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INTRACELLULAR SIGNAL TRANSDUCTION:
A Journey from the Plasma Membrane to the Nucleus
(with interesting stops along the way)
Paula Tracy, Ph.D.
Given C409
[email protected]
INTRACELLULAR SIGNALING:
Signal Transduction
Cell membranes, as well as the cell cytoplasm and even the cell nucleus, contain cell-specific
receptors for various ligands, which are involved in outside-inside signaling,
i.e. signal transduction.
Ligands include hormones, growth factors, cytokines, prostaglandins and proteases.
Hormones are involved in a variety of metabolic processes that maintain homeostasis
e.g. fuel metabolism. Particularly noteworthy in that regard are glucagon, insulin and
the catecholamines (epinephrine and norepinephrine).
Growth factors are involved in mitogenesis, whereas cytokines play critical roles in the
differentiation, proliferation and function of various cell lineages..
Interaction of such ligands with their membrane, cell-specific receptors or intracellular
receptors causes conformational changes in the receptor and, in many instances receptorassociated cytoplasmic proteins.
Such events result in the initiation of a cascade of important, but as yet incompletely
understood, events leading to e.g. enzyme activation, differentiation and/or cell division.
General Schemes of Intercellular Signalling
Extracellular signaling
molecules released by
cells occurs over distances
from a few microns - autocrine (c)
and paracrine (b) signaling to
several meters in endocrine (a)
signaling. In some instances,
receptor proteins attached to the
membrane of one cell interact
directly with receptors on an
adjacent cell (d).
© 2000 by W. H. Freeman and Company. All rights reserved.
FORCES DRIVING SELECTION OF CURRENT MECHANISMS
Need for coordinated intercellular communication
Need to translate extracellular signals into series of intracellular events,
while allowing for specificity
Specificity Determinants:
1. Specific receptors on or in the target cells recognize an appropriate ligand.
2. Specific response to receptor occupancy - effector pathways
Diversity of intercellular communication is achieved with hundreds of
signaling molecules, including...
Proteins, small peptides, amino acids, nucleotides, steroids,
fatty acid derivatives, and even dissolved gases such as NO and CO
Intracellular receptors:
Receptor Classes
- signaling molecules include
steroid hormones, retinoids,
thyroxine, etc
- receptor-hormone complex
acts a transcription factor
to alter transcription of certain
genes
Cell surface receptors:
- signaling molecules include
peptide hormones, catecholamines, insulin, growth
factors, cytokines, etc
-binding, and subsequent events,
triggers an  or  in the
cytosolic concentration of
a second messenger; or the
activated,bound receptor acts
as a scaffold to recruit and
activate other intracellular
proteins
© 2000 by W. H. Freeman and Company. All rights reserved.
ADVANTAGES
1. Each cell is programmed to respond to specific combinations of signaling molecules.
2. Different cells can respond differently to the same chemical signal.
Molecular Biology of the Cell, 2002
HORMONES - First class of signaling molecules defined
Secreted from endocrine cells - specialized signaling cells that
control the behavior of an organism as a whole:
1. Differ from other intracellular mediators
2. Usually stimulate metabolic activities in tissues remote from the secretory organ
3. Active at very low concentrations (pM - M)
4. Response to hormonal signal comes as a direct and rapid result of its secretion
5. Metabolized rapidly so effects are, in most instances, short-lived, leading to
rapid adaptations to metabolic changes
Categories:
1. Peptides or polypeptides - insulin, glucagon, growth hormone, insulin-like
growth factors, vasopressin, prolactin….
2. Glycoproteins - follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH)…
3. Steroids - glucocorticoids (aldosterone, cortisol), steroids (progesterone,
testosterone), retinoic acid…
4. Amino acid derivatives - epinephrine, norepinephrine, thyroxine, triidothyronine
AGONISTS vs. ANTAGONISTS
Agonist
mimics a hormone in binding productively to a receptor
Antagonist
mimics a hormone stereochemically, but binds to the receptor
non-productively, inhibiting the action of the natural hormone
Agonist
e.g. important therapy
in asthma
Hormone
binds 2 receptor in lung 
bronchial relaxation
binds 2 receptor in heart muscle 
increased heart rate
Antagonist
control heart beat
RECEPTOR CHARACTERISTICS
1. Participates in transduction of the signal from the external messenger to
some component of the metabolic machinery
2. Has at least one additional functional site which is altered by ligand binding
(allosteric site)
3. Ligand binding to receptors is saturable, resembling Michaelis-Menten kinetics
© 2000 by W. H. Freeman and Company. All rights reserved.
4. Ligand-receptor interaction characterized by tight binding (Kd = pM - M)
How to calculate a Kd and number of receptors
from direct binding data
Derivation of a Scatchard Plot
Direct Ligand Binding Plot and the Derived Scatchard Plot
Molecular Biology of the Cell, 2002
Simple Intracellular Signaling Induced by an Extracellular Signaling Molecule
A signaling molecule activates its receptor  activation of an intracellular signaling pathway, i.e. a series of signaling
proteins, which may interact with a target protein to change the behavior of the cell.
