Chapter 11 - Signal Transduction

184
CONTENTS
Introduction 184
General Principles of Signal Transduction 184
Cell Structures 185
General Principles of Receptors and Cell
Signaling 186
Mechanisms of Intracellular Signaling 187
Select Signaling Systems Relevant to Allergy 197
Additional Therapeutic Considerations 200
Conclusion 201
11
Signal Transduction
PAUL J. BERTICS* | CYNTHIA J KOZIOL-WHITE |
MONICA L. GAVALA | GREGORY J. WIEPZ
Introduction
The processes whereby various external and internal stimuli
(signals) serve to modulate cellular behavior are collectively
known as signal transduction. These events are critical for the
control of cell growth, differentiation, function, movement, and
adaptability. Cells can respond to diverse stimuli that range
from small molecules, such as ions and various nutrients, to
larger molecules, including hormones, cytokines, growth
factors, toxins, and allergens. In addition, processes such as cell-
cell contact, cell adhesion to matrix components, cell deforma-
tion, or even cell damage can all elicit specic signal transduction
processes, allowing the cell to respond appropriately to differing
situations and challenges. Furthermore, although many studies
have focused on the capacity of external factors to transduce
intracellular signals and alter cell behavior, a process termed
outside-in signaling, there is an increasing appreciation for the
reverse process, inside-out signaling, wherein intracellular events
control how the cell interacts with its external environment. For
example, certain intracellular signals can result in the activation
or inhibition of cell-cell or cell-matrix adhesion molecules such
as the integrins.
In general, signal transduction events are tightly regulated
and involve the coordinated action of numerous molecules to
affect the proper change in cell phenotype and function.
However, cells are exposed to numerous stimuli simultaneously
that culminate in the ultimate behavioral change in the cell. The
outcome depends on a multitude of interactions that regulate
the appropriate response or, because of an imbalance in media-
tors, can result in a disease state. Modulation of specic cellular
signaling events, cellular products, or interactions is the basis of
many pharmacologic interventions.
The study of cell signaling is a diverse, rapidly expanding
eld driven by an understanding of how cells respond to various
stimuli in order to develop therapeutics that can selectively
target cell-specic behaviors. Accordingly, knowledge of the
mechanisms by which various hormones, cytokines, chemo-
kines, and allergens can modulate immune cell function is
important to the eld of allergy. Although research into signal
transduction is extensive,
1
several major themes can be dened
at the cellular level and with respect to immune cell function.
Thus, this chapter rst focuses on general principles in cell
signaling, such as receptors, relevant cell structures, and
common signaling mechanisms. This is followed by a discus-
sion of select signaling systems that are key to immune func-
tion, including several families of activating and inhibitory
receptors, their downstream signaling cascades, and their mod-
ulation. Throughout, therapeutic aspects associated with certain
signaling processes are examined.
General Principles of Signal
Transduction
This chapter focuses on the signal transduction mechanisms
initiated at the plasma membrane (outside-in signaling).
*In memory of a great scientist, mentor, and teacherPaul John Bertics:
November 6, 1956, to December 22, 2011.
SUMMARY OF IMPORTANT CONCEPTS
Signal transduction is the process whereby various external and
internal stimuli (signals, ligands) initiate a series of events that
modulate cellular behavior.
Initiation of outside-in signaling begins with ligand binding of a
receptor that is present on the cell membrane for nonpermeable
ligands or at intracellular sites for lipophilic/cell-permeable
ligands.
Cell surface receptors often respond to ligand binding by modu-
lating intracellular enzymes (kinases, lipases, phosphatases), G
protein activity, ion channels, and gene transcription/mRNA pro-
cessing or by serving as scaffolding sites for other signaling or
regulatory proteins.
Intracellular receptors generally act as DNA/chromatin-binding
molecules that promote or inhibit specic gene transcriptional
activities.
Signal transduction is often amplied by the action of intermedi-
ates (second messengers), including small molecules (e.g.,
cAMP), proteins (G proteins), lipids (diacylglycerol), ions (Ca
2+
),
and gases (nitric oxide).
Receptor activation can regulate multiple cellular functions
(secretion/degranulation, migration, replication, differentiation,
apoptosis), offering numerous targets and tremendous possibili-
ties for modulation of distinct intracellular pathways for the phar-
macologic management of inammation and specic diseases.
11 Signal Transduction 185
Cell Structures
The cell is a sophisticated entity that can respond to a multitude
of stimuli and changes from its microenvironment, producing
a precise outcome that maintains the status quo of the body.
Cellular anatomy has developed in such a manner as to isolate
the internal workings from nonspecic stimulation and to com-
partmentalize the machinery by grouping specic molecules
that can interact within and between their location. Although
many structures make up a typical cell, the parts relevant to
signal transduction include the plasma membrane, cytoskele-
ton, and several organelles. (See Alberts and associates
2
for an
extensive treatise on the biology of the cell.)
PLASMA MEMBRANE
Eukaryotic cells are encapsulated by a plasma membrane that
makes the cell selectively permeable to many extracellular
factors, including nutrients, lipids, proteins, ions, and patho-
gens. The plasma membrane is a uid lipid bilayer containing
a complex mixture of phospholipids, glycolipids, sterols, and
proteins. This structure serves as an effective barrier to mole-
cules that are poorly lipid soluble and also plays a critical role
in the bidirectional ow of information. This conduction of
information includes the specic recognition of extracellular
factors, including hormones, toxins, adhesion molecules, and
pathogens, that function to modulate cellular responses. As pre-
viously noted, the recognition of these factors at the plasma
membrane is mediated by receptors, and receptor engagement
elicits changes in cell behavior through the alteration of
Because cell signaling involves many effector molecules and
cellular structures, it is important rst to dene several basic
concepts (Fig. 11-1).
For example, external stimuli that cannot freely enter the
cell, such as water-soluble factors (e.g., cytokines, chemokines)
or various externally tethered molecules (e.g., cell surface pro-
teins, extracellular matrix components) generally interact with
specic cellular recognition molecules (receptors) that possess
an externally facing ligand-binding site. These cell surface
receptors then transduce information into the cell through
various conformational and enzymatic activities that allow for
signal amplication and regulation of specic intracellular
enzyme activities, ion uxes, cytoskeletal reorganization, and
secretory events. Also, depending on the receptor, cell surface
initiated signaling may alter transcriptional activities, chroma-
tin structure, messenger RNA (mRNA) stability/processing
(e.g., microRNAs), translational activity, and posttranslational
processing. Furthermore, signaling through cell surface recep-
tors can result in receptor desensitization, internalization, and
recycling to achieve feedback control and to prevent excessive
stimulation that may lead to pathology.
With respect to lipid-soluble factors that can penetrate the
membrane, such as steroid hormones, the receptors for these
molecules are largely located within the cell, often in the nucleus,
wherein ligand-receptor complex formation generally serves to
modulate gene transcription. Once again, feedback pathways
exist to contain the magnitude and duration of the initiated
signals. Specic cellular structures, receptors, and processes are
common to numerous signal transduction systems associated
with allergy, as discussed next.
Figure 11-1 Overview of signal transduction. This model depicts many of the primary pathways initiated following ligand binding to its cognate
receptor. In general, receptor activation leads to signal amplication and modication of cellular events, including secretion, cytoskeletal changes,
and certain enzymatic activities and alterations in gene expression (transcription, mRNA stability, protein induction). All these processes exhibit
complex feedback controls. An understanding of these signaling systems is valuable in the development of therapeutics for allergic disorders.
Intracellular
receptors
Lipid soluble
(e.g., glucocorticoids)
Hormones/
ligands
Water soluble
(e.g., IL-5)
Cell surface receptors
Feedback controls
Amplification cascades
Regulation of enzyme receptor activity
Cytoskeletal reorganization
Ion fluxes, secretion, etc.
Regulation of transcription
Protein stability mRNA stability and transcription control
(e.g., microRNAs)
Modulation of processing/secretion
Nuclear receptors/
regulatory proteins
Extracellular
Plasma
membrane
Cytoplasm
Nucleus
186 SECTION A Basic Sciences Underlying Allergy and Immunology
potential or permeability that occur with certain signaling
events can mediate programmed cell death (apoptosis).
General Principles of Receptors
and Cell Signaling
The initiation of outside-in signaling begins with a receptor,
generally a protein(s) that selectively binds the signal-initiating
factor (e.g., cytokine, hormone). Receptors exist on the plasma
membrane for ligands that cannot readily enter the cell, as well
as at intracellular sites (e.g., cytoplasm, nucleus) for ligands that
are lipophilic or cell permeable (e.g., corticosteroids). Ligand-
receptor (LR) interaction is selective and is required for initiat-
ing the signaling response. The biologic response is often
proportional to LR complex formation, which can be described
by the following equilibrium expression:
[ ] [ ] [ ] L R LR +
where [L], [R], and [LR] are the concentrations of free ligand,
unoccupied receptor, and ligand-receptor complex, respectively.
The dissociation constant (Kd) for LR breakdown follows:
Kd L R / LR = [ ] [ ] [ ]
In general, ligand-receptor interaction is of high afnity
(Kd <10
9
M), and the receptor number per cell is small (satu-
rable), which limits the signal that is produced. Because [LR] is
proportional to output, the signal can be regulated by altering
ligand or receptor concentration or by modulating receptor
afnity (Kd). This is a perfect therapeutic target, primarily
through the use of antibodies that can bind with higher afnity
and block the binding of ligand to the receptor, thus inhibiting
activation or sequestering the ligand before it binds to the
receptor. For example, the humanized antiinterleukin-5 anti-
bodies mepolizumab and reslizumab block the availability of
IL-5 to activate IL-5 receptors on eosinophils and are being
tested for the treatment of asthma.
4
In some cases, multiple receptors for the same ligand permit
separate signaling behaviors, ligand sensitivities, and distinct
cell type responses. Receptor afnity and internalization/
turnover are often controlled by ligand binding, and these
events can regulate or desensitize LR formation and cell respon-
siveness. Furthermore, various end points may require different
degrees of receptor occupancy; for example, some effects are
detected at low receptor occupancy, because this induces enough
of one type of signal, whereas other effects may need full recep-
tor occupancy, requiring a larger or different signal. For example,
IL-5 or granulocyte-macrophage colony-stimulating factor
(GM-CSF) at nanogram/milliliter (ng/mL) levels can increase
eosinophil survival, but higher cytokine levels are needed to
enhance cell adhesion or degranulation.
The following sections detail how certain cell surface recep-
tors respond to ligand binding by activating enzymes such as
protein and lipid kinases, inducing changes in G protein activ-
ity, regulating ion channels, and serving as docking or scaffold-
ing sites for other signaling proteins. Conversely, intracellular
receptors often act as DNA/chromatin-binding molecules that
promote or inhibit specic gene transcriptional activities. These
events can alter protein expression or activation and induce a
change in cell behavior. These cascades are not usually initiated
by a single hormone or receptor system in vivo, but are often
inuenced by the concerted action of numerous factors that are
presented to the cell simultaneously or sequentially. However,
intracellular processes (signal transduction). These processes
entail changes in the action of various enzymes, structural pro-
teins, adapter molecules, and transcription factors. In addition,
the regulated ow of ions (e.g., Ca
2+
) across the plasma mem-
brane can modulate various signaling events.
Another important feature of the plasma membrane is the
presence of specialized microdomains (lipid rafts) that consist
of a unique composition of sterols, lipids, and proteins. These
localized differences in membrane structure promote the
recruitment of certain receptors and associated molecules to
these regions, facilitating the rapid activation of these signaling
complexes in response to appropriate stimuli.
CYTOSKELETON
The cytoskeleton consists of various bers and laments with
specialized functions that contribute to the cells shape, mobil-
ity, and function (e.g., endocytosis) as well as to the intracellular
movement of proteins, vesicles, and chromosomes. The major
components include microlaments (e.g., actin), intermediate
laments (e.g., laminin, vimentin), and microtubules (e.g.,
-tubulin). Because of their nature (negatively charged), the
cytoskeletal elements present a favorable surface that allows
their association with many signaling molecules (e.g., kinases,
low-molecular-weight guanine nucleotidebinding proteins [G
proteins], phospholipases), and these structures also provide
support for localized anchoring as well as directed transport
within a cell.
3
CELLULAR ORGANELLES
The endoplasmic reticulum (ER), nucleus, and mitochondria
also play key roles in intracellular signaling. The ER is a mem-
branous network of tubules and cisternae continuous with the
nuclear envelope that participates in many cell functions,
including protein synthesis, ion sequestration/release, and
processing/trafcking of membrane-associated and secreted
proteins. The ER, similar to the plasma membrane, contains
channels that allow for ions, especially calcium (Ca
2+
), to
undergo regulated release into the cytoplasm, where they mod-
ulate various signaling processes.
The nucleus is a membrane-enclosed organelle that contains
most of the cells genetic information (chromosomes) and is the
major site of gene regulation. Cell responses to external and
internal stimuli can lead to changes in gene expression through
the alteration of transcription factor activity and chromatin
structure. In this regard, DNA is tightly wrapped around pro-
teins called histones, thereby forming higher-order structures
known as nucleosomes. Modications such as acetylation and
methylation, in response to cellular, environmental, or develop-
mental cues, can modulate histone function and affect gene
expression, so-called epigenetics. Following gene transcription,
the mRNA is processed, transported out of the nucleus, and
translated into protein at sites such as the ER. These processes
are subject to regulation by distinct signal transduction
networks.
Mitochondria are major sites of metabolic function, includ-
ing lipid/carbohydrate metabolism, oxidative phosphorylation,
and adenosine triphosphate (ATP) synthesis. This organelle
participates in the metabolism (e.g., hydroxylation, sulfation)
of molecules for excretion and in the destruction of oxidative
free radicals. Also, alterations in mitochondrial membrane
dephosphorylates and inactivates extracellular signalregulated
kinases (ERKs) and p38 MAP kinases that are linked to
the control of gene transcription, cell cycle progression, and
stress responses. In contrast, protein dephosphorylation can
sometimes lead to activation, as with the tyrosine kinase Src
(see Fig. 11-2).
5
ASSEMBLY OF SIGNALING COMPLEXES
The orchestrated assembly of various proteins and lipids into
deliberate cascades is central to progression of signal transduc-
tion. Many mechanisms are used to achieve this goal, includ-
ing the promotion of protein-protein and/or protein-lipid
to some degree, activation of a specic pathway leads to a well-
documented series of choreographed steps that are comparable
across many cell types, although cell-specic differences do
exist.
Mechanisms of Intracellular Signaling
Signals emanating from an activated receptor are often medi-
ated or amplied through effector molecules known as intra-
cellular messengers or second messengers. These intermediates
may promote gene expression and protein synthesis, but often
they regulate proteins and factors already present in the cyto-
plasm, initiating a rapid signaling response. Common cell-
signaling processes and posttranslational modications include
protein phosphorylation and dephosphorylation, the assembly
of signaling complexes through protein-protein and protein-
lipid interactions, and protein modications (e.g., ubiquity-
lation, sumoylation, acetylation, methylation). Other signaling
processes promote the modication of membrane lipids and
the initiation of cytoplasmic ion uxes (often Ca
2+
). Effector
molecules are often assembled into modules and compart-
mentalized in cells, and these signaling systems are frequently
similar between cell types and across species. Interestingly, the
activation/deactivation of these common signaling modules
does not necessarily result in the same response between cell
types (because of the differential expression of intracellular
effector molecules) or even within a cell type (because a cell
often integrates multiple stimuli simultaneously). This dis-
cussion introduces major signaling pathways that operate in
many cells.
PHOSPHORYLATION/DEPHOSPHORYLATION
Activation or inhibition of signaling proteins can involve phos-
photransfer from ATP to specic amino acids (generally serine,
threonine, and/or tyrosine) by enzymes known as protein
kinases. These enzymes exhibit unique substrate specicities
and selectively regulate various pathways. Kinase specicity
arises from the recognition of certain amino acid sequences
surrounding the residue(s) to be phosphorylated, and these
recognition sequences are one of the parameters used to classify
kinases into different families. In general, the regulation of
protein kinases leads to the control of their protein substrates,
thereby transmitting the signal to downstream signaling targets.
In certain cases, phosphorylation can elicit a conformational
change that removes an allosteric inhibitor, allowing for protein
activation (Fig. 11-2). An example of this process is the phos-
phorylation of protein kinase C isoforms, whereby phosphory-
lation alters the enzyme conformation such that a pseudosubstrate
domain dissociates from the catalytic site. This process leads
to kinase activation and allows substrate access to the catalytic
site. Phosphorylation can also inhibit the function of certain
enzymes, such as myosin light chain kinase (MLCK). Phos-
phorylation desensitizes MLCK to activation by Ca
2+
and Ca
2+
-
dependent kinases, thereby preventing it from phosphorylating
myosin, which is necessary for force generation in muscle
contraction.
Protein phosphorylation is a transient modication, and
phosphoprotein phosphatases catalyze the removal of protein-
associated phosphates. Generally, dephosphorylation halts
protein activation and signal amplication; for example,
mitogen-activated protein (MAP) kinase phosphatase-1
Figure 11-2 Examples of intramolecular interactions that regulate
protein function. A, Intramolecular association of a pseudosubstrate
domain in the amino terminal domain of protein kinase C zeta (PKC)
sterically occludes the enzyme active site and blocks catalytic activity.
Nearby adenosine triphosphate (ATP) binding and phosphorylation of
this protein activates the enzyme by inducing a conformational change
that promotes pseudosubstrate dissociation and increased substrate
access to the catalytic site. B, Association of two domains within Src
family kinases (e.g., Src, Lyn) in the resting, primed, and active
states. In the inactive state (left) the SH2 domain of these kinases
associates with a phosphorylated tyrosine (P) on the kinase domain
C-terminal region. On dephosphorylation of this tyrosine by specic
phosphotyrosine phosphatases (middle), the protein unfolds and is
in a state suitable for activation, which occurs when another tyrosine
located in the kinase domain becomes phosphorylated (right).
ATP binding site
Cysteine rich site
ATP binding &
phosphorylation
ATP binding
COOH
COOH
Hinge
Protein kinase C
zeta chain
C4
Pseudosubstrate
Pseudosubstrate
Substrate
Bound to catalytic
site (kinase activation)
P P
Phosphoryl transfer site
A
B
C3
C3
C1
C1 NH
2
SH3
SH2
Kinase
NH
2
P
SH3 SH3
Fully active Primed Inactive
SH2
Kinase
SH2
Kinase
P
interaction via molecular colocalization and/or phosphory-
lation, protein modication or effector binding to alter turn-
over, and the regulated trafcking of signaling molecules to
discrete cellular compartments to restrict their movement and
increase their potential interaction. Many signaling molecules
contain specic motifs that mediate these protein-protein/lipid
interactions (Table 11-1). Examples include phosphotyrosine
interaction motifs known as Src homology type 2 (SH2), phos-
photyrosine binding (PTB), and immunoreceptor tyrosine-
based activation motifs (ITAMs), as well as phospholipid-binding
domains known as Pleckstrin homology (PH) domains, and
proline-richbinding domains such as Src homology type 3
(SH3) domains. Protein modication and complex formation
can also have a negative role in signaling, as when modication
(e.g., ubiquitylation) or effector molecule binding (e.g., c-Cbl)
targets a protein for degradation via cellular proteases /protea-
some, or when a recruited effector molecule either is an inacti-
vating enzyme (e.g., phosphotyrosine phosphatase SHP-1)
or recruits an inactivating enzyme (e.g., Dok adapter mole-
cules recruit protein phosphatases or G proteininactivating
molecules).
Another mechanism for controlling signaling molecules is to
modify trafcking of the molecules such that they are uniquely
localized even before an initiating signal. Examples include lipid
modications (e.g., palmitoylation, myristoylation, isoprenyl-
ation) of proteins (e.g, Src, certain G proteins) that allow for
their membrane localization and recruitment of receptors,
kinases/phosphatases, G proteins, and adapter molecules (that
link together various signaling molecules) into membrane
microdomains containing high levels of cholesterol, glycolipids,
and specic proteins (lipid rafts).
COMMON SIGNALING MECHANISMS
ASSOCIATED WITH PROTEIN
PHOSPHORYLATION
Signaling from an activated cell surface receptor often involves
its transduction and amplication through second messengers,
which include the regulated synthesis and turnover of cyclic
adenosine 3,5-monophosphate (cAMP), initiation of Ca
2+

