C h a p t e r
6
Signal Transduction and
Second Messengers
Karen Lounsbury
OUTLINE
6.1 Receptor Communication 103
6.1.1 Ion Channels 103
6.1.2 G-Protein Coupled Receptors 103
6.1.3 Receptor Tyrosine Kinases 107
6.1.4 Cytokine Receptors (Tyrosine Kinase-
Associated Receptors) 109
6.1.5 Intracellular Receptors 109
6.1.6 Other Classes of Receptors 110
6.2 Receptor/Second Messenger Crosstalk 110
6.3 Signal Transduction Targets for Drug Discovery 111
Central to the communication between cells is the pro-
cess of signal transduction. Signal transduction is the
mechanism by which cell surface receptors receive
information from extracellular signals such as hor-
mones and neurotransmitters, and amplify this infor-
mation through the actions of second messengers.
Second messengers in turn activate pathways intrinsic
to processes such as protein secretion, cell differentia-
tion, and cell division. Many pharmacological agents
elicit their clinical activity and side effects through
interactions with receptors and/or their downstream
signaling targets. As our understanding of receptor sig-
naling has progressed, intracellular signaling mole-
cules have become a major target of drug discovery.
This chapter will review the major receptor-mediated
signaling pathways and will outline current and future
therapeutic strategies to target these pathways.
6.1 RECEPTOR COMMUNICATION
Receptors are the primary sensors of the cell environ-
ment, but ligand binding alone is useless without sig-
nal transduction. The effect of a pharmacologic
agent on the ability of receptors to couple to down-
stream signals thus defines whether it will increase
(agonist) or decrease (antagonist) the physiologic
activity of the receptor. Selective regulation of the
magnitude, time-course, and spread of the signal
allows a single ligand to transmit a complex set of phys-
iological effects. Signal transduction by receptors can
be broken into four major categories:
n
Ion channels
n G protein-coupled receptors
n Receptor tyrosine kinases
n Intracellular receptors
6.1.1 Ion Channels
Ion channels can be regulated by receptors either
directly (ligand-gated) or indirectly (second-messen-
ger regulated) to modulate the passage of ions and
other hydrophilic molecules across the plasma mem-
brane. This regulated movement of ions can have
effects on the membrane potential (i.e., Kþ efflux) as
well as on downstream signaling pathways (i.e., Ca2þ
influx). The specifics of this regulation will be covered
in Chapter 7.
6.1.2 G-Protein-Coupled Receptors
G-protein-coupled receptors are the most abundant
class of receptors and are responsible for regulating a
multitude of physiologic processes including vision,
muscle contraction, secretion, and neurotransmission.
These receptors are inserted into the plasma mem-
brane in a serpentine fashion that results in seven
transmembrane domains. When a ligand binds to the
active site of the receptor, a conformational change
occurs within the intracellular domains resulting in
the activation of a guanine nucleotide-binding protein
(G-protein).
Prior to the knowledge that G-proteins existed,
there was a great deal of information regarding the
interactions of agonists and antagonists with receptor
103
Chapter 6 Signal Transduction and Second Messengers

proteins present within cell membranes, but there was
little understanding of how receptors communicated
with their downstream targets. A tremendous amount
of research from 1970 to 1990 revealed that there are
three separate proteins necessary for signal transduc-
tion including the receptor (R), which recognizes
external signals; the effector (E), which is responsible
for the production of second messengers; and the G-
protein (G), which is necessary for communication
between the receptor and effector (Figure 6.1). The
importance of this research on our understanding of
cell signaling is evidenced by the Nobel Prize for Phys-
iology or Medicine awarded to U.S. scientists Alfred
Gilman and Martin Rodbell for their pioneering
research in the discovery of G-proteins and the role
of these proteins in signal transduction in cells.
G-proteins function as signal transducers through
their cyclical regulation influenced primarily by recep-
tors and guanyl nucleotides (Figure 6.2). G-proteins are
heterotrimeric in structure, made up of an a subunit,
which confers both catalytic activity and signaling func-
tion, and a bg heterodimer subunit complex, which tar-
gets the G-protein to the appropriate membrane
receptor and can also interact with downstream effectors.
A G-protein is considered to be in an “inactive” state
when its a subunit is bound to GDP and associated with
its respective bg subunit. When a receptor is coupled to
a G-protein heterotrimer, it exhibits an increased affinity
for its corresponding agonist. An agonist binding to the
receptor induces activation of the G-protein by stimulat-
ing exchange of bound GDP for GTP on the a subunit,
which promotes release of the bg subunit. The free
GTP-a and bg subunits can functionally couple to their
downstream effector molecules (i.e., Gs coupling to ade-
nylyl cyclase). Because exchange of GTP for GDP is the
limiting step, ligand-mediated receptor activation is the
key activator of signaling by G-proteins.
The GTPase activity inherent to the a subunit
hydrolyzes the bound GTP to GDP, resulting in a
decreased affinity for effector and an increased affinity
for bg. The GTPase activity thus acts as a turnoff and
recycling mechanism for the a subunit. The turnoff
mechanism can be enhanced by G-protein interaction
with specific regulator of G-protein signaling (RGS)
isoforms, which act as both GTPase activating proteins
and as antagonists of the interaction between a sub-
units and their receptors.
The use of G-proteins as a relay between receptor
ligand binding and effector activation has important
implications toward effectiveness of signaling. First,
the time-course of the effect is not dependent on
ligand binding, but instead on the length of second
messenger activation. Thus, the relatively slow hydroly-
sis of GTP by the G-protein maintains the signal well
after ligand has dissociated. Second, the dispersion
of second messengers by G-protein coupling greatly
increases the spatial effectiveness of the signaling
throughout the cell. Together these mechanisms amplify
the ligand-initiated signal to broadcast a programmed
effect from a single source.
Heterotrimeric G-proteins can couple signals from
over 1000 receptor subtypes, and their effector molecules
include both ion channels and a variety of enzymes.
There are 16 a, 5 b, and 14 g genes, so a large number
of subunits are produced, and they can combine in vari-
ous ways. There are specific functions for both the a sub-
units and the bg heterodimers. The a subunits can be
divided into five families, Gs, Gi, Gt, Gq, and G12/13,
each with a relatively characteristic set of effectors. A com-
pilation of functions reported for specific receptor-G-
protein coupling can be found in Table 6.1.
6.1.2.1 G-Protein-Coupled Second Messengers
Despite the diversity of receptor subtypes, there is a rel-
atively small subset of G-protein-coupled second mes-
sengers that are the convergence point for multiple
receptors. This convergence allows for a complicated

