Chap. 15 Signal Transduction

1
Chap. 15 Signal Transduction G Protein-coupled
Receptors

• Topics
• Signal Trans. From Extracellular Signal to
  Cellular Response
• Cell-Surface Receptors Signal Transduction
  Proteins
• G Protein-coupled Receptors (GPCRs) Structure
  and Mechanism
• GPCRs That Regulate Ion Channels
• GPCRs That Regulate Adenylyl Cyclase
• GPCRs That Regulate Cytosolic Calcium

• Goals
• Learn the general properties of signaling
  molecules (ligands), cell-surface receptors,
  intracellular signal transduction components.
• Learn the G protein cycle of reactions involved
  in GPCR signaling.
• Learn the rhodopsin signal trans pathway used in
  vision.
• Learn the epinephrine receptor signal trans
  pathway used for control of glycogen degradation.
• Learn about the GPCR-stimulated IP3/DAG signaling
  pathway.

2
General Principles of Signal Transduction
Signal transduction refers to the overall process
of converting extracellular signals into
intracellular responses (Fig. 15.1). Key players
in signal transduction are signaling molecules,
receptors, signal transduction proteins and
second messengers, and effector proteins. Cells
respond to signals by changing the activity of
existing enzymes (fast) and/or the levels of
expression of enzymes and cell components
(slower) by gene regulation (Steps 7a 7b).
Receptors and signal transduction systems have
evolved to detect and respond to hormones, growth
factors, neurotransmitters, pheromones,
oxygen, nutrients, light, touch, heat, etc. There
are an enormous number of signal molecules and
receptors in cells. In contrast, there are
relatively few types of intracellular signal
transduction systems.
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General Principles of Signal Transduction
In animals, signaling systems are classified
based on the distance over which they act (Fig.
15.2). Endocrine signaling acts over long
distances within the organism (e.g., insulin).
Paracrine signaling acts over very short
distances, for example between neighboring cells.
Neurotransmitters and developmental signals
typically act in this manner. In autocrine
signaling, cells release ligands that bind to
their own surface receptors, modulating activity.
Many growth factors act in this manner. Finally,
signaling systems involving plasma
membrane-attached proteins act via direct
cell-to-cell contact.
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Signal Transduction Components Receptors
Cell surface receptors bind to their ligands
(signaling molecules) via their extracellular
domains (Fig. 15.3). In all cases, binding causes
a conformational change in the receptor that
leads to the transmission of an intracellular
signal. Binding specificity and affinity are
determined by the extent of molecular
complementarity between the ligand and the
receptor. A given receptor may exhibit
specificity for a certain ligand or a group of
closely related (structurally) ligands. A given
ligand may bind to a number of different types of
receptors, that exhibit different effector
specificity (different cell responses). Further,
two receptors that bind different ligands, may
signal via the same intracellular signal
transduction system, even within a single cell.
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Signal Transduction Components
Kinases/Phosphatases
Proteins that participate in intracellular signal
transduction fall into two main classes--protein
kinases/phosphatases and GTPase switch proteins.
Kinases use ATP to phosphorylate amino acid
side-chains in target proteins. Kinases typically
are specific for tyrosine or serine/threonine
sites. Phosphatases hydrolyze phosphates off of
these residues. Kinases and phosphatases act
together to switch the function of a target
protein on or off (Fig. 15.4). There are about
600 kinases and 100 phosphatases encoded in the
human genome. Activation of many cell-surface
receptors leads directly or indirectly to changes
in kinase or phosphatase activity. Note that some
receptors are themselves kinases (e.g., the
insulin receptor).
6
Model for Kinase-mediated Signal Trans.
Fig. 15.5 illustrates a simple signal
transduction pathway involving one kinase bound
to a receptor and one predominant target protein.
A number of signaling systems discussed in the
course function via this general model.
7
Signal Trans. Components GTPase Switches
GTPase switch protein also play important roles
in intracellular signal transduction (Fig. 15.6).
GTPases are active when bound to GTP and inactive
when bound to GDP. The timeframe of activation
depends on the GTPase activity (the timer
function) of these proteins. Proteins known as
guanine nucleotide-exchange factors (GEFs)
promote exchange of GTP for GDP and activate
GTPases. Proteins known as GTPase-activating
proteins (GAPs), stimulate the rate of GTP
hydrolysis to GDP and inactivate
GTPases. We will cover two classes of GTPase
switch proteins--trimeric (large) G proteins, and
monomeric (small) G proteins. Trimeric G proteins
interact directly with receptors, whereas small G
proteins interact with receptors via adaptor
proteins and GEFs.