Glucagon
Epinephrine
Thrombin
Insulin
Growth factors
Molecular Biology of the Cell, 2002
THREE LARGEST CLASSES OF CELL SURFACE RECEPTORS
GPCR Modeled after Rhodopsin
Intracellular Signaling Mediated by G protein-linked Membrane Receptors
e.g. Glucagon, Epinephrine and Thrombin as signaling molecules
1. Activates a chain of events  alterations in concentrations of signaling molecules; elaborate sets of
interacting molecules that can relay signals from cell surface to the nucleus
2. Components: Receptor; Transducer (G protein): Effector (membrane-bound enzyme);
Second messenger (e.g. cAMP); Response (cascade of highly-regulated protein phosphorylations, etc)
RECEPTOR:
typically a seven transmembrane
domain, integral membrane protein
Ligand binding domain
(extracellular site)
-adrenergic receptor
All G protein-coupled receptors
resemble the -adrenergic receptor
in their amino acid sequence and membrane
topography.
Gs binding site
(intracellular site)
Signal Transduction Pathway  Activation of Adenylate Cyclase by Gs
Receptor: 7 TM domain integral membrane protein
Transducer: Gs
Effector: adenylate cyclase
Second messenger: cAMP
Cycle of G protein dissociation/association:
Activation of G proteins involves GTP
displacement of GDP bound to the  subunit
and dissociation of the complex from the  subunits,
an exchange facilitated by a GEF protein(guanine
nucleotide exchange factor) specific for Gs.
G protein activation
The active  subunit binds to and activates/
stimulates membrane-associated adenylate cyclase
which catalyzes the conversion of ATP to
cyclic AMP (cAMP).
The GTPase activity associated with the  subunit
slowly hydrolyzes the bound GTP to GDP which is
facilitated by a GAP (GTPase activating protein)
specific for Gs.
GTP hydrolysis
The  subunit with GDP bound reassociates
with the  subunits, and membrane associated
adenylate cyclase returns to its basal activity level
Adenylate cyclase
activation
Characteristics of G proteins
1. G protein is an  trimeric protein which binds guanine nucleotides.
2. They function to couple integral membrane receptors to target
membrane-bound enzymes.
3. They can be considered molecular switches wherein…
GDP (inactive)  GTP (active) + 
4. The dissociated  subunit expresses GTPase activity.
5. GTPS blocks GTPase
activity of GTP.
i with
GDP bound
Region that changes
conformation when
GTP is hydrolyzed
Signal Transduction Pathway  Activation of Adenylate Cyclase by Gs
Receptor: 7 TM domain integral membrane protein
Transducer: Gs
Effector: adenylate cyclase
Second messenger: cAMP
Response: PKA regulated
protein phosphorylations
Molecular Biology of the Cell, 2002
Activation of cAMP-dependent protein kinase (PKA)
cAMP binds to the PKA regulatory subunits  conformational changes, which
causes their dissociation from the catalytic subunits  kinase activation. Release
of the catalytic subunits requires the binding of more than two cyclic AMP
molecules greatly sharpening the response of the kinase to changes in [cAMP].
PKA is a Ser/Thr kinase with discrete substrate specificity, thus facilitating a
cascade of highly regulated protein phosphorylations.
Signal Transduction Pathway  Activation of Adenylate Cyclase by Gs
Down stream effects:
1. Phosphorylation of
regulatory enzymes of
metabolic pathways.
2. Increase transcription
of certain proteins via
phosphorylation of CREB
leading to  protein synthesis of target proteins.
Activated PKA enters the
nucleus and phosphorylates
CREB (cAMP Response
Element Binding protein).
Once phosphorylated, CREB
recruits the coactivator CBP
(CREB Binding Protein).
This complex binds to the
CREB-binding element to
stimulate gene transcription.
Molecular Biology of the Cell, 2002
How Increased Intracellular cAMP Leads to Increased Gene Transcription.
Signal Transduction Pathway  Activation of Adenylate Cyclase by Gs
Early off signals:
1. Gs-associated GTPase
activity  GTP hydrolysis
to GDP  leading to reassociation of GsGDP
and dissociation of the
hormone/receptor complex
Down stream off signals:
1. cAMP hydrolyzed
by phosphodiesterase
2. Ser/Thr phosphatases
dephosphorylate the
phosphorylated target
proteins.
**
Desensitization or Endocytosis of GPCR’s
Effected by Phosphorylation
1. The ligand activated receptor can be phosphorylated on select Ser/Thr residues by
GRK (e.g. BARK -  adrenergic receptor kinase). These phosphorylated residues
provide a docking site for arrestin resulting in inactivation/desensitization.
2. In some instances, arrestin binding targets the receptor for clathrin-dependent
endocytosis.
3. In addition, if the occupied GPCR leads to  cAMP, the receptor can also be
phosphorylated by PKA leading to its inactivation/densensitization.
Clathrin-dependent Receptor-mediated endocytosis
Gs vs. Gi
Regulation of Adenylate Cyclase Activity
Gs stimulates adenylate cyclase
Gi inhibits adenylate cyclase
e.g. epinephrine can increase or decrease intracellular cAMP concentrations,
depending upon the receptor to which it binds
 adrenergic receptors couple to Gs, whereas
2 adrenergic receptors couple to Gi
Vasopressin binding to its GPCR activates Gs   cAMP and activation of PKA.