Second
R G E
messengers
Table 6.1 Heterotrimeric G protein
Input Transduction Output
Signaling Pathways
Figure 6.1 Amplification of signals by second messengers.
G protein Receptors Effectors
Gs b-adrenergic amines, Adenylyl cyclase
glucagon, cAMP
GTP GDP E histamine,
Agonist
βγ serotonin
R R* Gi a2-adrenergic Adenylyl cyclase
amines, Kþ channels
acetylcholine,
Gα -GDP Gα -GTP opiods, serotonin
βγ Golf Odorants Adenylyl cyclase
E* Go Neurotransmitters(?) Adenylyl cycase(?)
Gq Acetylcholine, Phospholipase C IP3,
βγ serotonin DAG, Ca2þ
RGS Gt Photons (rhodopsin cGMP
Pi and color opsins) phosphodiesterase
cGMP
Figure 6.2 GTPase cycle of G protein activation.

104

environment to be processed into a coordinated cellular
effect. Following are the primary G-protein-coupled sec-
ond messenger signaling pathways that have been char-
acterized in mammalian cells.
Gs/Gi Coupling to Adenylyl Cyclase Receptor cou-
pling through Gs or Gi results in a cascade of events
that begins with the stimulation or inhibition of adeny-
lyl cyclase activity (Figure 6.3). Adenylyl cyclase cata-
lyzes the conversion of ATP to 30 ,50 -cyclic AMP
(cAMP), which mediates a multitude of hormonal
responses including carbohydrate metabolism, ion
homeostasis, heart contractility, and neuronal signal-
ing. Many of these activities of cAMP have been attrib-
uted to the activation of cAMP-dependent protein
kinase (PKA), a ubiquitous protein with multiple sub-
strates for phosphorylation. The catalytic subunits of
PKA are held inactive through their interaction with
regulatory subunits. When cAMP binds to the regu-
latory subunits, the active catalytic subunits are
released and are able to promote phosphorylation of
proteins containing PKA consensus phosphorylation
sites. The specificity of cAMP effects is thus dependent
on the expression and proximity of kinase substrates.
Perpetuation of the PKA-mediated phosphorylation
signal is regulated by specific and nonspecific phos-
phatases that catalyze the removal of phosphate from
the PKA substrates. The termination of the cAMP sig-
nal is mediated by its degradation to 50 -AMP by cyclic
Sensory cortex
Brain stem
NE
βAR βAR*
Gαs -GDP Gαs -GTP
βγ
Adenylyl cyclase
ATP cAMP
PKA PKA*
Stimulate neurotransmitter release
Increase heart contractility
Decrease peripheral vasoconstriction
Increase carbohydrate metabolism
Figure 6.3 Signal amplification in the “fight-or-flight”
response.
6.1 Receptor Communication
nucleotide phosphodiesterases that are selective for
cAMP or able to degrade both cAMP and cGMP. Com-
petitive inhibition of phosphodiesterases is one mech-
anism by which the methylxanthines such as caffeine
and theophylline exert their effects.
The G-proteins Gs and Gi are direct targets for bac-
terial toxins produced by V. cholerae and B. pertussis,
respectively. Cholera toxin activates Gs independently
of receptor activation causing increased activation of
adenylyl cyclase and opening of ion channels in the
epithelial lining of the colon. The increased flow of
ions into the colon increases the osmotic transfer of
water, resulting in diarrhea and often death due to
dehydration. Pertussis toxin causes a modification of
Gi, preventing it from interacting with its receptors
or inhibiting adenylyl cyclase in the respiratory epithe-
lium and phagocytotic immune cells. The resulting
increase in adenylyl cyclase activity enhances cell inva-
sion and inhibits phagocytotic functions. The clinical
result is a pervasive cough, which has led to the refer-
ence to pertussis infection as whooping cough.
Research leading to the identification of the mechan-
isms for these toxins was not only important clinically,
but also fueled research to understand the receptor-G-
protein-effector links.
Other G-proteins that are related to Gs and Gi
include Golf and Go. Golf is structurally and function-
ally similar to Gs. Golf was originally discovered in the
olfactory neuroepithelium and striatum, but has subse-
quently been identified in several peripheral tissues as
well. Like Gs, Golf couples to activation of adenylyl
cyclase. Although its role in the periphery has yet to
be characterized, in the brain it is known to communi-
cate odorant signals by coupling to olfactory receptors.
Go has similar effector functions as Gi, including inhi-
bition of adenylyl cyclase, but it is somewhat selectively
expressed in the brain and heart. It is not clear why
diversification to Go is advantageous, but new research
suggests that Go is regulated by selective GTPase acti-
vating proteins that may fine-tune the response in a tis-
sue-selective manner.
A classic example of the physiological effects
mediated by the Gs/Gi-coupled signaling cascade is
the “fight-or-flight” response (Figure 6.3). Beginning
in the brain, the stressor signal (such as a loud noise
or bright light) is relayed from the sensory cortex to
the thalamus and brain stem. This signaling stimulates
release of catecholamine neurotransmitters such as
norepinephrine and dopamine in the locus ceruleus.
The neurotransmitters bind to G-protein coupled
receptors that activate adenylyl cyclase via Gs. The
resulting production of cAMP enhances the catalytic
activity of PKA. PKA then stimulates release of neuro-
transmitters that regulate neurons responsible for
spontaneous “flight” responses. A prolonged stimula-
tion of the locus ceruleus activates release of acetylcho-
line from the preganglionic neurons of the autonomic
nervous system. Acetylcholine binds to nicotinic acetyl-
choline receptors in the adrenal medulla causing Naþ