uxes, and generation of phospholipid metabolites.
Heterotrimeric G Proteins and cAMP Formation
Cyclic AMP is critical for the actions of many factors (e.g.,
epinephrine), and the ultimate response to cAMP depends on
the enzyme prole of the target cell. Intracellular cAMP levels
represent a balance between cAMP formation from ATP through
adenylate cyclases and its hydrolysis to 5-AMP by cAMP-
phosphodiesterases (PDEs). Human adenylate cyclases are
located at the plasma membrane, differentially expressed and
tightly regulated. These systems are composed of a receptor, a
guanine nucleotidebinding protein complex (G protein), and
an adenylate cyclase isoform. The complex spans the membrane
seven times (so-called 7-spanner receptors), with the G protein
coupled receptor (GPCR) ligand-binding site exposed to the
extracellular space and the G protein/adenylate cyclase on the
cytoplasmic side
6
(Figs. 11-3 and 11-4). The GPCR, depending
on its sequence, interacts with a specic heterotrimeric G
protein complex, which is composed of , , and subunits. At
rest, the G subunit contains a bound guanosine diphosphate
(GDP), but on ligand binding, a receptor-mediated conforma-
tional change promotes the exchange of guanosine triphosphate
Common Protein Motifs Associated with
Signal Transduction Molecules
Motifs Name and function Examples
DD Death domain; 80- to 100-
residue motifs that allow for
heterodimerization of
molecules containing death
domains; apoptosis by
caspases
TNFR1, TRADD,
IRAK4, Fas
DH DBL homology domain; confers
exchange activity to GTP
exchange proteins (e.g., Ras,
Rac, Rho)
Vav, Sos
ITAM Immunoreceptor tyrosine-based
activation motif; interacts with
tyrosine kinases to propagate
signals for receptors lacking a
cytoplasmic tail
CD3, FcRI,
Ig-/Ig-
ITIM Immunoreceptor tyrosine-based
inhibition motif; region
necessary for negative
regulation of some immune
receptor signaling
FcRIIB,
(CD326),
PECAM-1
(CD31),
Siglecs
PDZ Binds to the C-terminal 4 to 5
residues, frequently part of
transmembrane receptors, at a
Ser/Thr-X-Val sequence, or ion
channels
Na
+
/H
+
pumps
bind to
-adrenergic
receptors
through these
domains
PEST Prolineglutamic acidserine-
threoninerich sequence
Targets proteins for rapid
degradation to proteosome
Notch1
PH Pleckstrin homology domain;
binds phosphoinositides to
allow a protein to be
responsive to lipid messengers
Sos, PLC, Akt,
Btk
PTB Phosphotyrosine-binding
domain; binds Asn-Pro-X-Tyr
motifs
Shc and IRS-1
PX Phox homology domain; binds
PIP
3
, targeting the proteins to
the membrane
p40
phox
, p47
phox
RING
nger
Characteristic of ubiquitin
ligases; transfers ubiquitin to a
substrate protein
TRAF6
SH2 Src homology 2 domain; binds
specic phosphotyrosine-
containing peptide motifs
Shc, Grb2,
STATs
SH3 Src homology 3 domain; binds
to proline-rich sequences with
a minimal consensus site of
Pro-X-X-Pro (with proline
being preceded by aliphatic
residue)
Src, Crk
SLAM Signaling lymphocyte activation
molecules; inuence outcome
of T cellantigen-presenting
cell and natural killer cell
target cell interactions
CD84, CD48,
CD150,
CD229
SOCS
box
Suppressors of cytokine signaling
domain; involved in targeting
proteins for ubiquitylation
SOCS-1,
SOCS-3
UBA Ubiquitin-associated domain;
40-residue motif that interacts
with ubiquitinated residues
c-Cbl, E2
ubiquitin
conjugating
enzyme
TABLE
11-1
Cyclic AMP and Protein Phosphorylation
The effects of cAMP are mediated by protein phosphorylation
events catalyzed by the cAMP-dependent protein kinase (PKA).
PKA is composed of two catalytic (kinase) subunits (C), and
two regulatory subunits (R) that bind cAMP. Inactive PKA is a
complex of R
2
C
2
, but on binding two cAMPs to each R, the
complex dissociates and generates free C subunits that are cata-
lytically active.
Active PKA phosphorylates many enzymes, transcription
factors, and other proteins, and this amplication allows for a
small amount of hormone to stimulate the production of many
end products and effects. The signal can be reversed by protein
dephosphorylation of the target proteins via phosphatases,
which are also regulated. Additionally, the degradation of cAMP
by phosphodiesterases, which break the phospodiesterase bond,
also limits the extent of the induced signal. Conversely, cAMP
action can be prolonged by certain therapeutics that inhibit
specic PDEs. For example, the PDE4 inhibitor roumilast is
being tested for the treatment of chronic obstructive pulmonary
disease and asthma.
7
Increases in cAMP can modulate gene transcription
through PKA-mediated phosphorylation of transcription
factors, including the cAMP regulatory (response) element
binding (CREB) protein. Phosphorylation of CREB regulates
its interaction with DNA and other transcriptional control
(GTP) for bound GDP. The resulting G-GTP complex dissoci-
ates from the G/G subunits. The G-GTP complex, depend-
ing on the G isoform, may either stimulate (Gs) or inhibit
(Gi) adenylate cyclase. The free G/G subunits can also
interact with certain adenylate cyclases (as well as with other
effectors; see later). G protein effects are rapidly but only tran-
siently manifested because the G subunits possess intrinsic
GTPase activity that slowly hydrolyzes GTP to GDP. Thus, GTP
hydrolysis turns off the action of the G subunits and promotes
the reassociation of the G-GDP, G, and G subunits.
Many receptors are coupled to G proteins (e.g., chemokines;
see Chapter 7), and other classes of heterotrimeric G protein
isoforms besides Gs and Gi exhibit specic receptor coupling
proles and regulate downstream effectors other than adenylate
cyclases. These other heterotrimeric G proteincoupled systems
modulate signaling molecules such as phospholipases, nucleo-
tide exchange factors, or phosphodiesterases (see Fig. 11-4).
Additionally, there are other G protein classes, including the
low-molecular-weight (LMW; about 21 kD) G proteins (Ras,
Rac, Rho, Cdc42) that exist as monomers and are regulated by
other receptors/proteins that facilitate GDP-GTP exchange and
GTPase activity. As discussed later, these LMW G proteins regu-
late protein kinase cascades, such as the MAP kinases, and are
linked to cell growth control, differentiation, secretion, gene
expression, motility, and cytoarchitecture.
Figure 11-3 Heterotrimeric G proteins: receptor association, G protein cycling, and target effectors. Many signaling systems important for
immune cell function entail the extremely rapid activation of G proteincoupled receptors (GPCRs). Each GPCR can regulate one or more het-
erotrimeric G protein complexes, composed of , , and subunits. In the resting state, guanosine diphosphate (GDP) is bound to G in the
receptor-associated complex, but on ligand-induced changes in GPCR conformation, the GDP is replaced with guanosine triphosphate (GTP).
The G-GTP dissociates from G/G, and the subunits modulate specic effectors depending on the G protein isoform. G contains intrinsic
GTPase activity that hydrolyzes the bound GTP to GDP, and the G-GDP reassembles with G/G to return to the resting state. GEF, Guanine
nucleotide exchange factor; Lbc, oncogene that encodes a Rho-GEF.
No ligand
GPCR
Ligand
-GTP
GTP GDP
-GDP
GTPase
Pi
-GDP -GTP
Adenylate cyclase (cAMP increases)
Protein kinase A (PKA)
Axin
G
s
(G
s
,

