11/15/2013

M. Pareja
To be or not to be, that is the question
Hamlet, from Shakespeares Hamlet

survival depends on an elaborate intercellular

communication network that coordinates
growth, differentiation and metabolism
cells adjacent to one another frequently
communicate through cell-cell contact
other forms of communication cover larger
distances = extracellular signaling molecules
Cells are continuously responding to signals from the endogenous and exogenous
environment.

-extracellular signaling can occur over:

1. large distances or endocrine signaling signaling molecules are called
hormones
- act on target cells distant from their site of synthesis
-usually carried through the bloodstream
2. short distances or paracrine signaling affects target cells within proximity
to the cell that synthesized the molecule
-usually mediated by neurotransmitters and some growth
factors

-extracellular signaling can occur over:

3. no distance or autocrine signaling the signal feeds-back and affects
itself
-action of many growth factors
-these compounds generally act on themselves to
regulate proliferation
-seen frequently in tumor cells
-many compounds can act through two or even three types of cell signaling
e.g. growth factors (e.g. EGF) paracrine and autocrine and endocrine
-epinephrine endocrine and paracrine

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signaling molecules are released by signaling cells

the signal is called the ligand
the ligand binds to its specific receptor on a target
cell
this ligand-receptor interaction induces a
conformational or shape-change in the receptor
produces a specific response - called the cellular
response
can include a vast array of compounds
Example: signaling by T cells in the immune system

e.g. small amino acid derivatives, small peptides, proteins

Generic Signal Transduction

(1) synthesis of the signaling molecule by the signaling cell

(2) release of the signaling molecule by the signaling cell
(3) transport of the signal to the target cell
(4) detection of the signal by a specific receptor protein
receptor-ligand specificity
(5) a change in cellular metabolism, function, or
development = cellular response
triggered by the receptor-ligand complex specific to the ligandreceptor complex

(6) removal of the signal, which usually terminates the

cellular response degradation of ligand

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Signal Transduction Process

a.k.a. Ligand/Primary messenger

Can be a hormone, neurotransmitter,
antigen, bacteria, lipid and other molecules
that can interact with a cell membrane-bound
or intracellular receptors.

Circulating hormones
act on distant targets
travel in blood
endocrine hormones

Local hormones
paracrine hormones &
autocrine hormones

two types
lipid soluble
water soluble

Steroids
-lipid-soluble hormones can easily

enter a cell by diffusing through the

plasma membrane
-PROBLEM: how do they travel in the
water-based blood??
-SOLUTION: they are carried by
carrier-proteins
-these hormones then enter their
target cell where they result in a
specific cellular effect or response

lipids derived from cholesterol in SER

different functional groups attached to
core of structure provide uniqueness
interact with specific intracellular
receptors (within the cell) to turn specific
genes on or off
effective for hours or days

Thyroid hormones

tyrosine ring plus attached iodines are

lipid-soluble
activate enzymes involved in the
catabolism of fats and glucose
help set our basal metabolic rate

Retinoids

vitamin A derivatives
have dramatic effects on proliferation
and differentiation plus cellular death
(i.e. apoptosis)

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Hormone diffuses
through phospholipid
bilayer & into cell
Binds to receptor
turning on/off specific
genes
New mRNA is formed
& directs synthesis of
new proteins
New protein alters
cells activity

Amino acid derivatives, small

peptides and protein hormones
modified amino acids or amino
acids put together
serotonin, melatonin, histamine,
epinephrine

larger peptide hormones

insulin and glucagon

Eicosanoids
derived from arachidonic acid
(fatty acid)
prostaglandins or leukotrienes
prostaglandins despite being
lipidphilic bind to cell surface
receptors

-water soluble hormones can easily travel within the blood

-PROBLEM: how do they enter a cell and result in a cellular response??
-SOLUTION: binding to specific cell-surface receptors
-this binding activates the receptor and results in a series of cellular events called
the second messenger system

Can not diffuse through plasma membrane

Hormone receptors are integral membrane
proteins
act as first messenger

Receptor protein activates G-protein in

membrane
G-protein activates adenylate cyclase to
convert ATP to cAMP in the cytosol
Cyclic AMP is the 2nd messenger
Activates kinases in the cytosol to speed
up/slow down physiological responses
Phosphodiesterase inactivates cAMP
quickly
Cell response is turned off unless new
hormone molecules arrive

Agonists and Antagonists

If a molecule binds a receptor but cannot activate
it, i.e., cannot generate a signal, it acts as an
antagonist. It competes with and blocks the
activity of other endogenous ligands.

Cell membrane-bound
Intracellular

If a molecule binds to a receptor and activates the

downstream signal transduction pathways, it acts
as an agonist.