influx, membrane depolarization, and release of epi-
nephrine. Epinephrine has effects that are dependent
on the array of receptors expressed on the membrane
105
Chapter 6 Signal Transduction and Second Messengers
of target cells. Depending on the subtype, adrenergic
receptors can couple to Gs, Gi, or Gq. Actions through
b receptor/Gs coupling promote an increase in carbo-
hydrate metabolism, heart contractility and lung action
as well as dilation of blood vessels feeding the musculo-
skeletal system. Conversely, actions of epinephrine
through a receptor coupling results in vasoconstriction
of vessels feeding the gastrointestinal and renal systems.
The result of these opposing actions of epinephrine
allows the body to divert energy away from internal pro-
cesses to the muscles for an optimal fight-or-flight
response.
Gt Coupling to cGMP Phosphodiesterase In photore-
ceptor rod outer segments, light-activated rhodopsin
activates transducin (Gt), which stimulates a cGMP-
specific phosphodiesterase. This activation causes
rapid degradation of cGMP resulting in the closure
of cGMP-regulated sodium channels and membrane
hyperpolarization. The resulting hyperpolarization
reduces neurotransmitter release, thus the amount of
neurotransmitter released is reduced in bright light
and increases as light levels fall. The Gt signal is termi-
nated following GTP hydrolysis. This chain of signal-
ing events is called “the vertebrate phototransduction
cascade” and is critical for rapidly adjusting the visual
response to different light intensities.
Gq Coupling to Phosphoinositide Hydrolysis and
Calcium Signaling Receptor coupling through Gq
results in the activation of phospholipase C-b (PLC-b)
(Figure 6.4). PLC-b enzymatically cleaves the mem-
brane phospholipid phosphoatidylinositol-4,5-bisphop-
shate (PIP2) into diacylglycerol (DAG) and inositol
trisphosphate (IP3). Both DAG and IP3 act as important
second messengers. DAG remains in the membrane
where it recruits and activates protein kinase C.
IP3 stimulates the opening of IP3-mediated Ca2þ chan-
nels on intracellular organelles that store Ca2þ such as
the endoplasmic reticulum. The release of Ca2þ into
the cytoplasm regulates several signaling cascades inclu-
ding opening of ion channels and activation of Ca2þ/
calmodulin-dependent kinases.
Receptor
Phorbol ester
Gqα PKC
DAG
PLC Neurotransmitter release
cell proliferation
PIP2
Lithium IP3 Ca2+
Figure 6.4 Gq signaling through phospholipase C (PLC).
106
The termination of signaling by Gq-generated sec-
ond messengers is achieved by dephosphorylation of
IP3 and deacylation of DAG. Removal of Ca2þ from
the cytoplasm is achieved by Ca2þ-binding proteins
and Ca2þpumps. Ca2þ pumps in the plasma mem-
brane remove Ca2þ from the cell whereas pumps in
organellar membranes accumulate Ca2þ to replenish
intracellular Ca2þ stores.
Because of its more complicated signaling cascade,
Gq mediated signaling is more complex and cell-type
specific. There are many isoforms of protein kinase C
that have selective substrates and functions. In addi-
tion, different cell types express variable amounts of
Ca2þ-mediated kinases and ion channels and Ca2þ
can play a role in events ranging from cell contraction
and secretion to gene expression and cell division.
Examples of exogenous agents that alter the Gq sig-
naling pathway include phorbol esters, which mimic
DAG to activate protein kinase C, and lithium, which
blocks recycling of phosphoinositides. Phorbol esters
are recognized as tumor promoters due to their ability
to excessively activate protein kinase C-mediated sig-
naling to upregulate cell growth. Conversely, lithium
is used as a treatment for manic disorders because
blocking the phosphoinositide signaling pathway pre-
vents overactivity of signaling through excitatory
amine neurotransmitter receptors in the brain such
as dopamine and norepinephrine.
G12/G13 Coupling to Small GTPases G12 and G13
were discovered through their sequence homology to
previously characterized G-proteins, and their functions
are not yet completely known. Evidence thus far sug-
gests that their main effectors are small GTPases. G12
reportedly interacts with the Ras GTPase, thus activating
the MAP kinase cascade and cell proliferation
(described later). It is not surprising then that G12 has
been identified as a potential tumor-promoting onco-
gene. G13 couples to lysophosphatidic acid receptors
and activates the small GTPase Rho. The Rho GTPase
has several functions related to cytoskeleton rearrange-
ments and signaling through protein kinases, thus G13
likely has an important role in cell motility.
Signaling by bg Subunits Although initial reports were
not readily accepted, it is now well established that the
G-protein bg subunit complex also has effector func-
tions. Most cell types express multiple forms of b and
g subtypes, making individual assessments of physio-
logic function difficult. Although many functions have
been attributed to selective bg pairings, the best char-
acterized functions are activating/inhibiting adenylyl
cyclase, enhancing Kþ channel activity, and stimulating
activity of phospholipase C-b. Like the G-protein a sub-
units, the effector activity is related to localization of
targets, thus recruitment of scaffolding proteins that
link protein regulators to membrane-bound enzymes
is likely the most important role of the bg complex.
Although not directly targeted by pharmacologic
agents, the loss of bg subunits has been correlated to
diseases including atherosclerosis, hypertension, and
metabolic syndrome.