G
sXL
, G
solf
)
Adenylate cyclase (cAMP decreases)
Phospholipases
Phosphodiesterases
G
(G
1-3
,

G
o
, G
t
,

G
z
, G
gust
)
PLC (intracellular Ca
2+
increases)
Phosphoinositide turnover
Protein kinase C (PKC)
Rho
G
q
(G
q
,

G
14
, G
11
,

G
15
, G
15/16
)
p115-Rho GEF
Leukemia associated Rho-GEF (LARG)
Post-synaptic diversity protein (PDZ)-Rho GEF
A-kinase anchoring protein (AKAP)-Lbc
Rho
G
12
(G
12
,

G
13
)
P13 Kinase
PLC
Ion channels
Cytoplasm
Plasma
membrane
GPCR ligands (examples)
Peptides and proteins (chemokines)
Biogenic amines (epinephrine, histamine)
Lipids (prostaglandins, leukotrienes)
Amino acids (glutamate)
Ions (calcium)
Nucleotides/nucleosides (ATP)
Extracellular
Phospholipases
Phospholipid metabolites can act as intracellular and intercel-
lular signaling molecules. Many factors activate phospholipases
involved in the hydrolysis of either the head group or the fatty
acids from the glycerol backbone of specic phospholipids (Fig.
11-5, A). Different phospholipases hydrolyze distinctive por-
tions of the phospholipid; phospholipases A
1
(PLA
1
) and A
2