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GPLRs are involved in a range of signaling pathways, including

light detection, odorant detection, and detection of certain
hormones and neurotransmitters
Many different mammalian cell-surface receptors including
GPLRs are coupled to a trimeric signal-transducing G protein
made of an alpha, beta and gamma subunit complex
Ligand binding activates the receptor, which activates the G
protein, which activates an effector enzyme to generate an
intracellular second messenger
e.g. adenylyl cyclase converts ATP to cAMP
depending on regulation at the effector enzyme this pathway
can be either activated or inhibited
by the type of G protein activated by the hormone-receptor
complex
Gs proteins result in stimulation of the effector enzyme
Gi proteins result in inhibition of the effector enzyme

adenylyl cyclase (AC)

3.

Ion channel
receptors
Structure:
Protein pores in the
plasma membrane

-ligand binding changes the confirmation of the receptor so that specific ions flow
through it
-the resultant ion movement alters the electric potential across the plasma
membrane
-found in high numbers on neuronal plasma membranes
e.g. ligand-gated channels for sodium and potassium
-also found on the plasma membrane of muscle cells
-binding of acetylcholine results in ion movement and
eventual contraction of muscle

-lack intrinsic catalytic activity

-binding of the ligand results in the formation of a receptor dimer (2 receptors)
-this dimer than activates a class of protein called tyrosine kinases
-this activation results in the phosphorylation of downstream targets by
these tyrosine kinases (stick phosphate groups onto tyrosines within
the target protein)
-receptors for cytokines such as XXXX, interferons (XXXXXXX)

2004-2005

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Signal
transduction
Cascade

-also called receptor tyrosine kinases OR ligand-triggered protein kinases

-similar to tyrosine-linked receptors - ligand binding results in formation of a dimer
-BUT: they differ from tyrosine-linked receptors intrinsic catalytic activity
-means that ligand binding activates it and the activated receptor acts as a
kinase
-recognize soluble or membrane bound peptide/protein hormones that act as growth
factors
e.g. NGF, PDGF, insulin
-binding of the ligand stimulates the receptors tyrosine kinase activity,
-results in phosphorylation of multiple amino acid residues within its target
such as serine and threonine residues
-this phosphorylation activates downstream targets
-its targets are generally other protein kinases which phosphorylate their
own downstream targets (other kinases??)

Intracellular receptors
Not all signal receptors are located on the plasma membrane. Some are proteins
located in the cytoplasm or nucleus of target cells.

The signal molecule must be able to pass through plasma membrane.
Examples:
~Nitric oxide (NO)
~Steroid (e.g., estradiol, progesterone, testosterone)
and thyroid hormones of animals).

Classes of membrane receptors

WHAT HAPPENS WHEN A LIGAND

BINDS TO A RECEPTOR?

Receiving the Signal: G-protein-linked Receptors (GPLRs)

GPLRs are an important and ubiquitous class of eukaryotic
receptors (>700 in humans)
The extracellular domain connects to the intracellular domain
through 7 transmembrane spans
The intracellular domain is coupled to a heterotrimeric Gprotein
The heterotrimeric g-protein is composed of 3 subunits: G ,
G , and G
When the G subunit is bound to GDP it is OFF; when it is
bound to GTP it is ON
When the extracellular domain binds to
the signal molecule, it causes a
conformational change relayed through
the transmembrane spans to the
intracellular domain
The conformational change relayed to
the intracellular domain causes the G
subunit to release GDP and bind to GTP
thereby activating both the G and
G /G subunits

produced by the activation of GPLRs and

RTKs

second messenger systems allow for amplification

of an extracellular signal
one epinephine molecule can bind one GPLR
this can result in the synthesis of multiple cyclic
adenosine monophosphate(cAMP) molecules
which can go on to activate and amplified number
of PKAs
a blood level as low as 10-10M epinephrine can
raise blood glucose levels by 50%

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NH2

cAMP
cAMP has a wide variety of effects depending on
the cell type and the downstream PKAs and other
kinases
in adipocytes, increased cAMP activates a PKA
that stimulates production of fatty acids
in ovarian cells another PKA will respond to cAMP
by increase estrogen synthesis
Hormone stimulation of Gs protein-coupled
receptors leads to activation of adenylyl cyclase
and synthesis of the second messenger cAMP
most commonly studied second messenger
cAMP and other second messengers activate
specific protein kinases (cAMP-dependent protein
kinases or PKAs)

N
H2

5' C

O
O

4'

H
P

1'

H
OH

3'

2'

O
O-

hormone
signal
outside
GPCR

plasma
membrane

GDP
GTP

GTP

GDP

OH + ATP

Protein

Transmitting the Signal: Protein Kinases

O + ADP

O
Pi

KINASES

ATP cAMP + PP i

Protein Kinase
Protein

cytosol

AC

It activates a class of proteins (enzymes)

called...............
Remember this when you go to med
school..............