6.1.3 Receptor Tyrosine Kinases
Receptor tyrosine kinases (RTKs) mediate signaling by
insulin and a variety of growth factors such as epider-
mal growth factor (EGF) and platelet-derived growth
factor (PDGF). RTKs were discovered in 1980 and,
because of their importance in the regulation of onco-
genes and cell growth, there has been a flurry of prog-
ress in characterizing the mechanisms underlying their
activity and regulation. Unlike G-protein-coupled
receptors, RTKs are characterized by their intrinsic
tyrosine kinase activity that becomes activated upon
ligand binding. The majority of RTKs exist on the cell
surface as monomers with a single transmembrane
domain. When activated, these receptors dimerize and
transfer phosphate to hydroxyl groups on tyrosines of
target proteins within their vicinity (Figure 6.5). For
most RTKs, autophosphorylation of the intracellular
domain of the receptor is a key element to communica-
tion with second messengers.
The phosphorylated tyrosines on the RTK function
as specific binding sites for proteins that contain SH2
(Src homology 2) domains. These proteins include
enzymes such as phosphoinositide-3 kinase (PI3K)
and phospholipase C (PLC) and adaptor proteins such
as growth receptor binding protein-2 (Grb-2). The
specificity of growth factor signaling is determined by
the proximity of substrates that are directly phosphory-
lated by the receptor or that bind to adaptor proteins
through SH2 domains. These proteins are often tar-
geted to the receptor through specific scaffolding pro-
teins that localize the appropriate targets to the
receptor.
There are three primary mechanisms that have
been identified for activation of effector proteins by
RTKs as illustrated for PI3K signaling in Figure 6.5.
The first mechanism involves recruitment of proteins
to the membrane to mediate interactions with
enzymes and lipid activators. The second mechanism
is by mediating conformational change, which pro-
motes activation; the third involves direct activation
of enzyme activity by tyrosine phosphorylation. These
three mechanisms work synchronously to accomp-
lish localization, protein interaction, and enzyme
activation.
RTK
SH2
Y--p
Y--p
Tyr phos Y--p SH2
Target proteins
Figure 6.5 Growth factor signaling through receptor tyrosine kinases.
6.1 Receptor Communication
6.1.3.1 Receptor Tyrosine Kinase-coupled Second
Messengers
There has been rapid progress in multiple fields
toward the understanding of signaling pathways acti-
vated by RTKs, and the picture that emerges is one
that includes multiple signaling networks that modu-
late the flow of information to destinations in the
membrane, cytoplasm, and nucleus. Described below
are the RTK-coupled second messenger signaling net-
works that have been characterized in mammalian
cells.
Ras/MAP Kinase Signaling Cascade All known RTKs
stimulate activation of the small GTPase, Ras. Like
heterotrimeric G-proteins, Ras activation requires
exchange of GTP for GDP. Unlike heterotrimeric G
proteins, Ras does not have a bg subunit and has much
less efficient intrinsic GTPase activity. For this reason,
once activated by a GTP exchange factor, Ras remains
active until it interacts with a GTPase activating protein
(GAP). RTKs recruit the Ras GTP exchange factor,
son-of-sevenless (SOS), via the SH2 and SH3 domains
on the Grb-2 adaptor protein (Figure 6.6). Once at
the membrane, SOS mediates exchange of GTP on
Ras, thus stimulating its interaction and activation of
downstream targets.
The downstream targets of Ras include both PI3K
and the mitogen activated protein kinase (MAPK) cas-
cade. Ras activates the first MAPK in the cascade, Raf
kinase, which then stimulates MAP kinase (MEK) by
phosphorylating a critical Ser residue in its activation
domain. MEK then phosphorylates and activates the
MAP kinase ERK (extracellular signal regulated
kinase), which has functional targets in the plasma
membrane, cytoplasm, and nucleus. MAP kinase sig-
naling cascade is highly conserved throughout evolu-
tion and has an important role in the control of cell
metabolism, cell division, and cell motility.
Termination of MAP kinase signaling can be accom-
plished by receptor downregulation, Ras interaction
with its GAP, which catalyzes the hydrolysis of GTP on
Ras, and by tyrosine and serine phosphatases that dis-
rupt the kinase cascade. Gain of function mutations that
Lipid
PH
Binding
p-Tyr Membrane
recruitment
107
Chapter 6 Signal Transduction and Second Messengers

RTK

DAG PIP2 PIP3 PIP3

PIP3 PH
SH2 PH
PH Y--p PDK AKT
Y--p PI3K
IP3 PLC Y--p SH2

Cell survival
Cell division

Figure 6.6 Ras/MAP kinase signaling cascade.