(PLA
2
) hydrolyze the ester bonds of the intact phospholipid at
the C-1 and C-2 positions of the glycerol backbone, respectively.
Phospholipase C (PLC) hydrolyzes the phosphodiester bond
between the phosphate of the head group and the glycerol back-
bone, whereas phospholipase D (PLD) hydrolyzes the phospho-
diester bond between the phosphate and the head group.
Multiple isoforms of each phospholipase exist; however, certain
isoforms will act on only one type of phospholipid (e.g., phos-
phatidylcholine or phosphatidylinositol) while others are less
specic. Additionally, some isoforms are not tightly regulated,
whereas other isoforms are under strict control. For example,
several PLA
2
isoforms are activated by phosphorylation or by
Ca
2+
or Ca
2+
-calmodulin binding.
Phospholipase A
2
Activation
Many systems that induce Ca
2+
mobilization result in enhanced
PLA
2
activity and the release of arachidonic acid, a 20-carbon
fatty acid usually found at the C-2 position of membrane phos-
pholipids. Arachidonic acid is a precursor for a group of poorly
proteins; for example, CREB interacts with specic cAMP-
regulated enhancer (CRE) regions in the IL-6 and inducible
nitric oxide synthase genes and induces their expression.
Ions in Cell Signaling
Fluxes in intracellular ion concentrations can affect many cell
processes,
8
including membrane depolarization, protease acti-
vation, and the activity of numerous phospholipases and
protein and lipid kinases. As such, ion uxes are integral to the
dissemination of signals from the plasma membrane and can
control events such as secretion/degranulation, gene transcrip-
tion, and cytoskeletal reorganization. These events can affect
processes associated with immune function, including chemo-
taxis, survival, and the degranulation of cytotoxic proteins that
contribute to inammatory responses.
Cytoplasmic free [Ca
2+
] is normally about 10 to 100 nmol/L
but can be increased rapidly in response to stimuli, such as
epinephrine and various chemoattractants (see Fig. 11-4). This
change in cytoplasmic free [Ca
2+
] can be achieved by two major
mechanisms, the release of intracellularly stored Ca
2+
, such as
from the ER, and the inux of extracellular Ca
2+
(generally
about 1 mmol/L). These events can elevate intracellular free
[Ca
2+
] to 1 to 10 mol/L, which is enough to activate proteins
such as the Ca
2+
-dependent protein kinases and certain phos-
pholipases. In some cases, these Ca
2+
effects are mediated by the
Ca
2+
-binding protein calmodulin.
Figure 11-4 Common signaling pathways. Several widespread systems involved in outside-in signaling are linked to the generation of second
messengers, such as cAMP, cytoplasmic free calcium ion (Ca
2+
), and phospholipid metabolites. Left, Activation of single transmembrane-spanning
receptors that possess intrinsic tyrosine kinase activity and/or activate/recruit nonreceptor kinases that regulate downstream kinases and phos-
pholipases such as PLC- isoforms. These PLCs catalyze the breakdown of PIP
2
to IP
3
and DAG, which promote Ca
2+
release from intracellular
stores (enoplasmic reticulum) and protein kinase C (PKC) activation, respectively. Middle, GPCRs are seven transmembrane receptors whose
intracellular domains interact with specic heterotrimeric G proteins. Multiple G protein subfamilies exist (see Fig. 11-3) that regulate adenylate
cyclase (with changes in cAMP levels and PKA activation) and/or phospholipases (PLCs or PLA
2
) that modulate Ca
2+
uxes or the production of
proinammatory eicosanoids. Right, Ligand-gated ion channels can permit ions (e.g., Ca
2+
) to traverse the plasma membrane into the cell down
their concentration gradient, which activates signaling networks that can involve Ca
2+
-binding proteins (calmodulin) and various phospholipases
(e.g., PLA
2
) and protein kinases (e.g., PKCs).
PLC-1,2 PLC
P
P
PIP
2
IP
3
+ DAG
Intracellular
Ca
2+
mobilization
Ca
2+
+
calmodulin
Ca
2+
Cytoplasm
ER
PKC activation
(>12 isoforms)
Regulation of various downstream enzyme activities, ion channels, and transcriptional processes
Regulation of
adenylate cyclase
PLA
2
Ion channel
activation
G proteincoupled
receptor
Cytokine/growth factor
receptor
Receptor/non-receptor
Tyrosine kinases
Ion channel
Heterotimeric
Ligand-gated
G proteins
Eicosanoids
PKA
Protein kinase
activation
Alterations
of cAMP levels
Plasma
membrane
Extracellular
family (protein kinase C, PKC) that can bind tightly to plasma
and nuclear membranes. PKC phosphorylates serine-threonine
residues of specic proteins (e.g., receptors, transcription
factors), thus modulating their activity.
Phosphoinositide 3-Kinase
Besides serving as a precursor for DAG and IP
3
, PIP
2
can
also be phosphorylated by phosphoinositide 3-kinase (PI3K)
at the 3 position of the inositol ring to form PIP
3
. PI3K
possesses phosphotyrosine-binding sites (SH2 domains) and
is recruited to certain plasma membrane receptors after
their ligand-induced phosphorylation. At the membrane,
PIP
2
is converted to PIP
3
by PI3K, and PIP
3
activates
3-phosphoinositide-dependent kinases (PDKs). In turn, the
PDKs can phosphorylate/activate other protein kinases, such as
Akt (Table 11-2 and Fig. 11-5, C). Activated PI3K and Akt
appear essential for mediating many hormone and cytokine
effects, including nutrient uptake, gene expression, and cell sur-
vival. Subsequently, active Akt can be regulated by dephosphor-
ylation via protein phosphatase 2A.
Low-molecular-weight G Proteins
The Ras and Rho families of LMW G proteins function as
molecular switches, cycling between an inactive GDP-bound
state and an active GTP-bound state, and serve to regulate the
activation of various protein kinase cascades (e.g., MAP kinase
cascades; Fig. 11-6). The cycling between the active and inactive
forms of these G proteins is controlled by (1) a group of
water-soluble factors known as the eicosanoids. These factors
include the prostaglandins, prostacyclins, thromboxanes, and
leukotrienes such as LTC
4
, which can induce many effects,
including bronchospasm, mucus secretion, and eosinophil
recruitment. These lipid derivatives act locally as intercellular
mediators that can affect inammation, smooth muscle con-
traction, and platelet aggregation. In turn, the therapeutic
effects of decreased PLA
2
expression and the attenuated action/
production of various eicosanoids are important in treating
certain inammatory responses associated with allergy.
9
Phospholipase C Activation and Phosphoinositide
Hydrolysis
Phosphatidylinositol (PI) metabolites are often important for
cell responses to stimuli that mobilize intracellular Ca
2+
. In this
regard, a small pool of PI in the plasma membrane is sequen-
tially phosphorylated to phosphatidylinositol-4-phosphate
(PIP) and phosphatidylinositol-4,5-bisphosphate (PIP
2
) by
several PI kinases (Fig. 11-5, B). PIP
2
can be hydrolyzed to
diacylglycerol (DAG) and 1,4,5-trisphosphoinositol (IP
3
)
by phosphoinositide-specic PLC isoforms (PI-PLCs). With
appropriate stimuli, ligand-receptor complex formation can
activate PI-PLCs by either tyrosine phosphorylation (e.g., PLC-
1,PLC-2) or heterotrimeric G proteins (Gq) that stimulate
a PIP
2
-specic PLC (e.g., PLC-). Both DAG and IP
3
serve as
intracellular messengers; IP
3
interacts with Ca
2+
channels in the
ER and rapidly promotes Ca
2+
release, whereas DAG activates
members of a Ca
2+
/phospholipid-dependent protein kinase
Figure 11-5 Examples of phospholipid metabolism in signal
transduction. A, Site-specic cleavage of membrane phos-
pholipids by phospholipases modulates the production of
metabolites that serve either as second messengers (DAG, IP
3
)
or as lipid mediators in inammation (e.g., arachidonic acid
metabolites released via PLA
2
). B, Modication of phospholip-
ids by lipid kinases (e.g., phosphoinositide [PI] kinases) and
phospholipases (PI-PLCs) can also generate second messen-
gers (IP
3
, DAG). C, Phosphorylation of PIP
2
by PI3 kinase
results in, PIP
3
, a lipid product that activates phosphoinositide-
dependent kinases (PDKs). In turn, the PDKs phosphorylate/
activate Akt, which is a protein kinase that induces many cel-
lular effects, including cell survival. This effect can be inhibited
by protein phosphatase 2A (PP2A), which would result in the
dephosphorylation and inactivation of Akt.
Phospholipase A
1
O
O
O
C
C
P
O
R
1
Phospholipase A
2
Arachidonic acid
(Eicosanoids)
(Head group)
Phospholipase D Phospholipase C
Tyrosine
Phosphorylation
R
2
X
CH
2
CH
2
CH
O
O
O
Pl-PLC
Pl-4-kinase Pl-5-kinase
Pl3 kinase
Pl
A
B
C
PlP PlP
2
PlP
3 PDK 1,2 PP2 A
Akt
Phosphorylated Akt
Cell survival
Gq/11
Mobilize Ca
2+
from
intracellular stores
IP
3
+ DAG
Activate PKC isoforms
activated in response to cell stimulation by agents such as
growth factors, cytokines, chemotactic factors, and phorbol
esters.
10
The MAP kinase family includes the ERKs, the c-Jun
NH2-terminal kinases (JNKs), and the p38 stress-activated
protein kinases. These kinases are regulated by members of the
LMW G protein family, such as Ras and Rac (see Fig. 11-6). For
example, active Ras can interact with effector molecules includ-
ing the serine/threonine kinase Raf-1. Active Ras recruits Raf-1
to the membrane, whereon it phosphorylates and activates
certain dual-specicity kinases (MAPK/ERK kinases, or MEKs),
which phosphorylate and activate specic members of the
MAP kinase family (e.g., ERK1 and ERK2). Substrates for ERKs
include cytoplasmic PLA
2
, the p90 ribosomal S6 kinase (p90
Rsk), cytoskeletal proteins, membrane-localized receptors, and
certain transcription factors. As such, the accumulation of
active Ras and the consequent stimulation of ERK1 and ERK2
lead to the control of many cellular processes, including the
production of lipid mediators, cytoskeletal changes, and tran-
scriptional events. In fact, the ERKs, together with the other
MAP kinase family members, can trigger the activation of
numerous transcription factors (e.g., Elk-1, CREB, ATF2, C/
EBP-, NFAT, c-Jun/c-Fos), which can modulate cytokine and
inammatory mediator expression.
Cytoplasmic Tyrosine Kinases
Cytokine and chemoattractant signaling in immune cells
has been shown to be critically dependent on receptor inter-
action and activation of multiple cytoplasmic tyrosine kinases,
accessory/nucleotide exchange factors (e.g., Sos or Vav) that are
often recruited to tyrosine phosphorylated receptors by adapter
molecules such as Shc and Grb2, at which point they promote
the accumulation of the active GTP-loaded form of the G
protein, and (2) proteins that induce G protein inactivation
through stimulation of intrinsic GTPase activity (the GTPase-
activating proteins [GAPs] are examples of this class of modula-
tors). Many of these accessory proteins are regulated by tyrosine
kinasedependent pathways and are localized in proximity to
these G proteins through association with adapter proteins. In
turn, there are many downstream effectors of the LMW G pro-
teins, which allow them to be critically linked to the control of
various biologic end points, such as gene transcription and
cytoskeletal reorganization.
For example, active Ras can interact with multiple effector
molecules such as the protein kinase Raf-1 and PI3 kinase. The
recruitment of Raf-1 to the plasma membrane by active Ras and
the initiation of MAP kinase cascades (see Fig. 11-6) comprise
a well-characterized signaling cascade crucial for transcription
factor regulation.
MAP Kinase Cascades
The MAP kinase family is composed of serine/threonine protein
kinases that are highly conserved throughout evolution and are
Figure 11-6 Examples of low-molecular-weight (LMW) G protein
MAP kinase signaling cascades frequently used by immune stimuli.
Many signaling cascades progress through a series of kinase (phos-
phorylation) reactions. Activation of MAP kinases (ERKs, JNKs, p38)
is a widely distributed cellular response. Signal initiation often occurs
via cell surface receptors that activate low MW G proteins (Ras, Rho)
through receptor recruitment of adapter molecules (Grb2, Shc) and
nucleotide exchange factors (Sos, Vav), resulting in the cascade of
MAPKKKs, MAPKKs, and MAPKs. In the case of ERK activation, Ras
activation leads to several steps of protein phosphorylation/activation,
resulting in specic substrate activation such as transcription factors.
Similar cascades of LMW G proteins are involved in regulating the
JNK and p38 MAP kinases, which control the expression of many
inammatory gene products. GPCRs, G proteincoupled receptors;
Ser, serine; Thr, threonine; Tyr, tyrosine.
MAPKKK
Low Mol Weight G-Protein Ras
Raf, PI3K
GDP-GTP exchange
Activation/phosphorylation
Ser phosphorylation
Thr/Ser phosphorylation
MAPKK MEK1, MEK2
Tyrosine Kinases, GPCRs, Cytokine Receptors Receptors
ERK1, ERK2 MAPK
Rac
ASK1
MKK3/6
JNK p38
MKK4/7
MLK3/6
Substrates in the cytoplasm and nucleus
Tyr/Thr phosphorylation
Examples
Shared Intracellular Cascade Pathways with
Functions in Signal Transduction
Modules/Components Select Functions
RasRaf-1MEK-ERK
(MAP kinase)
Receptor regulation, cytoskeletal
changers, cPLA
2
activation,
transcription factor activation
Rac/MLK/MKK/p38
(MAP kinase)
Transcription factor activation
Rac/ASK1/MKK/JNK
(MAP kinase)
Transcription factor activation
PLC/IP
3
-Ca
2+
/DAG-PKC Secretion, contraction, motility
changes, regulation of wide variety
of enzymes (e.g., protein kinases,
phospholipases, proteases)
cAMP/PKA Response to many receptors for
modulation of signaling
Shc/Grb2/Sos Dynamically associated Ras-activating
module
Calcineurin/NFAT Ca
2+
-dependent phosphatase in NFAT
activation
mTOR/S6 kinase Mammalian target of rapamycin in
cell proliferation and growth
IP
3
/Akt/PDK Activation of transcription factors for
cell survival
Pyk2/FAK Cytoskeletal regulation; adhesion
moleculeimmunoreceptor crosstalk
JAK/STAT JAK2/STAT5 (example of Src shared
cytokine-signaling pathway)
NF-B/RelA/IB DNA-binding factors/transcriptional
regulators
ITIM/SHIP/SHP-1 Immunoreceptor inhibitory pathway
ITAM/Src family
kinases/SYK/ZAP70
Immunoreceptor stimulatory pathways
SMADs 1 to 7 TGF- superfamily intracellular
signaling
TABLE
11-2
which allows CREB to interact with DNA at cAMP-responsive
elements (CREs), resulting in the transcription of CREB-
responsive genes. (e.g., IL-2, COX-2).
13
JAKs/STATs
The Janus kinasesignal transducer and activator of transcrip-
tion (JAK-STAT) module is important for cell responses to
various cytokines, chemoattractants, and growth factors
14
(Fig.
11-7). Many cytokine/hematopoietic receptors can activate spe-
cic members of the Janus cytoplasmic tyrosine kinases (JAKs
1 to 3, TYK2). Depending on the receptor, specic members
of the JAK family are constitutively, but noncovalently, associ-
ated with a specic hematopoietic receptor subunit. Ligand-
induced receptor activation and dimerization of the JAKs
results in their reciprocal phosphorylation/activation, and
among the substrates of the active JAKs are the receptor sub-
units and members of the STAT family of transcription factors
(STATs 1 to 4, 5a/b, and 6). The tyrosine phosphorylation of
receptors by JAKs creates binding sites for the SRC homology
2 (SH2) region of STATs. Once bound, the STATs are subject to
JAK-dependent tyrosine phosphorylation, which promotes
STAT homo- or heterodimerization. The activated STAT dimer
then translocates into the nucleus, where it binds to consensus
STAT-binding sequences and modulates transcription. The
ability of STATs to activate transcription is enhanced by their
phosphorylation on serines located near the C terminus in the
transactivational domain by serine-threonine kinases. Con-
versely, JAK/STAT activity is attenuated by phosphatases (e.g.,
SHP-1) and the cytokine-inducible SH2-containing proteins
(e.g., CIS-1). Several tyrosine-phosphorylated STATs also may
act as cytoplasmic adapter proteins, thereby widening their
possible role(s) in cell signaling.
14
STAT6 is of particular
including Syk, ZAP-70, Lyn, and other members of the Src
family of tyrosine kinases.
11
These enzymes are noncovalently
associated with various receptors, and their activity is thought
to be critical for mediating many immune responses. The regu-
lation of these enzymes generally involves their phosphoryla-
tion and recruitment into signaling complexes, as previously
discussed. Alterations in these signaling pathways leads to
impaired immune function and disease states such as severe
combined immunedeciency (SCID; ZAP-70 deciency) and
X-linked agammaglobulinemia (Brutons tyrosine kinase
mutation).
12
TRANSCRIPTIONAL REGULATION AND PROTEIN
PHOSPHORYLATION
Many signaling events can promote changes in gene transcrip-
tion and mRNA turnover, including the activation of PKA,
PKC, and the MAP kinases, changes in Ca
2+
uxes, and the regu-
lation of intracellular tyrosine kinases (e.g., Src, Syk, Lyn).
These events are mediated largely by the phosphorylation of
transcription factors and/or proteins involved in controlling
mRNA stability. There are many additional modes of regulating
gene expression, but several common gene transcription path-
ways involve protein kinase cascades and are key to immune cell
regulation.
CREB
The transcription factor CREB is phosphorylated by serine/
threonine kinases on serine 133. Many receptor systems activate
CREB leading to the activation of various signaling cascades
including PKA and PKC. After phosphorylation, CREB seeks
out a cofactor, CREB-binding protein (CBP) and/or p300,
Figure 11-7 General signaling pathways by the JAK-STAT module. Upon ligand activation of a JAK-associated receptor, the JAKs undergo
reciprocal phosphorylation and tyrosine-phosphorylate the receptor to create STAT-binding sites. The bound STATs are then tyrosine-
phosphorylated by the JAKs, undergo homo- or heterodimerization, and translocate into the nucleus, where they bind to consensus STAT
recognition sequences (ISRE/GAS, interferon-stimulated response elements/interferon- activation sites) and modulate gene transcription.
JAK/STAT activity is opposed by phosphatases (e.g., SHP-1), the protein inhibitor of activated STATs (PIAS), and induced gene products such
as CIS-1. P, Phosphorylated tyrosine.
ISRE/GAS
STAT
STAT
STAT
STAT
STAT
STAT
JAK JAK
PIAS
CIS, SOCS, Pim-1
Nucleus
Ligand
(e.g., cytokine,
growth factor)
P P P P
P
P
P
P
SMAD signaling and subsequent propagation of probrotic
mediators have been linked to airway remodeling seen in asthma
patients. Similar to STATs, the phosphorylation of receptor
SMADs (R-SMADs) leads to their dimerization and transport
into the nucleus to regulate gene transcription. Interestingly,
some SMADs facilitate nuclear import of activated R-SMADs,
and others inhibit SMAD-dependent transcription.
17
Glucocorticoid Signaling
Glucocorticoids (GCs; e.g., prednisone, dexamethasone, hydro-
cortisone) are some of the most effective antiinammatory
therapeutics used for controlling a wide variety of inamma-
tory diseases such as asthma and allergies. Because of their
lipophilic nature, GCs are able to traverse through the plasma
membrane and bind glucocorticoid receptor (GR) in
the cytoplasm. After binding, the GC/GR complex trans-
locates into the nucleus, and homodimerizes to bind to gluco-
corticoid response elements (GREs) on the promoters of a
number of antiinammatory genes to elicit transcription
9
(see
Chapter 99).
Histone Acetylation/Methylation
and Gene Transcription
DNA-histone interactions are important for chromatin struc-
ture and gene regulation. Histones are subject to modications
that inuence their activities. For example, histone acetylation
can loosen the tightly wound DNA structure and allow for
increased DNA access to transcription factors, thus allowing
gene transcription to occur.
18
Histone acetyltransferases (HATs)
catalyze this process and act as transcriptional coactivators.
Histone acetylation is reversible, and histone deacetylases
(HDACs) are often associated with the repression of transcrip-
tion. Similarly, histone methylation by histone methyltransfer-
ases (HMTs) is another modication that can either repress or
activate gene expression and is regulated by signaling pathways
that impinge on transcription.
MEMBRANE MICRODOMAINS
The maintenance of specialized plasma membrane microdo-
mains (detergent-resistant membranes, lipid rafts, glycolipid-
enriched microdomains, caveolae) that are composed of high
local concentrations of cholesterol and sphingolipids allow
many eukaryotic cells to organize a subset of their receptor-
signaling systems into these compartments
19
(Fig. 11-10). This
arrangement facilitates the temporal and spatial regulation
of cellular functions. For example, studies using neutrophils
have revealed that IL-8 priming promotes the recruitment
of NADPH oxidase components to lipid rafts, and on stimula-
tion with chemoattractants, superoxide production is greatly
enhanced.
20
Many signaling molecules are localized to membrane micro-
domains, including Ras, Src family kinases, -arrestins, GPCRs,
and glycosyl-phosphatidylinositolanchored proteins (e.g.,
uPAR, CD16). The movement of molecules in and out of these
microdomains, together with interactions between micro-
domains, appears important in the control of cell signaling. In
many cases, disrupting these domains by altering plasma mem-
brane cholesterol levels or by treating membranes with sphin-
gomyelinases can result in the attenuation or potentiation of
certain signaling events, such as ERK activation and PLD activ-
ity, respectively.
21
importance in allergic diseases, because its activation is critical
for IL-4 and IL-13mediated events such as helper T cell type
2 (Th2) chemokine production and has been targeted for
asthma therapeutics.
15
Nuclear Factor-B
One signaling event initiated by many immune stimuli (e.g.,
TNF-) is the activation of the NF-B family of transcriptional
regulators (Fig. 11-8). In fact, the expression of many survival
factors, cytokines, chemokines, and enzymes involved in inam-
matory mediator production is associated with NF-B activa-
tion. Briey, the NF-B or Rel family of transcription factors
exists in the cytoplasm basally and consists of various homodi-
meric or heterodimeric pairings between family members. The
activity of these dimers in the cytoplasm is suppressed by the
binding of members of an inhibitory protein family, IB, such
that one IB molecule binds each dimer. On the initiation of
signaling events that activate cytoplasmic IB kinases (IKKs),
the serine phosphorylation of IB isoforms by the IKKs targets
IB for proteasome-mediated degradation. IB removal allows
the NF-B complex to translocate into the nucleus, where it
binds to regulatory elements present in various gene promoter
regions. NF-B activation and nuclear import are also associ-
ated with the subsequent induction of IB isoforms such that
the system exhibits a self-regulatory behavior. Interestingly,
activated NF-B can also bind to CBP. When both CREB and
NF-B are present, CREB will act as an inhibitor of NF-B by
competing for a restricted pool of CBP.
16
SMADs
Activation of the SMAD family of transcription factors is the
primary signaling cascade initiated by the transforming growth
factor- (TGF-) superfamily (Fig. 11-9). TGF-induced
Figure 11-8 General signaling mechanisms associated with the
nuclear factor-B (NF-B) module. In the inactive state, cytoplasmic
NF-B/RelA dimers are associated with an IB isoform that maintains
NF-B. Upon cell stimulation, IB is phosphorylated by the IKKs, is
ubiquitylated, and undergoes proteosomal degradation, allowing the
NF-B/RelA complex to translocate into the nucleus. NF-B is further
processed in the nucleus (e.g., phosphorylation, acetylation) and asso-
ciates with coactivators to initiate transcription or corepressors to
inhibit transcription.
Tyrosine kinases
Ubiquitylation
and proteosomal
degradation
Nuclear membrane
Modified by:
Phosphorylation
Acetylation
Dephosphorylation
Inhibition of
transcription
Activation of
transcription
RelA
RelA
Hormonal
activation
IKKs
NF-B
NF-B
IB
IB
Inactive complex
in cytoplasm
P
P
IB
P
P
Caveolins
In some cells, there exists Triton Xresistant plasma membrane
microdomains termed caveolae that are rich in cholesterol and
proteins (caveolins) critical for transport processes, such as cho-
lesterol and receptor trafcking.
23
Caveolae are enriched for
various receptors, effector proteins, and lipids important for cell
signaling. The caveolins are integral membrane proteins of 21
to 24 kD that act as the coat protein of caveolae. Interestingly,
a cytosolic N-terminal juxtamembrane region, the caveolin-1
scaffolding domain, can interact with certain lipid-modied
signaling molecules such as heterotrimeric G proteins, Ras, and
Src family tyrosine kinases. These interactions appear to seques-
ter the proteins within caveolae and to modulate or suppress
their activities until proper ligand stimulation leads to signaling
complex formation.
MECHANISMS FOR SIGNAL DOWNREGULATION
Receptor Internalization
Ligand-induced receptor internalization through clathrin-
coated pits is one mechanism by which cells downregulate
SELECT SCAFFOLDING MOLECULES
-Arrestins
The GPCRs are a large family of signaling molecules that are
known to activate heterotrimeric G proteins to regulate down-
stream effectors. The GPCRs are typically desensitized by recep-
tor phosphorylation through GPCR-associated kinases (GRKs)
as well as other kinases, followed by the binding of a class of
molecules known as the -arrestins. The arrestins desensitize
GPCRs and attenuate G protein binding but can also redirect
GPCR signaling to pathways involving Src family kinases and
the ERKs. Arrestin interaction with a GPCR is mediated by its
selectivity for the phosphorylated and activated (ligand-bound)
form of the receptor. The phosphate-sensing core of arrestin
interacts with phosphorylated GPCRs and facilitates their traf-
cking into clathrin-coated pits for internalization and degra-
dation. Arrestin-GPCR interaction also involves a conformational
change of the arrestin molecule that allows for the binding of
Src, MAP kinases (ERKs, JNKs, p38) and associated molecules
such as Raf-1, Ask1, MEKs, and MKKs, leading to MAP kinase
activation.
22
Figure 11-9 Example of SMAD signaling. Dimeric ligands of the transforming growth factor- superfamily (e.g., TGF-, activins) bind to type
II membrane receptor serine-threonine kinases and transphosphorylate the Gs domains of type I receptors to promote docking of receptor-
associated SMADs (R-SMADs). Activated type I receptors subsequently phosphorylate recruited R-SMADs on C-terminal serines, and this asso-
ciation is stabilized by adapter proteins (e.g., SARA, Dab2). Upon phosphorylation, R-SMADs dissociate from type I receptors, dimerize, and
form a complex with a co-SMAD (e.g., SMAD4), translocate into the nucleus, and initiate transcription, binding to SMAD-binding elements (SBEs)
in the promoters of target genes. The R-SMADs are eventually dephosphorylated, which results in the dissociation and export of inactive SMADs
to the cytoplasm. SMAD signaling is also regulated by inhibitory SMADs (I-SMADs), and SMAD ubiquitylation-regulatory factors (SMURF) 1 and
2 target R-SMADs for degradation.
P
P
II
I
II
II
II
P
P
Dab2
R-SMAD
R-SMAD
R-SMAD
P
R-SMAD
R-SMAD
R-SMAD
co-SMAD
P
P
R
-
S
M
A
D
c
o
-
S
M
A
D
co-SMAD
D
im
e
riz
a
tio
n
I-SMAD
e.g., Type 1 collagen
I-SMAD
FKBP12
Gs domain
Kinase
domains
Adapter
proteins
SMURF
SMAD-binding elements
SMURF 1,2
SARA
Dab2
SARA
I
I
Sumoylation has been associated with various cellular events,
including signal transduction, nuclear transport, cell cycle pro-
gression, and stress responses. Unlike ubiquitylation, sumoyla-
tion does not promote protein degradation but may augment
protein stability and control protein localization. Although
SUMO 1 to 3 exhibit a wide tissue distribution, SUMO4 expres-
sion appears restricted to the kidney and immune cells. Interest-
ingly, sumoylation of IB with SUMO1 and SUMO4 makes
IB more resistant to proteasome-mediated ubiquitylation
and degradation, leading to attenuated NF-B activity.
Attenuation of JAK-STAT Signaling
Signaling through the JAK-STAT pathway by hematopoietic
receptors, such as various interferon and interleukin receptors,
is also subject to feedback control (see Fig. 11-7). For example,
certain STAT-induced genes serve as JAK-STAT inhibitors,
including a family of proteins known as the suppressors of cyto-
kine signaling (SOCS) that interacts either with the receptor to
inhibit JAK or STAT binding or with JAK family members to
inhibit their catalytic activity.
27
SOCS-mediated inhibition
can also involve the recruitment of the ubiquitin-transferase
system that targets proteins for degradation.
28
Eight members
of the SOCS family have been identied, including SOCS 1
to 7 and CIS. Interestingly, the SOCS1 knockout mouse
exhibits a phenotype consistent with augmented signaling by
interferon (IFN)-, and administration of IFN-neutralizing
antibodies corrected the phenotype, suggesting that SOCS1
serves as a critical modulator of cytokine signaling. Besides the
SOCS proteins, members of the protein inhibitor of activated
STAT (PIAS) family also appear to regulate the STATs, as well
as NF-B and the tumor suppressor protein p53. The PIAS
proteins can modulate transcription through multiple mecha-
nisms, including antagonizing the DNA-binding activity of
signaling pathways.
24
Other mechanisms include protease-
mediated shedding of cell surface receptors and the desensitiza-
tion of receptors through complex formation with certain
effector molecules. As discussed for -arrestins, the latter mech-
anism can be seen with the -adrenergic receptors, which are
GPCRs that exhibit ligand-stimulated phosphorylation (via
GRKs), followed by interaction with the -arrestins and recruit-
ment into endosomal vesicles, where they are either recycled to
the cell surface or degraded (resulting in decreased surface
expression).
22
Ubiquitylation
Ubiquitin is a 76amino acid peptide that can be conjugated to
select proteins to modulate their turnover and signaling.
25

