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

BC368Biochemistry of the Cell II

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

Amplification

FeedbackRegulation

Types of signals

Intercellular signaling

Intercellular signaling can occur over long distances (endocrine) or short distances.

hormone

neurotransmitter some growth factors

some cytokines

Properties of signal transduction

Fig. 12-1

Properties of signal transduction

Fig. 12-1

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.



Agonists bind to the receptor and mimic the effects of the normal signal.



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

Thyroidhormones

Nuclear receptors

Hormone-receptor complex acts in the nucleus to affect gene expression.

Nonpolar signal molecules can pass through the plasma membrane.

Receptor can be in cytosol or nucleus.

Nuclear receptors

Females who produce excess testosterone can have hyperandrogenism.

Problems with steroid signaling

Caster Semenya2009 World 800-Meter

Champion

Individuals who have a defective testosterone receptor have androgen insensitivity.

Jazz singer Eden AtwoodX,Y Genotype

Their use in sports was pioneered by East Germany, which had a systematic governmental doping program from 1965 to 1990.

Anabolic steroids are testosterone agonists used to build skeletal muscle and stimulate bone growth.

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 SlupianekShot Put World Record Holder

1980 –1984

Anabolic Steroids


✓

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G-protein coupled receptors

GS-protein pathway: β-adrenergic receptor

Animation

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.

GS-protein activation

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

Overview

G-protein summary

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.

Toxins that Target G proteins

•Vibrio cholerae produces the cholera toxin, which ADP-ribosylates GSα, inhibiting GTPase activity.

Box 12-2

Net result: cAMP is high

Toxins that Target G proteins

•Bordetella pertussis produces the pertussis toxin, which ADP-ribosylates GIα, inhibiting nucleotide exchange.

GI

GI

Pertussistoxin

GI is kept off, so AC is on and cAMP levels increase


Drugs that Enhance cAMP

•Caffeine and theophylline inhibit cAMP phosphodiesterase.

cAMPphosphodiesterase


Termination of Response

Removal of signal

Desensitization of receptor

Hydrolysis of GTP (promoted by GTPase activator proteins [GAPS])

Degradation of 2nd messenger

Hydrolysis of phosphates

Pathway can be terminated at any step!

Mechanisms of DesensitizationFig. 12-8

Strategy #1: Receptor Level

Strategy #2: Downstream Effects

Morphine receptor works through GI.

Body responds to morphine by increasing AC and PKA expression.

Mechanisms of Desensitization

Gq activates phospholipase C

DAG and IP3

DAGIP3

DAG and IP3

Gq acts through DAG and IP3

~Fig. 12-10

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

“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. MillardChemistry Honors Thesis

Amherst College, 1984

Ca2+ as Second Messenger

~Fig. 12-11 Ca2+ as Second Messenger

Calmodulin Animation

Receptor Tyrosine kinases

Receptors for many growth factors, cytokines, and hormones.

Binding of signal initiates a kinase cascade, beginning with autophosphorylation.


Activated receptor is a dimer.

Monomer that dimerizes (e.g., epidermal growth factor receptor)

Starts out as dimer (e.g., insulin receptor)


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.

This pathway is an excellent example of signal amplication.


Growth Factor Signaling


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).

•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

ERKAnimation


•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-factor- receptor-binding protein 2, and SRF, serum response factor.

Regulation of GTP-Switch Proteins

Guanine nucleotide Exchange Factor

GTPase Activating ProteinRegulator of G-protein 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

RTK Example 2: Insulin ReceptorFig. 12-15

•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.

•One target is IRS-1.

RTK Example 2: Insulin ReceptorFig. 12-16

•Insulin signaling has many effects. Here’s another one.

Erythropoietin (EPO) is a peptide hormone that stimulates red blood cell production in the bone marrow.

RTK Example 3: EPO Receptor

An estimated 70% of professional cyclists in Europe used EPO in the mid 1990s.

EPO receptor has no intrinsic protein kinase activity, but recruits a tyrosine kinase (JAK).


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.


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.

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:Single- transmembrane pass receptorCytosolic 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.

signaling chapter 12, pp. 433-460, 471-472 february 19, 2015 bc368 biochemistry of the cell ii

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