Table 6.2 Pipeline Targets of Growth Factor Signaling in Development

Drug/compound Target Mechanism Clinical Phase

PTK/ZK
VandetanibZD6474
Axitinib/AG-013736
Vatalanib/PTK787
Lapatinib/GW572016
Dasatinib/BMS-354825
Nilotinib/AMN-107
CP-751871
Flavopiridol
PD332991
PD0325901
ARRY-142886
Sorafenib/BAY 43-9006
VEGFR Tyr kinase inhib. Phase I/II, colon, liver cancer
VEGFR, EGFR Tyr kinase inhib. Phase I/II, multiple studie
VEGFR, PDGFR Tyr kinase inhib. Phase I/II, multiple studie
VEGFR, PDGFR, c-Kit Tyr kinase inhib. Phase I/II, multiple studies
EGFR, ErbB2 Tyr kinase inhib. Phase I/II, multiple studie
Src, Abl Tyr kinase inhib. Phase I/II, multiple studie
Abl Tyr kinase inhib. Phase III, Leukemia
IGFR Antibody Phase II, combination therapy
Pan-cdk Cell cycle Inhib. Phase I/II, solid tumors
Cdk4 Cell cycle Inhib. Phase I/II Advanced cancer
MEK Kinase inhibitor Phase I/II Advanced cancer
MEK Kinase inhibitor Phase II, Melanoma
RAF, VEGFR, PDGFR, c-Kit Kinase inhibitor Phase III, Kidney Cancer

disrupt these termination signals can lead to uncon-

trolled cell growth that leads to many pathologies Table 6.3 Kinase Inhibitors Used
including inflammation, fibrosis, and cancer. These Clinically
mutations are prevalent in leukemia, gastrointestinal,
and gynecologic cancers and have also been linked to
proliferative fibrotic diseases of the kidney, lung, and Disease
cardiovascular system. Drug Target Indication
Several anti-cancer agents, either approved or in
Small Molecules
development, target the kinase activity of RTKs and
components of the MAP kinase cascade (Tables 6.2 Imatinib Abl, PDGFR, c-Kit Leukemia, GI
(Gleevec) Cancer
and 6.3). The drug imatinib (Gleevec) was initially Gefitinib EGFR Lung Cancer
approved based on its inhibition of the tyrosine kinase (Iressa)
activity of the intracellular RTK, Abl, which is asso- Erlotinib EGFR Lung Cancer
ciated with leukemia. Its dramatic success and relative (Tarceva)
Sorafenib VEGFR, PDGFR, Kidney
selectivity for tumors has led to its further use as an (Nexavar) c-Kit B-raf, Raf-1
RTK inhibitor in other cancers. Other successful anti- Sunitinib VEGFR, PDGFR,
cancer agents that target RTKs in cancer include (Sutent) c-Kit
tamoxifen (Nolvadex) and trastuzumab (Herceptin),
which target the estrogen receptor and ErbB2 RTKs,
respectively, in breast cancer and bevacizumab Biologics
(Avastin) that targets the VEGF RTK pathway in colon Trastuzumab ErbB2 Breast Cancer
cancer. Drugs in the pipeline include multitargeted (Herceptin) (HER-2/neu)
Cetuximab EGFR
tyrosine kinase inhibitors and inhibitors of the Raf (Erbitux)
and MEK kinases. Newer methods of inhibiting these Bevacizumab VEGF Colon, Pancreatic
signaling pathways also are being developed that (Avastin) Cancer
include selective knockout of RTK signaling