Ubiquitylation involves ubiquitin conjugation to a lysine residue
of a target protein or to an already-bound ubiquitin molecule,
thereby forming a branching structure. This modication can
serve to target the protein for degradation by the 26S protea-
some if the multiple ubiquitin molecules are conjugated by
lysine-48. Conversely, protein conjugation to ubiquitin lysine-
63 can promote protein-protein interaction between the
monoubiquitinated substrate and proteins with ubiquitin-
binding domains. This modication can also be transient,
because there are enzymes that remove the ubiquitin modica-
tion, such as ubiquitin-specic proteases.
Sumoylation
Another posttranslational event relevant to cell signaling is
sumoylation, which is a protein modication involving the
addition of a member of the small, ubiquitin-related modier
family (SUMO 1 to 4).
26
These proteins are approximately 100
amino acids in length, but unlike ubiquitin, which can form
branching structures, SUMO cannot be conjugated to itself.
Figure 11-10 Scaffolding of intracellular signaling cascades. Effector molecule complexes are frequently assembled before or in response to
cell stimuli and are mediated through protein-protein and protein-lipid interactions. Depicted here are several common groupings for cytokine
receptors (left) and the G proteincoupled receptors (GPCRs; right). These complexes can form around the seed of a specic receptor type or
may inherently be associated with membrane microdomains (lipid rafts), which often contain a diverse array of receptor types. This spatial
association promotes not only rapid responses but also the capacity for crosstalk between receptor systems. This includes the recruitment and
activation of scaffolding molecules (e.g., -arrestins) to GPCRs that can serve to link separate systems together to coregulate specic pathways
(e.g., ERKs and Src family kinases) and thus allow for one system to prime the other.
Ras
Raf
MEK
ERK
1 & 2
-arrestins
-arrestins
Cytokine receptor
Mediator production
Gene expression
Survival
Cytoskeleton reorganization
Mediator/granule release
Motility
Lipid rafts
(cholesterol rich)
GPCR
JAKs
STATs
Lyn
Lyn
Ras
GRK
Src
Syk
Grb2
Sos
Shc
increased cytoplasmic IP
3
/Ca
2+
levels, respectively. Similarly,
CysLT1 promotes bronchial smooth muscle contraction through
increased Ca
2+
uxes and kinase activation (PKC, MAP kinases).
Antagonists of CysLT1 include montelukast, pranlukast, and
zarlukast and these agents can reduce IL-4/IL-13 production,
attenuate eosinophil numbers, and decrease airway remodeling
9