PKA phosphorylates various proteins  the ultimate aggregation of microtubular
subunits, which insert as water channels in the luminal plasma membrane to
increase the reabsorption of water by free diffusion.
Figure 21.38 – Devlin, Textbook of Biochemistry
Images of cAMP Transients in Cultured Aplysia Sensory Neurons.
The cell was loaded with a fluorophore that would allow for the quantification of
cAMP concentrations within the cell.
A: Free cAMP in the resting cell is < 5 X 10-8 M.
B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplasmic
cAMP to ~ 1 X 10-6 M (red), especially within fine processes with a high
surface to volume ratio. Thurs, within 20 sec of stimulation, the intracellular
[cAMP] increased ~ 20-fold.
Inhibition of Gs and Gi by Bacterial Toxins
Cholera toxin effects on Gs:
ADP ribosylation of an Arg residue
in the s subunit of Gs inhibition of
associated GTPase activity
Pertussis toxin effects on Gi:
ADP ribosylation of a Cys residue
in the i subunit of Gi  an inability
to inhibit adenylate cyclase activity.
Thus, both toxins cause increased
intracellular cAMP concentrations!
© 2000 by W. H. Freeman and Company. All rights reserved.
Gs vs Gi vs Gq
Gs and Gi coupled to adenylate cyclase   [cAMP]
G q coupled to phospholipase C   [Ca2+]
INTRACELLULAR Ca2+ AS A “SECOND” MESSENGER
Cells must work very hard to maintain low intracellular [Ca2+]
in order for Ca2+ to result in effective intracellular signaling.
**
Molecular Biology of the Cell, 2002
Cellular mechanisms that maintain very low intracellular Ca2+ concentrations
A: Ca2+ is actively pumped out of the cytosol.
B: Ca2+ is pumped into the ER and mitochondria.
Ligand Binding to a GPCR Linked to Gq  Phospholipase C Activation
Phospholipase C (PLC)-catalyzed hydrolysis of PIP2 Formation of 2 second messengers
**
**
Phospholipase C (PLC)-catalyzed hydrolysis of PIP2 Formation of 2 second messengers
IP3  release of Ca2+ from intracellular stores by binding to an IP3-gated Ca2+ channel
in the endoplasmic reticulum.
DAG with released Ca2+ and membrane-associated phosphatidylserine activates Protein
Kinase C (PKC - a Ser/Thr kinase). PKC directly phosphorylates intracellular proteins
some of which  gene transcription.
Phosphoinositide-activated Second Messenger System 
 Intracellular [Ca2+]
Ca2+ “Second” Messenger System
Release from intracellular stores subsequent to PLC-catalyzed hydrolysis of PIP2
1. Must be distinguished from cAMP-induced effects where cAMP activates a variety of
Ca2+ channels   Ca2+ influx from extracellular milieu (e.g. -adrenergic receptor occupancy
in muscle cells   Ca2+ influx   rate and force of heart beat).
2. Increased intracellular [Ca2+] - Third messenger: Immediate vs. Sustained responses
- Ca2+ binds to its ubiquitous intracellular receptor, calmodulin, thereby a) activating
Ca2+/calmodulin-dependent kinases (CaM kinases), Ser/Thr kinases b) that have their own
sets of substrate proteins, some of which when phosphorylated can  or  gene transcription.
Increased Intracellular Ca2+: A Third Messenger
Calmodulin in a ubiquitous intracellular Ca2+ receptor
Ca2+/calmodulin (CaM) complexes activate CaM-dependent kinases
(e.g. CaM-kinase II)
Molecular Biology of the Cell, 2002
Structure of Ca2+/calmodulin complex based on
X-ray diffraction and NMR spectroscopy studies
Molecular Biology of the Cell, 2002
Ca2+ Second Messenger System
Release from intracellular stores subsequent to PLC-catalyzed hydrolysis of PIP2
1. Must be distinguished from cAMP-induced effects where cAMP activates a variety of
Ca2+ channels   Ca2+ influx from extracellular milieu (e.g. -adrenergic receptor occupancy
in muscle cells   Ca2+ influx   rate and force of heart beat).
2. Increased intracellular [Ca2+] - Third messenger: Immediate vs. Sustained responses
- Ca2+ binds to its ubiquitous intracellular receptor thereby a) activating Ca2+/calmodulindependent kinases (CaM kinases), Ser/Thr kinases b) that have their own sets of substrate
proteins, some of which when phosphorylated can  or  gene transcription.
- Ca2+ works in concert with DAG and PS to stimulate PKC, a Ser/Thr kinase, that like the
CaM kinases has multiple substrates.
Protein Kinase C Activation of Gene Transcription
Two Independent Pathways
Molecular Biology of the Cell, 2002
Down-regulation of Phosphoinositide-activated
Second Messenger System
Off-signals:
1. IP3 rapidly dephosphorylated by phosphatases.
2. DAG rapidly hydrolyzed.
3. Ca2+ rapidly pumped out.
4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.