108

components using gene silencing technology such as
small interfering RNA (siRNA).
Phosphoinositol Metabolism RTKs stimulate phos-
phoinositol metabolism through their activation of
phospholipase C-g (PLC-g) and phosphoinositide-3
kinase (PI3K) through binding of their SH2 domains
to phospho-tyrosines on the receptor. Similar to Gq acti-
vation of PLC-b, activation of PLC-g results in hydrolysis
of PIP2 to form DAG and IP3, which promote PKC and
Ca2þ signaling to their downstream cellular targets. Acti-
vated PI3K phosphorylates phosphoinositides to gener-
ate the second messengers, PI(3,4)P2 and PI(3,4,5)P3.
PI(3,4,5)P3 induces membrane translocation of several
proteins including soluble protein tyrosine kinases, pro-
tein kinase B (Akt), and nucleotide exchange factors for
the small GTPases Arf and Rac. These proteins interact
with the phosphoinositides through plextrin-homology
domains (PH domains). These signaling proteins are
important for the modulation of proteins that directly
regulate cell survival by blocking apoptosis pathways
(Figure 6.7).
Inactivation of these signaling pathways is mediated
by phosphoinositide-specific phosphatases such as
phosphatase and tensin homolog (PTEN). PTEN is a
tumor suppressor protein that is mutated in several
human cancers. Loss of PTEN results in unregulated
cell survival, and thus aberrant cell growth.
6.1.4 Cytokine Receptors (Tyrosine
Kinase-Associated Receptors)
Tyrosine kinase-associated receptors signal by
recruiting activated cytosolic enzymes to the cell
membrane. The resulting activation is similar to
RTK signaling, but the kinase activity is not related
directly to the receptor molecule. Classic examples
of this type of receptor are cytokine receptors that modu-
late gene expression in immune cells as well as many
RTK
SH2
Y--p
Y--p Grb-2
SH2 SH3
Figure 6.7 Phosphoinositide signaling by receptor tyrosine kinases.
6.1 Receptor Communication
other cell types. Cytokine receptors include receptors
for interleukins, erythropoietin, and interferons.
When bound to ligand, these receptors dimerize and
gain binding affinity for members of the Janus-kinase
(JAK) family. This noncovalent binding increases the
kinase activity of JAK, which sets into motion phosphor-
ylation of multiple tyrosines on substrates including the
receptor. Key substrates are the signal transducers and
activators of transcription (STAT) proteins that dimer-
ize via their phosphorylated tyrosines/SH2 interactions,
and then translocate to the nucleus where they regulate
gene transcription. The majority of identified gene tar-
gets are related to immune cell function. Overactivity
of this pathway can lead to inflammatory disorders,
whereas lack of pathway function results in immune sup-
pression and susceptibility to infection.
The pathway is negatively regulated by protein tyro-
sine phosphatases and by feedback inhibition through
STAT-mediated expression of suppressors of cytokine
signaling (SOCs). Agonists for cytokine receptors are
currently used to stimulate red blood cell maturation
in anemia (erythropoietin) and to stimulate the
immune system (interferons). The cascade nature of
the cytokine pathway lends itself to future agents that
specifically target components of the pathway.
6.1.5 Intracellular Receptors
Intracellular receptors require ligands that are mem-
brane permeable and include receptors for steroid
hormones, lipophilic vitamins, and small molecules
such as nitric oxide and hydrogen peroxide. Members
of the steroid hormone receptor family are structur-
ally similar and exhibit similarities in their molecular
mechanisms (Figure 6.8). In the absence of ligand,
the receptor is retained in the cytoplasm by binding
to heat shock protein 90 (HSP90), which conceals
the receptor’s nuclear localization signal. Following
ligand binding, HSP90 is released and the receptor
rapidly translocates to the nucleus. Steroid receptors
Ras Ras Raf
GDP GTP
SOS MEK --p
ERK --p
Metabolism
Cell division
Cell motility
109
Chapter 6 Signal Transduction and Second Messengers
Cytoplasm
HSP90
GR
GR
Nucleus
Figure 6.8 Glucocorticoid receptor signaling directly modulates gene transcription.
function as transcription factors, thus once in the
nucleus they bind to specific steroid response ele-
ments on the DNA. These sequences are found
in the regulatory region of genes, and binding by
steroid receptors can either increase or decrease
transcriptional activity of the gene. For example,
glucocorticoids promote nuclear import of the gluco-
corticoid receptor, which results in an upregulation
of anti-inflammatory genes and a downregulation of
pro-inflammatory cytokines. Changes in gene tran-
scription mediated by steroid receptors are relatively
slow in onset yet result in long-term changes in gene
expression. Thus pharmacologic agents that mimic
steroid actions have a slow onset of effects, yet last a
long time.
Nitric oxide is a small diffusible gas that has localized
effects. Once inside the cell, NO activates soluble guany-
lyl cyclase, an enzyme that catalyzes the production of
cGMP from GTP. The cGMP produced has the primary
effect of activating cGMP-dependent protein kinase,
which has a number of effects including smooth muscle
cell relaxation. This mechanism of vasodilation is
manipulated by a number of important vasodilating
drugs that either mimic NO (such as nitroglycerin) or
prevent degradation of cGMP (such as sildenefil).
6.1.6 Other Classes of Receptors
Although the majority of receptors fit into the classes
described previously, there are some receptors that
do not. Smaller classes of receptors include receptor
110
Glucocorticoid
HSP90
HSP90
HSP90
GR
GRE
X
Inflammatory
genes
tyrosine phosphatases that are found primarily in
immune cells. These receptors can negate RTK signal-
ing by catalyzing the removal of phosphate from tyrosines
and can mediate immune suppression. Another small
class of receptors has intrinsic serine/threonine kinase
activity (transforming growth factor b receptor) and
guanylyl cyclase activity (B-type natriuretic peptide recep-
tor). A recombinant form of B-type natriuretic peptide
(Nesiritide) is used in the treatment of heart failure to
enhance vasodilation through the resulting increase in
cGMP production as described for NO earlier.
6.2 RECEPTOR/SECOND MESSENGER
CROSSTALK
Almost all second messenger signaling involves reversible
phosphorylation, so it is easy to see how interactions
between kinases across different second messenger sys-
tems can occur. Consensus sequences for phosphoryla-
tion can overlap leading to common substrates and, as
demonstrated by the MAP kinase pathway, many kinases
are themselves substrates for phosphorylation.
The phosphorylation modification lends itself to
both signal amplification and to flexible regulation.
Phosphorylation (-PO3) is a relatively dramatic modifica-
tion in that it adds three negative charges to the protein,
enough to significantly alter the three-dimensional con-
formation of the protein. The covalent phosphobond is
also quite stable when compared to allosteric bond
between receptor and ligand.