(see Chapters 9 and 100).
Histamine Receptors
Histamine can induce symptoms associated with rhinitis, bron-
chospasm, or cutaneous wheal and are responses.
31
Histamine
released from mast cells and basophils acts on vascular endo-
thelium and bronchial smooth muscle cells. Histamine recep-
tors (HR1 to HR4) are GPCRs widely distributed across tissue
types. The role of HR2 to HR4 in inammatory/allergic pro-
cesses is less clear than HR1, which is coupled to Gq/11 and
stimulates IP
3
production and Ca
2+
uxes/PKC activation. These
events increase nitric oxide generation and leukotriene produc-
tion. HR1 also promotes NF-B activation and inammatory
cytokine/chemokine production.
31
Many therapeutic antihista-
mines, including loratadine, fexofenadine, and cetirizine, are
inverse agonists of HR1 and potently inhibit allergen-induced
responses in the skin, nose, and airways
32
(see Chapter 94).
Adrenergic Receptors
Widely distributed -adrenergic receptors (BAR 1 to 3) are
linked to Gs and the activation of adenylate cyclase. BAR2 is
found in the respiratory tract, including airway smooth muscle,
epithelial and endothelial cells, and mast cells. Interestingly, an
inverse relationship exists between forced expiratory volume in
1 second (FEV
1
) and BAR2 density in the lungs.
33
When stimu-
lated, these receptors allow for bronchiole smooth muscle relax-
ation. Common BAR2 agonists include the short-acting agonists
albuterol and procaterol and the long-acting agonists for-
moterol and salmeterol.
Proposed mechanisms for BAR2-induced smooth muscle
relaxation and bronchospasm alleviation include PKA-
dependent phosphorylation of proteins that control muscle
tone and inhibiton of Ca
2+
release from intracellular stores or
reduced Ca
2+
entry into the cell. Other studies suggest BAR2
agonists relax airway smooth muscle by modulating potassium
channels through Gs to counteract the excitatory response of
Ca
2+
currents, as well as altering MLCK phosphorylation pro-
les, thus reducing smooth muscle contractile force.
34,35
The use of BAR2 agonists is benecial, despite possible
adverse side effects of increased asthma exacerbations and
decreased agonist ability to produce bronchodilation. Patients
become refractory to prolonged or repeated agonist exposure,
possibly because of receptor desensitization and internalization.
In addition, BAR2 polymorphisms may alter receptor behavior,
resulting in greater receptor downregulation
36
and airway
hyperreactivity.
33
Adverse effects may also be linked to agonist
action on cells other than airway smooth muscle. BAR2 agonists
can suppress eosinophil apoptosis
37
and upregulate HR1 expres-
sion,
38
thus aggravating this condition. BAR2 agonists can also
promote positive effects, such as the inhibition of histamine and
leukotriene release from mast cells.
9
(See Chapter 95 for more
information on -adrenergic therapies.)
Nucleoside/Nucleotide Receptors
Adenine nucleotides can be released from cells in a regulated
manner (platelet degranulation) or as a consequence of cell
transcription factors, promoting protein sumoylation, and
recruiting transcriptional corepressors or coactivators. Target-
ing the JAK-STAT pathway with tofacitinib has been effective in
treating rheumatoid arthritis.
29
Select Signaling Systems Relevant
to Allergy
For the proper organization and effective mediation of a spe-
cic immune response, multiple signaling events must be inte-
grated. The examples discussed here address select receptor
types and downstream signaling pathways important in cellular
and physiologic responses to allergens, as well as current thera-
pies used to modulate these responses.
G PROTEINCOUPLED RECEPTORS
G proteincoupled receptors are plasma membrane localized
and composed of seven transmembrane segments. The specic
G proteins associated with these receptors can either activate or
inhibit various signaling pathways (see Fig. 11-3). Besides their
linkage to heterotrimeric G proteins, GPCRs may associate with
other effector molecules, such as JAK2 and -arrestins.
22
These
effectors can then facilitate receptor signaling or desensitization
and downregulation. Because of their role in initiating signal
transduction pathways, many therapeutics target the GPCRs, as
discussed next.
Chemokine Receptors
Chemokine receptors are a superfamily of GPCRs that control
immune cell behavior; they promote chemotaxis, cell adhesion,
and mediator release (see also Chapter 7). The chemokine
receptor superfamily is divided into four classes based on the
chemokine/ligand interaction motif to which they bind (CC,
CXC, CX3C, or XC). Many chemokine-induced events are
inhibited by cell treatment with Bordetella pertussis toxin, which
ADP ribosylates and inactivates the subunit of Gi/Go, sug-
gesting that certain chemokine receptors are coupled to these G
proteins. However, some chemokine-induced events are pertus-
sis toxin insensitive; IL-8induced chemotaxis does not require
Gi but is mediated by G/G after receptor activation. Che-
mokine receptors can also activate PLC isoforms, leading to IP
3

production and increased cytoplasmic Ca
2+
, which can regulate
various kinases and promote cytoarchitectural changes. In
certain cases, chemokine receptor signaling also activates LMW
G proteins (Ras/Rho), which mediate many actin-dependent
processes, such as degranulation and chemotaxis. For example,
the chemokines RANTES, eotaxin, and MCP-1 have been linked
to eosinophil activation and recruitment to the lungs of allergic
asthmatic patients.
30
Leukotriene Receptors
Leukotrienes are lipid-based mediators derived from arachi-
donic acid liberated from membrane phospholipids after
PLA
2
activation and are separated into two groups: LTB
4
and
cysteinyl leukotrienes, including LTC
4
, LTD
4
, and LTE
4
. These
agents are implicated in inammatory diseases, including
asthma, because of their potent action as immune cell chemoat-
tractants and bronchospasm stimulators/inducers. LTB
4
has
two identied receptors, BLT1 and BLT2; the cysteinyl leukot-
rienes also have two, CysLT1 and CysLT2. LTB
4
-induced signal-
ing activates Gi and Gq, leading to decreased cellular cAMP and
allergic disease, whereas GM-CSF and IL-3 regulate the devel-
opment, activation, and survival of many immune cells.
43,44

