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.
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.
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.
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.
 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).
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.
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.
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.
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 Gs-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.
 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 = [R][L] / [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
occurs atbinding (Fig. 15.12). Namely, 50% of full response often
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.
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.
 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.
 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 G and G 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 G subunit activates it, and causes its
dissociation from the receptor and the Gß complex (Step 4).
G-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 G becomes inactive. It then dissociates from the
effector and recombines with Gß (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 G decreasing the time-
period during which G is active (not shown).
 Trimeric G Proteins & Their Effectors
There are 21 different G 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.
 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 Gs, which
activates adenylyl cyclase and raises intracellular [cAMP]. The
2-adrenergic GPCR signals via Gi, decreasing adenylyl cyclase
activity and intracellular [cAMP]. The 1-adrenergic GPCR is
coupled to Gq, which activates phospholipase C (PLC) and
signaling via the IP3/DAG pathway (see below). 1-adrenergic
GPCRs are present in the liver and blood vessels in peripheral
organs. Binding to 1-adrenergic GPCRs stimulates glycogen
breakdown in the liver, while blood flow to peripheral organs
is decreased. Cholera toxin produced by Vibrio cholera, locks
Gs-GTP in the active state, increasing [cAMP] in the large
intestine, causing electrolyte and water loss. Pertussis toxin
produced by Bordetella pertussis, locks Gi-GDP in the inactive
state, increasing [cAMP] in the airway epithelium, causing
mucus secretion into bronchial tubes, etc.
 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 Gi-GTP from Gß, 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.
 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.
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.
 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 Gt subunit, and dissociation of this trimeric G protein
(Steps 1 & 2). Gt-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.
 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.
 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.
 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.
 GPCRs that Regulate Adenylyl Cyclase
Adenylyl cyclase is an effector enzyme that synthesizes cAMP.
G-GTP subunits bind to the catalytic domains of the cyclase,
regulating their activity. Gs-GTP activates the catalytic
domains, whereas Gi-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 Gs-GTP, while PGE1 and
adenosine inactivate the cyclase via Gi-GTP.
 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.
 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 Gs-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.
Tissue-specific Responses to cAMP Signaling
 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.
 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 G, 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.
 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1-adrenergic GPCR signaling in the liver.

*
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.
 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.