Although each signaling pathway has distinct “cas-
settes” of kinases and phosphatases, the downstream
effects of second messenger pathways are not linear.
Signal integration (crosstalk) occurs creating a com-
plex network of communication between the different
signaling pathways. Activation of G-protein-coupled
receptors often results in transactivation of RTKs, and
conversely signaling by RTKs can affect G-protein-
coupled receptor activity. For example, activation of
the angiotensin II receptor leads to activation of Gq,
which couples to PLC-b activity. The resulting increase
in Ca2þ and protein kinase C activity has direct effects
on signaling components of the RTK pathway, namely,
Ca2þ-mediated activation of the Raf kinase and protein
kinase C phosphorylation of scaffolding proteins that
enhance recruitment of the Ras GTP exchange factor,
SOS. Conversely RTK signaling can lead to tyrosine phos-
phorylation of G-protein-coupled receptors and also has
effects on the RGS proteins that stimulate GTPase activ-
ity. The overall result of a signal such as angiotensin II,
therefore, is a combination of effects on multiple signal-
ing pathways. These complicated effects make it more dif-
ficult to establish a single target for drug development,
yet provide the potential for selectivity of pharmacologi-
cal response depending on the cell environment and
intracellular signaling networks.
6.3 SIGNAL TRANSDUCTION TARGETS
FOR DRUG DISCOVERY
There has been an exhaustive quest to transfer the vast
amount of information related to signal transduction
that has been acquired over the last 60 years into specific
molecularly targeted agents. Enzymology was the focus of
drug discovery in the 1950s, which led to the develop-
ment of many agents including the anticancer folate inhi-
bitors and methotrexate. Both of these agents affect DNA
synthesis, thus cells entering the cell cycle cannot
undergo division and follow programmed cell death
(apoptosis). Although relatively selective for rapidly
dividing cells, the side-effects of these agents have signifi-
cant morbidity including severe immune compromise.
With the advent of precise ligand binding studies and
receptor cloning in the 1980s, many drugs were devel-
oped, or their mechanisms discovered, that act as ago-
nists or antagonists of G-protein-coupled receptors.
The disadvantage of many of these agents is the lack of
specificity for receptor subtypes and the tendency to
cause receptor downregulation and drug tolerance.
A better understanding of the genetics and cell
signaling pathways involved in the regulation of cell
division has led to molecular targets that are down-
stream of the receptor and are selective to second mes-
senger pathways. Some current therapies, such as
lithium to treat mania, had their mechanisms discov-
ered long after they were used therapeutically. This
modern approach to drug targets has especially bene-
fited the cancer chemotherapy field. Several agents tar-
get signaling molecules such as tyrosine kinases and
have shown efficacy in the treatment of a variety of
cancers as described earlier (Table 6.3).
References and Further Reading
The future challenge of therapy targeted at signal
transduction pathways entails both identifying the best
targets and determining its overall effect on outcome.
It is these two goals that have led to an increase in
demand for clinical translational research. Patient-
based research is necessary to reveal the effects of sig-
nal transduction crosstalk in a disease setting where
both the targets and the outcome can be identified,
measured, altered, and then correlated with preven-
tion or progression of the disease state.
REVIEW QUESTIONS
1. What is the advantage of signaling by way of a
receptor coupling to an enzyme?
2. How can a neurotransmitter elicit the opposite
response in different cells? For example: epineph-
rine elicits heart muscle contraction and gastroin-
testinal smooth muscle relaxation.
3. What is the turn-off mechanism for Gq-mediated
signaling?
4. If lithium blocks recycling of phosphoinositides,
what receptor/G protein pathways will it affect?
5. What changes in the tyrosine kinase/Ras pathway
could cause overactivity of the receptor pathway,
possibly leading to unregulated cell division?
6. What is the leading target of newly approved anti-
cancer drugs that act on the growth factor recep-
tor/MAP kinase pathway?
7. The cytokine receptor signaling pathway in
immune cells is referred to as the JAK/STAT path-
way. How do JAK and STAT coordinately regulate
the immune response?
8. How are steroid receptors regulated differently
than plasma membrane receptors?
9. Explain the concept of transactivation between
receptor signaling pathways. How does this affect
individual signaling pathways?
10. What are the advantages and disadvantages of tar-
geted therapies based on knowledge of signal
transduction pathways?
REFERENCES AND FURTHER READING
Hubbard, K. B., & Hepler, J. R. (2006). Cell signalling diversity of the
Gqalpha family of heterotrimeric G proteins. Cell Signal, 18(2),
135–150.
Many receptors for neurotransmitters and hormones rely upon
members of the Gqalpha family of heterotrimeric G-proteins to exert
their actions on target cells. Galpha subunits of the Gq class of G-pro-
teins (Gqalpha, G11alpha, G14alpha, and G15/16alpha) directly link
receptors to activation of PLC-beta isoforms, which in turn, stimulate
inositol lipid (i.e., calcium/PKC) signaling. Although Gqalpha family
members share a capacity to activate PLC-beta, they also differ mark-
edly in their biochemical properties and tissue distribution that pre-
dicts functional diversity. Nevertheless, established models suggest
that Gqalpha family members are functionally redundant and that
their cellular responses are a result of PLC-beta activation and down-