8
Signal Trans. Components 2nd Messengers
While there are a large number of extracellular
receptor ligands ("first messengers"), there are
relatively few small molecules used in
intracellular signal transduction ("second
messengers"). In fact, only 6 second messengers
occur in animal cells. These are cAMP, cGMP,
1,2-diacylglycerol (DAG), and inositol
1,4,5-trisphosphate (IP3) (Fig. 15.8), and
calcium and phosphoinositides (covered later).
The functions of cAMP, cGMP, DAG, and IP3 are
summarized in the figure. Second messengers are
small molecules that diffuse rapidly through the
cytoplasm to their protein targets. Another
advantage of second messengers is that they
facilitate amplification of an extracellular
signal.
9
Signal Amplification in Signaling Pathways
At each step of many signal transduction
pathways, the number of activated participants in
the pathway increases (Fig. 15.9). This is
referred to as signal amplification, and hormone
signaling pathways are often referred to as
amplification cascades. For example, one
epinephrine-activated GPCR activates 100s of
Gas-GTP complexes, which in turn activate 100s of
adenylyl cyclase molecules, that each produce
hundreds of cAMP molecules, and so on. The
overall amplification associated with epinephrine
signaling is estimated to be 108-fold.
10
Ligand Binding and Receptor Activation
The reversible kinetic equation for ligand (L)
binding to a receptor (R) is R L ? RL The
dissociation constant for this reaction is Kd
RL / RL.
When L Kd, the receptor is 50 saturated.
When L 10Kd, the receptor is 90 saturated
at L 0.1Kd, the receptor is 10 saturated.
Typically, the Kd for ligand binding is higher
than the basal concentration of ligand. This is
needed for cells to optimally respond to changing
ligand concentration. Interestingly, the level of
physiological response typically does not
strictly parallel binding (Fig. 15.12). Namely,
50 of full response often
occurs at only 10-20 receptor occupancy. The
number of receptors in a cell is very important
in setting the physiological response. A decrease
in receptor number reduces the response, and vice
versa. You are not responsible for the additional
mathematical treatment of ligand-receptor binding
covered in the text.
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Ligand Agonists Antagonists in Medicine
Synthetic analogs of receptor ligands are widely
used in medicine. Compounds called agonists mimic
the function of the natural ligand by binding to
the receptor and inducing the normal response.
Antagonists bind to the receptor but induce no
response. Instead, they typically block binding
and signaling by the natural ligand. Examples of
an epinephrine agonist (isoproterenol) and
antagonist (alprenolol) are shown in Fig. 15.11.
Isoproterenol binds to bronchial smooth muscle
cell epinephrine receptors with 10-fold higher
affinity than epinephrine, and is used to treat
asthma, etc. Alprenolol is a beta-blocker that
binds to cardiac muscle cell epinephrine
receptors, blocking epinephrine action and
slowing heart contractions. It therefore helps
treat cardiac arrhythmias and angina.
12
Structure of GPCRs
G protein-coupled receptors (GPCRs) are the most
numerous class of receptors in most eukaryotes.
Receptor activation by ligand binding activates
an associated trimeric G protein, which in turn
interacts with downstream signal transduction
proteins. All GPCRs are integral membrane
proteins that have a common 7 transmembrane
segment structure (Fig. 15.15). The
hormone/ligand binding domain is formed by amino
acids located on the external side of the
membrane and/or membrane interior (Fig. 15.16a).
Likewise in rhodopsin, its light absorbing
chromophore 11-cis-retinal is located within the
transmembrane segment interior of the protein.
GPCRs interact with G proteins via amino acids in
the C3 and C4 cytoplasmic regions.
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G Protein Activation of Effectors
The trimeric G protein cycle of activity in
hormone-stimulated GPCR regulation of effector
proteins is summarized in Fig. 15.17 (next
slide). Initially, the G protein complex is
tethered to the inner leaflet of the cytoplasmic
membrane via lipid anchors attached to the Ga and
Gg subunits. The trimeric GDP-bound form of the G
protein is inactive in signaling. The binding of
a hormone to the GPCR triggers a conformational
change in the receptor (Step 1) which promotes
its binding to the trimeric G protein (Step 2).