H2O

Protein Phosphatase
A protein kinase transfers the terminal phosphate of
ATP to a hydroxyl group on a protein.
A protein phosphatase catalyzes removal of the Pi by
hydrolysis.

Activated receptors frequently transmit

signals through through intracellular
signaling proteins called kinases
Protein kinases are enzymes that add a
phosphate group from ATP onto a
substrate protein; this reaction is called
phosphorylation
Phosphorylation frequently serves to
activate the substrate of the kinase, but
can also target the substrate for
degradation

Kinase 1
P

Kinases are often themselves activated by

other kinases via phosphorylation and can
organize into phosphorylation cascades

Kinase 2
P

Kinase 3
Phosphorylation Cascade

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AND WHAT DO THESE KINASES DO?

The response of a particular cell to a signal

depends on the type of proteins it contains.

It activates effector proteins that

leads to cellular responses

Responding to the Signal: Effector Proteins

signal

The final step in cell signaling is activation of the

effector proteins
The effector proteins carry out the cellular response to
the signal

Effector Protein

Transcription factors are proteins that bind to specific

DNA sequences called promoters that are upstream of
the genes that are turned on
Promoters that are upstream of genes that are only
activated during specific cellular responses are called
response elements

Cytoskeletal Rearrangement

Effector protein

Effector proteins can also directly act on proteins that

regulate cell shape to induce changes in morphology by
rearranging the cytoskeleton
Other types of effector proteins directly regulate cell
growth by arresting the cell cycle or altering cellular
metabolism

activation of multiple kinases results in

a cascade of phosphorylation/
activation
-this cascade is frequently called a
signal-transduction cascade
-this cascade eventually leads to a
specific cellular response
e.g. changes in cell physiology
and/or patterns of gene
expression, cell proliferation and
differentiation, promotion of cell
survival, and modulation of cellular
metabolism

hormone
signal
outside
GPCR

plasma
membrane

GTP

GDP

GTP

AC

KINASE #1

p
KINASE #2

p
KINASE #3

TARGET

effect

Cell Cycle Arrest

Example: Protein Kinase A (PKA)

GDP

-the successive phosphorylation/

Changes in gene expression

Often the cellular response involves expression of

previously inactive genes which requires effector
proteins called transcriptional activators or transcription
factors

cytosol

Effects of PKA
Target Cell

Response

Skeletal muscle & liver cells

Breakdown of glycogen

cardiac

Strengthen heart contraction

Smooth muscle

Inhibition of contraction

Intestinal epithelium cells

Secretion of water and salts into the

lumen of the gut

ATP cAMP + PP i

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Biomedical
Importance

hormone
signal

Cholera
Toxin
Pertussis
Toxin

Secondary messengers
outside

GPCR

plasma
membrane

GDP

GTP

GTP GDP

AC

cytosol

ATP cAMP + PPi

other secondary messengers include:

IP3 and DAG breakdown products of phosphotidylinositol
(PIP2)
produced upon activation of multiple hormone receptor types
(GPLRs and RTKs)

calcium IP3 production results in the opening of calciumchannels on the plasma membrane of the ER release of
calcium
a rise in calcium in pancreatic beta cells triggers the exocytosis of
insulin
a rise in intracellular calcium also triggers contraction of muscle
cells
much study has been done on the binding of calcium to a protein
called calmodulin and the effect of this complex on gene
expression

O
O
R1

H2C
O

CH
H2C

R2

IP3

O
O

OH
2

Ca++

H
1

H
OH

OH
H

OH
5

OH

calmodulin

Ca++-release channel

phosphatidylinositol

Ca++

endoplasmic
reticulum

Ca++-ATPase
ATP
ADP + Pi
Ca++

Protein Kinase C (Secondary

messenger is DAG and IP3)
Regulated function

Target Tissue

Messenger

Platelet activation

Blood platelets

Thrombin

Muscle contraction

Smooth muscle

Acetylcholine

Insulin secretion

Pancreas, endocrine

Acetylcholine

Amylase secretion

Pancreas, exocrine

Acetylcholine

Glycogen degradation

Liver

ADH

Antibody production

B lymphocytes

Foreign antigens

OTHER PATHWAYS

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MAPK kinase paths

Stress, cytokines, hormones & mitogens
signal through cdc42/rac

cdc42/rac then activates one of three

MAPK paths:

Stress
Cytokines

p38MAPK: stress response &

apoptosis (MAPKAP-2, HSP27)

Stress
Hormones
Cytokines

Mitogens
Hormones

cdc42/rac

grb2

JNK: stress response & proliferation (jun)

MEKKs

Activation of ras/MEK/ERK path:

proliferation & differentiation
MAPK kinase

transcription
factors (nucleus)

MAPKAP-2
HSP27

ras

MEKKs

MEK 3/6

JNKK 1/2

p38MAPK

JNK 1/2

ATF

ATF

elk

jun

sos

raf-1

MEK 1/2

ERK 1/2
elk

elk

RSK

2004-2005

fos

Turn off of the signal:

1. G hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on G causes it to rebind
to the inhibitory complex.
Adenylate Cyclase is no longer activated.