stream calcium/PKC signaling. Growing evidence, however, indi-
cates that Gqalpha, G11alpha, G14alpha and G15/16alpha are
functionally diverse and that many of their cellular actions are inde-
pendent of inositol lipid signalling. Recent findings show that
111
Chapter 6 Signal Transduction and Second Messengers
Gqalpha family members differ with regard to their linked receptors
and downstream binding partners. Reported binding partners dis-
tinct from PLC-beta include novel candidate effector proteins, vari-
ous regulatory proteins, and a growing list of scaffolding/adaptor
proteins. Downstream of these signaling proteins, Gqalpha family
members exhibit unexpected differences in the signaling pathways
and the gene expression profiles they regulate. Finally, genetic stud-
ies using whole animal models demonstrate the importance of cer-
tain Gqalpha family members in cardiac, lung, brain, and platelet
functions among other physiological processes. Taken together,
these findings demonstrate that Gqalpha, G11alpha, G14alpha, and
G15/16alpha regulate both overlapping and distinct signaling path-
ways, indicating that they are more functionally diverse than previ-
ously thought.
Milligan, G., & Kostenis, E. (2006). Heterotrimeric G-proteins: A short
history. British Journal of Pharmacology, 147 (Suppl. 1), S46–S55.
Some 865 genes in man encode G-protein-coupled receptors
(GPCRs). The heterotrimeric guanine nucleotide-binding proteins
(G-proteins) function to transduce signals from this vast panoply of
receptors to effector systems including ion channels and enzymes
that alter the rate of production, release or degradation of intracellu-
lar second messengers. However, it was not until the 1970s that the
existence of such transducing proteins was even seriously suggested.
Combinations of bacterial toxins that mediate their effects via cova-
lent modification of the alpha-subunit of certain G-proteins and
mutant cell lines that fail to generate cyclic AMP in response to ago-
nists because they either fail to express or express a malfunctional
G-protein allowed their identification and purification. Subsequent
to initial cloning efforts, cloning by homology has defined the
human G-proteins to derive from 35 genes, 16 encoding alpha-subu-
nits, five beta and 14 gamma. All function as guanine nucleotide
exchange on-off switches and are mechanistically similar to other
proteins that are enzymic GTPases. Although not readily accepted
initially, it is now well established that beta/gamma complexes medi-
ate as least as many functions as the alpha-subunits. The generation
of chimeras between different alpha-subunits defined the role of dif-
ferent sections of the primary/secondary sequence and crystal struc-
tures, and cocrystals with interacting proteins have given detailed
understanding of their molecular structure and basis of function.
Finally, further modifications of such chimeras have generated a
range of G-protein alpha-subunits with greater promiscuity to inter-
act across GPCR classes and initiated the use of such modified G-pro-
teins in drug discovery programs.
112
Natarajan, K., & Berk, B. C. (2006). Crosstalk coregulation mechan-
isms of G protein-coupled receptors and receptor tyrosine
kinases. Methods in Molecular Biology, 332, 51–77.
G-protein-coupled receptors (GPCRs) and receptor tyrosine
kinases (RTKs) are transmembrane receptors that initiate intracellu-
lar signaling cascades in response to a diverse array of ligands.
Recent studies have shown that signal transduction initiated by
GPCRs and RTKs is not organized in distinct signaling cassettes,
where receptor activation leads to cell division and gene transcrip-
tion in a linear manner. In fact, signal integration and diversification
arises from a complex network involving cross-communication
between separate signaling units. Several different styles of crosstalk
between GPCR- and RTK-initiated pathways exist, with GPCRs or
components of GPCR-induced pathways being either upstream or
downstream of RTKs. Activation of GPCRs sometimes results in a
phenomenon known as transactivation of RTKs, which leads to the
recruitment of scaffold proteins, such as Shc, Grb2, and SOS in addi-
tion to mitogen-activated protein kinase activation. In other cases,
RTKs use different components of GPCR-mediated signaling, such
as beta-arrestin, G protein-receptor kinases, and regulator of G-pro-
tein signaling to integrate signaling pathways. This chapter outlines
some of the more common mechanisms used by both GPCRs and
RTKs to initiate intracellular crosstalk, thereby creating a complex
signaling network that is important to normal development.
Neer, E. J. (1995). Heterotrimeric G proteins: Organizers of trans-
membrane signals. Cell, 80(2), 249–257.
Neves, S. R., Ram, P. T., et al. (2002). G protein pathways. Science, 296
(5573), 1636–1639.
The heterotrimeric guanine nucleotide-binding proteins (G-pro-
teins) are signal transducers that communicate signals from many
hormones, neurotransmitters, chemokines, and autocrine and para-
crine factors. The extracellular signals are received by members of
a large superfamily of receptors with seven membrane-spanning
regions that activate the G-proteins, which route the signals to sev-
eral distinct intracellular signaling pathways. These pathways interact
with one another to form a network that regulates metabolic
enzymes, ion channels, transporters, and other components of the
cellular machinery controlling a broad range of cellular processes,
including transcription, motility, contractility, and secretion. These
cellular processes in turn regulate systemic functions such as embry-
onic development, gonadal development, learning and memory, and
organismal homeostasis.
Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases.
Cell, 103(2), 211–225.