Binding to the activated GPCR triggers the
dissociation of GDP (Step 3). Subsequent binding
of GTP to the Ga subunit activates it, and causes
its dissociation from the receptor and the Gßg
complex (Step 4). Ga-GTP then binds to the
effector protein regulating its activity. The
hormone eventually dissociates from the receptor
(Step 5). Over time (often less than 1 min), GTP
is hydrolyzed to GDP and Ga becomes inactive. It
then dissociates from the effector and recombines
with Gßg (Step 6). A hormone-bound GPCR activates
multiple G proteins, until the hormone
dissociates. Proteins known as regulators of G
protein signaling (RGS) accelerate GTP hydrolysis
by Ga decreasing the time-period during which Ga
is active (not shown).
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15
Trimeric G Proteins Their Effectors
There are 21 different Ga proteins encoded in the
human genome. The G proteins containing these
subunits are activated by different GPCRs and
regulate a variety of different effector proteins
(Table 15.1). The most common effectors
synthesize second messengers such as cAMP, IP3,
DAG, and cGMP. In the case of cAMP, a stimulatory
G?s subunit activates adenylyl cyclase and cAMP
production, whereas an inhibitory G?i subunit
inhibits adenylyl cyclase and cAMP production.
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GPCRs That Bind Epinephrine
Epinephrine is a hormone that signals the
"fight-or-flight" response. It elevates heart
rate, dilates the airway, and mobilizes
carbohydrate and lipid stores of energy in liver
and adipose tissue. In the heart, liver, and
adipose tissue, these effects are mediated via
binding to ß1- ß2-adrenergic GPCRs. Both
ß-adrenergic GPCRs signal via Gas, which
activates adenylyl cyclase and raises
intracellular cAMP. The a2-adrenergic GPCR
signals via Gai, decreasing adenylyl cyclase
activity and intracellular cAMP. The
a1-adrenergic GPCR is coupled to Gaq, which
activates phospholipase C (PLC) and signaling via
the IP3/DAG pathway (see below). a1-adrenergic
GPCRs are present in the liver and blood vessels
in peripheral organs. Binding to a1-adrenergic
GPCRs stimulates glycogen breakdown in the liver,
while blood flow to peripheral organs is
decreased. Cholera toxin produced by Vibrio
cholera, locks Gas-GTP in the active state,
increasing cAMP in the large intestine, causing
electrolyte and water loss. Pertussis toxin
produced by Bordetella pertussis, locks Gai-GDP
in the inactive state, increasing cAMP in the
airway epithelium, causing mucus secretion into
bronchial tubes, etc.
17
GPCRs that Regulate Ion Channels Muscarinic
Acetylcholine Receptor
The neurotransmitter, acetylcholine (ACH) binds
to two types of receptors known as the nicotinic
and muscarinic acetylcholine receptors. The
nicotinic receptor is itself a ligand-gated ion
channel that opens on ACH binding. This receptor
is located in the neuromuscular junctions of
striated muscle. The muscarinic ACH receptor, is
a GPCR found in cardiac muscle cells that is
coupled to an inhibitory G protein
(Fig. 15.20). The binding of ACH to this receptor
triggers dissociation of Gai-GTP from Gßg, which
in this case, directly binds to and opens a K
channel. The movement of K down its
concentration gradient to the outside of the
cell, increases the positive charge outside the
membrane, hyperpolarizing the cell. This results
in the slowing of heart rate.
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GPCRs that Regulate Ion Channels Rhodopsin
Rhodopsin is a light-activated GPCR found in the
rod cells of the eye. Rhodopsin molecules are
located within membrane disks in the outer
segments of rod cells (Fig. 15.21). About 107
copies of rhodopsin occur per cell. Rod cells are
important in capture of low intensity light
having a broad range of wavelengths. Closely
related color pigment receptors that respond to
more limited regions of the visual spectrum
(i.e., blue, green, red light) are present in
cone cells.
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Mechanism of Rhodopsin Activation by Light
Rhodopsin consists of the protein opsin bound to
the visual pigment, 11-cis-retinal. Like other
GPCR family members, rhodopsin is a
7-transmembrane segment protein. Rhodopsin
signaling is initiated when the retinal
chromophore absorbs a photon of light. Light
absorption causes an electronic rearrangement and
isomerization from 11-cis- to all-trans-retinal
(Fig. 15.22). The isomerization triggers a
conformational change in opsin, leading to
activation of a bound G protein known as
transducin (Gt). All-trans-retinal is released
and recycled to 11-cis-retinal which later
recombines with opsin.