Therapy in subsets of asthmatic patients with humanized anti
IL-5 antibody has been promising.
45
Interleukin-1
The IL-1 family ligands and receptors (IL-1Rs) are primarily
associated with autoinammatory conditions and are viable
therapeutic targets in diseases such as rheumatoid arthritis.
IL-1 promotes secretion of additional IL-1 and leads to a pro-
inammatory response. The IL-1Rs contain a cytoplasmic Toll/
IL-1 receptor domain that leads to downstream signaling
through association of MyD88, an adapter protein, and activa-
tion of IRAKs (IL-1Rassociated kinases) and IKKNF-B,
similar to Toll-like receptors. These signaling cascades activate
caspase 1, a cysteine protease responsible for the nal processing
of pro-IL-1 into its secretable/active form. This proinamma-
tory response is associated with diseases such as rheumatoid
arthritis and can be treated by blocking access of IL-1 to the
receptor (e.g., anakira) or by using neutralizing antibodies (e.g.,
canakinumab) against IL-1. Rilonacept, a fusion of the extra-
cellular portions of IL-1R1/IL-1RAcP and the Fc fragment of
immunoglobulin (Ig) G, is used in the treatment of cryopyrin-
associated periodic syndrome (CAPS).
46
OTHER INTERLEUKIN RECEPTORS
The IL-2 receptor family, which includes the IL-4, -7, -9, and
-15 receptors, is composed of ligand-specic chains and a
common subunit important for cell signaling.
47
The IL-2 and
IL-15 receptors also contain a subunit that contributes to
signaling.
48
Stimulation of receptors sharing the common
subunit promotes JAK1/3 phosphorylation,
49
whereas the Src
family kinases Lck,
50
Lyn, and Fyn
51
are associated with the
subunit. The IL-2 receptor also activates other signaling mole-
cules, including PI3 kinase, PLC-, Ras, Raf-1, and MAP
kinases.
47
The IL-13R shares the IL-4 subunit and both recep-
tors signal through JAK1 and Tyk2, activating STAT3/6.
52
IL-13
has been shown to be a viable target in the treatment of asthma
patients with lebrikizumab, a neutralizing antibody to IL-13,
which resulted in an increase in lung function.
53
In mice, neu-
tralizing antibodies against IL-13 suppress ovalbumin-induced
airway hyperresponsiveness, eosinophil inltration, inamma-
tory cytokine production, serum IgE levels, and airway remod-
eling. Similarly, neutralizating IL-9 in mice reduces airway
inammation, hyperresponsiveness, and bone marrow eosino-
philia, suggesting that both IL-13 and IL-9 may be therapeutic
targets in asthma (see Chapter 51).
The IL-6 receptor family includes receptors for IL-6, IL-11,
leukemia inhibitory factor, oncostatin M, and ciliary neuro-
trophic factor. All are known to require a common gp130 sig-
naling subunit that is linked to the activation of JAK1, JAK2,
and TYK2.
54
Stimulation with IL-6 promotes STAT1 and STAT3
activation and heterodimerization, as well as activation of
protein tyrosine phosphatase 2 (SHP2), which links this system
to Ras-ERK signaling and signal attenuation.
Interleukin-12 signals through two receptors (IL-12R1,
IL-12R2) that regulate the activation of Lyk, Tyk2, JAK2,
and STAT4. Although administered to alleviate asthma symp-
toms, IL-12 induces several toxic effects, including malaise and
cardiac arrhythmia, suggesting that this treatment may not be
practical.
55
damage or death. EctoATPases and nucleotidases modulate the
bioavailability of extracellular nucleotides and generate metab-
olites that are ligands for receptors on many cells. Nucleotide
receptors are divided into P1 and P2 classes. P1 receptors bind
adenosine/AMP, whereas P2 receptors bind ATP, adenosine
diphosphate (ADP), and the pyrimidine nucleotides uridine
triphosphate (UTP) and uridine diphosphate (UDP). The P2
family is divided into P2Y and P2X subclasses based on their
predicted structures. The P1 and P2Y receptors are GPCRs,
whereas P2X receptors are ligand-gated ion channels.
39
Mast cells, eosinophils, lymphocytes, neutrophils, and
macrophages express P1 (adenosine) receptors and exhibit
inammatory mediator elaboration on stimulation.
39
Inhaled
adenosine elicits bronchoconstriction in asthma and induces
mast cell release of histamine, leukotrienes, and inammatory
cytokines/chemokines. Adenosine receptor subtypes include
A1, A2A/A2B, and A3. A1 receptors are ubiquitously expressed,
whereas A2 receptors are expressed in mast cells, neutrophils,
platelets, pancreas, smooth muscle, and the vasculature. Activa-
tion of A2 receptors promotes Gs-induced elevations in cAMP
and IL-8 release. A3 receptors are expressed in many tissues,
especially the lung and liver. Its activation results in decreased
cAMP, increased Ca
2+
uxes, and histamine release. Elevated
adenosine levels activate A3 and promote eosinophil apoptosis
and inhibit eosinophil and neutrophil degranulation, reactive
oxygen species (ROS) release, and chemotaxis.
Widely expressed in immune cells, the P2Y receptors are
coupled to Gq/11, Gs (stimulator) or Gi (inhibitor), and regu-
late IP
3
generation, Ca
2+
uxes, and cAMP formation. Notably,
the leukotriene receptor antagonists montelukast and pranlu-
kast can attenuate P2Y-mediated activation of PLC and intra-
cellular Ca
2+
mobilization, thus modulating P2Y-mediated
inammatory processes in allergy.
The P2X receptors, widely expressed on immune cells, are
ATP-gated ion channels that elevate intracellular Ca
2+
and
mediate fast permeability to cations, including Na
+
and K
+
.
Interestingly, P2X
7
has been implicated in asthma-associated
airway inammation.
40
P2X
7
activation can enhance endotoxin-
induced responses in macrophages, promote cytoskeletal reor-
ganization and membrane blebbing (via Rho and p38), activate
transcription factors (e.g., NF-B, CREB, AP-1), and lead to the
elaboration of mediators (e.g., IL-1, VEGF, ROS).
41
Thus,
modulating P2X and P2Y receptors may effectively reduce
inammatory responses associated with allergy.
42
HEMATOPOIETIC RECEPTORS
Hematopoietic cytokines and their respective receptors control
the differentiation, maturation, priming, and activation of
many immune cells. Hematopoietic receptors are grouped into
families that share common subunits. Below some of the signal-
ing cascades induced by these receptors are highlighted.
Granulocyte-Macrophage Colony-Stimulating
Factor/Interleukin-3/Interleukin-5
The GM-CSF/IL-3/IL-5 cytokine receptor family shares a
common chain necessary for signaling and forms a high-
afnity receptor when associated or dimerized with a ligand-
specic chain. Signaling through these receptors includes the
activation of cytoplasmic tyrosine kinases (e.g., Lyn, Syk), Ras-
ERK, and JAK2-STAT3/5. IL-5 primarily targets eosinophils
and contributes to eosinophilic differentiation and survival in
these receptors initiates formation of TLR homodimers and
heterodimers, resulting in production of proinammatory
cytokines/chemokines and other mediators. TLRs are expressed
in monocytes/macrophages, dendritic cells, B cells, and mast
cells, but some are found in certain nonimmune cells, including
cells lining mucosal surfaces (e.g., intestinal epithelial cells).
Also, receptor expression is modulated by microbial invasion,
microbial components, and cytokines
58
(see Chapter 1).
INHIBITORY AND STIMULATORY RECEPTORS
Balancing stimulatory and inhibitory networks is important in
providing appropriate activation thresholds and sensitive
tuning mechanisms for cell responses. Many cell surface recep-
tors and signaling molecules are critical for this balance, includ-
ing Fc receptors (FcRs) and their homologs, leukocyte
immunoglobulin-like receptors (LIRs), and paired Ig-like
receptors and signal-regulatory proteins (SIRPs). These pro-
teins belong to a large class of receptors characterized by
common cytoplasmic tyrosine-based signaling motifs: immu-
noreceptor tyrosine-based activating motifs (ITAMs) that
contain two repeats of the consensus sequence Y-X-X-L/I spaced
by six to eight amino acids, or the immunoreceptor tyrosine-
based inhibitory motifs (ITIMs) with a sixamino acid consen-
sus sequence (I/V/L/S)-X-Y-X-X-(L/V) (see Table 11-1). Ligand
binding to activating receptors leads to rapid phosphorylation
of ITAM tyrosines by Src family kinases, which then initiate
For more information on cytokines and their receptors, see
Chapter 5.
TUMOR NECROSIS FACTOR
Tumor necrosis factor (TNF), a major cytokine released pri-
marily from macrophages but also from many other cells (e.g.,
lymphoid cells, mast cells, broblasts) and involved in proin-
ammatory responses, is associated with several diseases,
including Crohn disease and rheumatoid arthritis. TNF signals
through homotrimeric receptors 1 and 2 (TNF-R1, TNF-R2),
which activate caspases (involved in apoptosis), NF-B, and
activator protein 1 (AP-1) signaling cascades. TNF is a potential
therapeutic target implicated in airway inammation and bron-
chial hyperresponsiveness, but late stage clinical trials were
disappointing.
56
TOLL-LIKE RECEPTORS
Toll-like receptors (TLRs), a superfamily of transmembrane
IL-1Rlike molecules, function in the innate immune system by
distinguishing different pathogens based on molecular signa-
tures; TLRs are pattern recognition receptors (Fig. 11-11). They
contain leucine-rich motifs and a cytoplasmic region, Toll/
IL-1R (TIR) domain, that is necessary for cell signaling.
57
These
receptors are largely expressed on the cell surface, although
TLRs 7 to 9 have been found intracellularly. Signaling through
Figure 11-11 Examples of multimember families of cell-surface molecules that are involved in pattern recognition behavior, cell-cell interaction
responses, and modulation of inhibitory and stimulatory cell signals. Toll-like receptors (TLRs) are a superfamily of transmembrane interleukin-1
receptor (IL-1R)like molecules that function in the innate immune system by distinguishing different pathogens based on their molecular sig-
natures. Many other immune cell surface receptors and signaling molecules are critical in mediating a balance between inhibitory and stimulatory
cell activities, including the Fc receptors (FcRs) and their homologs, the leukocyte immunoglobulin-like receptors (LIRs), and signal-regulatory
proteins (SIRPs). These proteins belong to a large class of receptors (e.g., T cell receptors, Siglecs, paired Ig-like receptors) and are character-
ized by the possession of common cytoplasmic tyrosine-based signaling motifs: the ITAMs for stimulatory systems and ITIMs for inhibitory
systems. Ligand binding to activating receptor complexes promotes ITAM tyrosine phosphorylation and the recruitment of Src family kinases
that induce positive cell responses. Ligand binding to inhibitory receptor complexes promotes ITIM tyrosine phosphorylation and the recruit-
ment of SH2 domaincontaining phosphatases that attenuate cellular activation.
P
+
P
P
+
S
H
P
-
1
,
2
S
H
P
-
1
S
H
I
P
Z
a
p