2. Phosphodiesterases catalyze hydrolysis of

cAMP
AMP.
hormone
signal
outside
GPCR

plasma
membrane

GDP
GTP

GDP

GTP

cytosol

AC

ATP

cAMP + PP i

2004-2005

Methylxanthines
Caffeine
Theophylline (treatment of asthma)

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Target Cell

Response

Skeletal muscle & liver cells

Breakdown of glycogen

cardiac

Strengthen heart contraction

Smooth muscle

Inhibition of contraction

Intestinal epithelium cells

Secretion of water and salts into the

lumen of the gut

Receptor
desensitization

RTK ST- PI3K pathway

Overview of MAPK Signaling Pathways

P

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Therefore, Homeostatic mechanisms

of the human body are actually
molecular pathways

APPLICATION:
PHARMACODYNAMICS OF DRUGS

Any disruptions to the pathway

(physical, genetic, bacterial, viral etc)
can cause a disorder or disease

Drugs
Sildenafil citrate

Ca++
IP3

A drug can be an agonist (promoter) or antagonist

(inhibitor) of a homeostatic molecular pathway

calmodulin

Ca++-release channel
Ca++

endoplasmic
reticulum

Ca++-ATPase
ATP ++ ADP + Pi
Ca

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C-abl: Tyrosine Kinase activation by Translocation

Kinases:
Bcr-Abl

The prototypic TK oncogene is bcr/abl. The bcr/abl fusion

oncogene i s formed when the c-abl TK gene on
chromosome 9 is translocated to chromosome 22 and fused
with part of the bcr gene on that chromosome. The resulting
hybrid chromosome, the Philadelphia chromosome, encodes
a new protein called Bcr/Abl. New protein has increased
kinase
activity
and
drives
proliferation
causing
transformation. Charatceristic of CML a nd some forms of
ALL.

Cell-Cell and Cell-Matrix interactions can

activate signal transduction cascades

J Biomed Sci. 2006 Feb 23; Crosstalk between hepatocyte growth factor and integrin signaling pathways.
Chan PC, Chen SY, Chen CH, Chen HC

J Neurochem. 2006 Jan;96(1):148-59

Fibronectin promotes brain capillary endothelial cell survival and proliferation through alpha5beta1 and
alphavbeta3 integrins via MAP kinase signalling. Wang J, Milner R.

Immunopharmacol Immunotoxicol. 2005;27(3):371-93.

Interleukin-3, -5, and granulocyte macrophage colony-stimulating factor induce adhesion and
chemotaxis of human eosinophils via p38 mitogen-activated protein kinase and nuclear factor kappaB.
Ip WK, Wong CK, Wang CB, Tian YP, Lam CW.

Cell Commun Adhes. 2004 Sep-Dec;11(5-6):137-53.

ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on
collagen I and vitronectin. Salasznyk RM, Klees RF, Hughlock MK, Plopper GE.

Microsc Microanal. 2005 Jun;11(3):200-8.

Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart organogenesis:
implications for cardiac birth defects.Linask KK, Manisastry S, Han M.

Short quiz on lessons 1 and 2

Take home long quiz

In the schoolbook, you will be assigned a
particular drug. Illustrate & discuss the
molecular pathway (flowchart) of the drug.
(50pts)
To be given on Sunday (one week)

References
Alberts et al. Molecular Biology of the Cell, Chapter 15

Schoolbook (Friday to Monday)

20 points

Dohlman, H. and Thorner, J. Regulation of G-Protein

initiated signal transduction in yeast: paradigms and
principles. Annu. Rev. Biochem. 2001. 70:70354
Bao et al. Pheromone-dependent destruction of the Tec1
transcription factor is required for MAP kinase signaling
specficity in yeast. Cell. 2004. 119: 991
Schwartz and Madhani. Principles of MAP kinase signaling
specificity in Saccharomyces cerevisiae. Annu. Rev. Genet.
2004. 38: 725
Park, Zarrinpar and Lim. Rewiring MAP kinase pathways
using alternative scaffold assembly mechanisms. Science
2003. 299:1061

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