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Mechanism of Rhodopsin Signaling I
The rhodopsin signal transduction pathway is
shown in Fig. 15.23. Light absorption by
rhodopsin triggers GTP/GDP exchange on the
transducin Gat subunit, and dissociation of this
trimeric G protein (Steps 1 2). Gat-GTP binds
to and activates a cGMP phosphodiesterase,
reducing intracellular cGMP level (Steps 3 4).
This indirectly results in the closing of
non-selective Na/Ca2 ion channels in the
cytoplasmic membrane and hyperpolarization of the
membrane potential (Step 6). This results in
decreased release of neurotransmitter from the
cells. Thus, light is perceived by the brain due
to a decrease in nerve impulses coming from rod
cells. Studies have shown that only 5 photons
must be absorbed per human rod cell to transmit a
signal. A single activated molecule of rhodopsin
activates 500 transducin molecules in a classic
example of signal amplification.
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Mechanism of Rhodopsin Signaling II
Rhodopsin signaling must be rapidly shut down in
order for the eye to detect rapid movement and
other changes in objects in our surroundings. The
shut down of signaling is accomplished in about
50 milliseconds, and involves several
contributing processes. First, G?t-bound GTP is
rapidly hydrolyzed.The hydrolysis of GTP by G?t
is stimulated by a dimeric GAP protein consisting
of the RGS9/Gß5 subunits (Step 7, preceding
slide). Second, Ca2-sensing proteins that detect
a fall in intracellular Ca2 stimulate the
activity of guanylate cyclase, leading eventually
to re-opening of ion channels (Fig. 15.23).
Finally, the ability of activated rhodopsin to
stimulate transducin is down-regulated by the
phosphorylation of rhodopsin by rhodopsin kinase
(Fig. 15.24). Signaling by triphosphorylated
rhodopsin is completely blocked by the binding of
a protein called arrestin.
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23
Visual Adaptation
Rod cell signaling actually is reduced after
prolonged exposure to high light intensity. This
is apparent as a time delay during which vision
is compromised when we move from bright light to
a dark room. The change in sensitivity of our
eyes to high and low light levels is known as
visual adaptation. The biochemical mechanism by
which adaptation primarily occurs is shown in
Fig. 15.25. In the dark, transducin molecule are
transported to the outer rod segments, whereas
arrestin molecules are transported elsewhere in
the cell. In bright light, the distributions of
transducin and arrestin are reversed. Through the
distribution of these proteins, visual signaling
is desensitized at high light levels and
sensitized at low light intensities. Visual
adaptation allows rod cells to perceive contrast
over a 100,000-fold range of ambient light levels.
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Synthesis and Hydrolysis of cAMP
In the next few slides, we will cover signaling
by the second messenger, cAMP. As shown in Fig.
15.26, cAMP is synthesized from ATP by the enzyme
adenylyl cyclase. cAMP is broken down to AMP via
the enzyme cAMP phosphodiesterase.
25
GPCRs that Regulate Adenylyl Cyclase
Adenylyl cyclase is an effector enzyme that
synthesizes cAMP. Ga-GTP subunits bind to the
catalytic domains of the cyclase, regulating
their activity. Gas-GTP activates the catalytic
domains, whereas Gai-GTP inhibits them. A given
cell type can express multiple types of GPCRs
that all couple to adenylyl cyclase. The net
activity of adenylyl cyclase thus depends on the
combined level of G protein signaling via the
multiple GPCRs. In liver, GPCRs for epinephrine
and glucagon both activate the cyclase. In
adipose tissue (Fig. 15.27), epinephrine,
glucagon, and ACTH activate the cyclase via
Gas-GTP, while PGE1 and adenosine inactivate the
cyclase via Gai-GTP.
26
Adenylyl Cyclase Protein Kinase A
Adenylyl cyclase is an integral membrane protein
that contains 12 transmembrane segments (Fig.
15.28a). It also has 2 cytoplasmic domains that
together form the catalytic site for synthesis of
cAMP from ATP. One of the primary targets of cAMP
is a regulatory kinase called protein kinase A
(PKA), or cAMP-dependent protein
kinase. PKA exists in two different states inside
cells (Fig. 15.29a). In the absence of cAMP, the
enzyme forms a inactive tetrameric complex in
which 2 PKA catalytic subunits are non-covalently
associated with 2 regulatory subunits. When cAMP
concentration rises, cAMP binds to the regulatory
subunits which undergo a conformational change,
releasing the active catalytic subunits.
