Further optimization lead to a single candidate, which
underwent clinical trials but proved to be ineffective for angina.
During these trials, several patients noted experiencing enhanced penile
erections. Subsequently, PDE-5 was identified as the main cGMP-degrading
enzyme in the corpus cavernosm. Researchers at Pfizer then reshifted their
focus towards developing a drug for erectile dysfunction. Sildenafil (Viagra)
was approved by the FDA in 1998. In 2013, Viagra generated $1.88 billion of
revenue.