27
Regulation of Glycogen Degradation
Glycogen is a polysaccharide that serves as the
main store of glucose in many organisms. The
liver stores glycogen for 1) release to the CNS
during overnight fasting, and 2) release to
skeletal muscle in response to epinephrine.
Skeletal muscle stores glycogen for energy
metabolism, which is accelerated by epinephrine.
The reactions catalyzed by the key enzymes of
glycogen synthesis (glycogen synthase) and
degradation (glycogen phosphorylase) are shown in
Fig. 15.31a.Epinephrine activates glycogen
breakdown and blocks synthesis via activation of
glycogen phosphorylase and inhibition of glycogen
synthase. Epinephrine exerts these effects via
raising cAMP levels through Gas-GTP signaling.
The key target of cAMP is PKA. The activation of
PKA leads to phosphorylation and activation of
glycogen phosphorylase kinase and ultimately
glycogen phosphorylase (left). In contrast, PKA
inactivates glycogen synthase by phosphorylation.
PKA also phosphorylates an inhibitor of
phosphoprotein phosphatase, ensuring that protein
phosphatase remains off (right). Hydrolysis of
phosphates by protein phosphatase reverses the
effects of PKA.
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Tissue-specific Responses to cAMP Signaling
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Activation of Gene Transcription by GPCR Signaling
GPCRs regulate gene transcription by cAMP and PKA
signaling. As shown in Fig. 15.32, cAMP-released
PKA catalytic domains enter the nucleus and
phosphorylate the CREB (CRE-binding) protein,
which binds to CRE (cAMP-response element)
sequences upstream of cAMP-regulated genes. Only
phosphorylated p-CREB has DNA binding activity.
p-CREB interacts with other TFs to help assemble
the RNA Pol II transcription machinery at these
promoters. In liver, glucagon signaling via this
pathway activates transcription of genes needed
for gluconeogenesis.
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Down-regulation of GPCR/cAMP/PKA Signaling
A number of events contribute to the termination
of signaling by a GPCR. These include
dissociation of the hormone from the receptor,
hydrolysis of GTP by Ga, hydrolysis of cAMP via
cAMP phosphodiesterase, and phosphorylation and
desensitization of receptors by kinases such as
PKA and ß-adrenergic receptor kinase (BARK). In
addition, GPCRs can be removed from the membrane
by vesicular uptake.
31
GPCRs That Activate Phospholipase C
Another common GPCR signaling pathway involves
the activation of phospholipase C (PLC). This
enzyme cleaves the membrane lipid,
phosphatidylinositol 4,5-bisphosphate (PIP2) to
the second messengers, inositol
1,4,5-trisphosphate (IP3) and diacylglycerol
(DAG) (Fig. 15.35). In this case, the G?o and G?q
G? proteins conduct the signal from the GPCR to
PLC. This is the pathway used in a1-adrenergic
GPCR signaling in the liver.

32
IP3/DAG Signaling Elevates Cytosolic Ca2
The steps downstream of PLC that make up the
IP3/DAG signaling pathway are illustrated in Fig.
15.36a. IP3 diffuses from the cytoplasmic
membrane to the ER where it binds to and triggers
the opening of IP3-gated Ca2 channels (Steps 3
4). Another kinase, protein kinase C (PKC) binds
to DAG in the cytoplasmic membrane and is
activated (Step 6). In liver, the rise in
cytoplasmic Ca2 activates enzymes such as
glycogen phosphorylase kinase, which
phosphorylates and activates glycogen
phosphorylase. Glycogen phosphorylase kinase is
activated by Ca2-calmodulin. In addition, PKC
phosphorylates and inactivates glycogen synthase.
33
Nitric Oxide (NO)/cGMP Signaling
A related signaling pathway involving
phospholipase C operates in vascular endothelial
cells and causes adjacent smooth muscle cells to
relax in response to circulating acetylcholine
(Fig. 15.37). In the NO/cGMP signaling pathway,
the downstream target of Ca2/calmodulin is
nitric oxide synthase, which synthesizes the gas
NO from arginine. NO diffuses into smooth muscle
cells and causes relaxation by activating
guanylyl cyclase and increasing cGMP. As a
result arteries in tissues such as the heart
dilate, increasing blood supply to the tissue. NO
also is produced from the drug nitroglycerin
which is given to heart attack patients and
patients being treated for angina.