7
0
Src family
kinases
ZAP70
TRAFs
TAK, TABs
TRIF, TRAM
TLRs/IL-1R
SIRP
(stimulatory)
LIR
(stimulatory)
FcRs
chains

SIRP
(inhibitory)
LIR
(inhibitory)
IRAK
IKK
NF-B
NF-B
IB
My D88
D
A
P
1
2
Syk
ITIMs
ITAMs
P
P
P
the these receptors. ITIM phosphorylation creates docking sites
for SH2 domaincontaining phosphatases such as SHP-1 and
the inositol phosphatase SHIP, which then deliver an inhibitory
signal to receptor systems such as FcRs or cytokine receptors
that use tyrosine kinases (e.g., Src family kinases, Syk, JAKs) or
inositol phosphates (IP
3
, PIP
3
) as stimulatory signals. The utility
of LIRs containing multiple cytoplasmic ITIMs is undened but
may allow for signal amplication or recruitment of signaling
molecules.
Stimulatory LIRs possess short cytoplasmic domains devoid
of ITIMs and a positively charged arginine within their trans-
membrane domains. This arginine may direct LIR association
with other surface receptors, such as FcR common chain,
which interacts with FcRs for IgA, IgE, and IgG, and has a nega-
tively charged aspartate within its transmembrane region. The
interaction of these stimulatory LIRs with FcR- may be
required for cell surface expression and assembly of a signaling-
capable multimeric receptor. FcR- possesses a small extracel-
lular domain but contains an ITAM (see Table 11-1 and Fig.
11-11). Receptor activation may lead to recruitment of protein
kinases that phosphorylate tyrosine residues within the ITAM,
creating binding sites for SH2 domaincontaining kinases Syk
and ZAP-70, thus promoting initiation of stimulatory signals.
Signal-regulatory Proteins
The regulation of immune responses also involves SIRP family
members; expressed by myeloid cells that can interact with cell
surfacetethered proteins, either amplifying or attenuating cell
signaling.
60
The three SIRP members each contain three highly
homologous Ig-like domains in their extracellular regions but
differ in their cytoplasmic regions by the presence or absence
of ITIMs.
The SIRP family comprises , , and types. SIRP, found
in myeloid cells and neurons, utilizes CD47 and surfactant pro-
teins associated with lung inammation (e.g., SP-A) as ligands.
SIRP, also known as CD172b and found in macrophages and
neutrophils, has no known ligand. SIRP, also known as CD172g
or SIRP2, binds CD47 and is expressed in lymphocytes and
natural killer (NK) cells. SIRP initiates inhibitory signals such
as reduced macrophage phagocytosis and TNF production
through cytoplasmic ITIMs. Conversely, SIRP has a basic
amino acid side chain in its transmembrane region essential for
binding the activating adapter protein DAP12, which contains
a single ITAM that provides stimulatory signals. SIRP has a
short cytoplasmic domain with no known motifs for recruiting
signaling molecules and no charged residue to mediate associa-
tion with DAP12 or other adapter proteins; thus its signaling
potential is unclear.
Because of their interaction with cell surfacetethered pro-
teins, SIRPs are sometimes termed paired receptors. Additional
examples of inhibitory/stimulatory receptor systems include
CD40/CD40L, CD200 receptor, cytotoxic T lymphocyte antigen
4, intercellular adhesion molecules (ICAMs), sialic acidbinding
Ig-like lectins (Siglecs),
61
and the T cell receptors (TCRs).
Additional Therapeutic
Considerations
Therapeutics affecting protein kinases and transcription factors
are used to treat asthma, airway inammation, and other
inammatory diseases. Many kinase inhibitors, including those
targeting Src kinases, JAKs, and PI3 kinase isoforms, have been
signaling cascades leading to cellular activation. In contrast,
binding to the inhibitory receptors results in tyrosine phos-
phorylation of ITIMs, which generates docking sites for SH2
domaincontaining phosphatases that can attenuate cellular
activation. The balance between these activating and inhibitory
receptors modulates cell responses to numerous stimuli.
Fc Receptors
The FcRs are multi-subunit cell surface molecules that recog-
nize the Fc portion of immunoglobulin (Ig) molecules and
antibodies. Accordingly, receptors exist for each antibody class:
FcR binds IgG, FcR binds IgA, FcR binds IgE, Fc/R binds
IgM, and FcR binds IgD. Various leukocytes and other immune
cells express subsets of these receptors. There are two major FcR
types; one triggers cell activation, and one leads to inhibitory
signals. Activating FcR subtypes possess cytoplasmic ITAMs
(see Table 11-1 and Fig. 11-11) and can be divided into two
subclasses; one is composed of a ligand-binding subunit asso-
ciated with one or two ITAMs containing intracellular signaling
molecules, and the second consists of two related single-chain
IgG receptors that have a single cytoplasmic ITAM. Inhibitory
FcRs contain cytoplasmic ITIMs and, after phosphorylation,
recruit phosphatases that inhibit cell activation through protein
dephosphorylation. Thus, negative regulation of FcRs results
from coaggregation with receptors containing ITIMs.
Of relevance to allergy, the high-afnity IgE receptor (FcRI)
is primarily expressed by mast cells and basophils; its activation
leads to the release of histamine, eicosanoids, and proteases that
result in airway obstruction, smooth muscle contraction, vas-
cular leakage, and mucus secretion. Upon IgE binding, FcRI
aggregates, promoting rapid ITAM phosphorylation of the
receptor portion and subsequent activation of Syk and Src
family kinases (e.g., Lyn, Fyn) With the aid of adapter proteins
(e.g., LAT and NATL), these kinases activate multiple signaling
cascades, including PLC/PKC, PI3K/PLD, and MAPKs, as well
as the transcription factors NF-B, AP-1, and nuclear factor of
activated T cells (NFAT). Many patients with allergic disease
present with an elevated IgE level that correlates with disease
symptoms and therefore makes inhibiting FcRI signaling an
important therapeutic target. For example, omalizumab, a
humanized monoclonal antibody, binds circulating IgE, pre-
venting activation of the FcRI, and is used to treat moderate
to severe allergic asthma. In general, FcRs are key targets in
treating allergic inammation (see Chapters 3, 23, and 92).
Leukocyte Immunoglobulin-like Receptors
The LIR family, also known as immunoglobulin-like transcripts
(ILTs), are expressed in myeloid and lymphoid cells. Although
their ligands are not completely dened, some may recognize
major histocompatibility complex (MHC) class I molecules.
59

LIRs contain two or four Ig-like domains in their extracellular
regions and are either inhibitory cell surface transmembrane
receptors with larger intracellular domains (LIRs 1, 2, 3, 5, 8),
stimulatory cell surfaceassociated receptors with short cyto-
plasmic sequences (LIRs 6a, 6b, 7), or soluble molecules (LIR4).
Although the role of the rst two LIR classes have been charac-
terized, the function of LIR4 is unclear, but it may act as a
soluble ligand triggering other cell-surface receptors, or as an
antagonist of membrane-bound LIRs by competing for ligands.
It is thought that when inhibitory ITIM-containing LIRs are
brought into close proximity with activating receptors, the
ITIMs become tyrosine phosphorylated by kinases recruited to
Conclusion
Targeting various components of signaling pathways should
allow for potent treatments of allergic disease. Because of the
ubiquitous nature of these molecules, however, these therapies
face greater challenges in terms of safety and selectivity than
therapies targeted toward individual receptors that are more
restricted in their expression.
Acknowledgments
Paul J. Bertics, who held the endowed Robert Turell Profes-
sorship in the Department of Biomolecular Chemistry at
the University of Wisconsin (UW) School of Medicine and
Public Health, died unexpectedly on Dec. 22, 2011, at the age
of 55.
Paul was regarded across multiple disciplines in the scien-
tic community as a person with outstanding insight and
intelligence. His scientic interests focused on inter- and intra-
molecular mechanisms that translate ligand/receptor interac-
tions into physiologic outcomes. Pauls research career began
with examining signal transduction events through EGF recep-
tors on cancer cells, and expanded to purinergic and IL-5 family
receptors on innate immune cells, as well as state-of-the-art
research utilizing liquid crystal and nanostructured surfaces to
develop innovative detection methods for the rapid character-
ization of disease states. Although the extent of his academic
and scientic contributions was immense, he was and always
remained an unassuming and humble man. Paul was a natural
leader who brought out the best in people by sharing his
wisdom, empathy, compassion, concern, and most importantly,
his humor and infectious laugh.
As a scientist, teacher, colleague, friend, and above all,
a loving father and husband, Paul will be greatly missed,
but his legacy will continue in those who knew and learned
from him.
examined in inammation models and are in development for
possible human use. In terms of kinases, the p38 inhibitors
VX-702 and HEP 689 have been evaluated in clinical trials for
psoriasis and other chronic inammatory disorders. BIRB796,
another p38 inhibitor, is being evaluated in Crohn disease,
because CNI-1493, a dual inhibitor of JNK and p38, was effec-
tive in a small study of patients with Crohn disease.
62
A Syk
inhibitor, R-112 (Rigel), has been effective against allergic rhi-
nitis symptoms.
63
Another Syk antagonist, fostamatinib, has
entered a phase III study for rheumatoid arthritis. Because Syk
is involved in many processes and cell types, the selectivity,
safety, and mode of delivery of these drugs are areas of intense
analysis. Furthermore, inhibitors targeting Brutons tyrosine
kinase (BTK) are in clinical trials for B cell malignancies, and
also inhibit degranulation from basophils.
12
In terms of transcription factors, in mouse models an oral
small-molecule inhibitor (BMS345541) that blocks IKK2-
dependent phosphorylation of IB and attenuates NF-B-
dependent transcription reduces the incidence and severity of
arthritis. However, NF-B is a ubiquitous transcription factor,
giving rise to concerns that its inhibition may cause increased
susceptibility to infections. Similarly, one of the most common
therapies prescribed for asthmatic patients to control symp-
toms is inhaled or systemic glucocorticoids, which signal
through intracellular receptors to regulate gene transcription.
64

For example, corticosteroid-induced upregulation of IB
attenuates NF-B action and inhibits mediator production.
65

This subsequent decrease in cytokine and chemokine produc-
tion attenuates lymphocyte and macrophages proliferation
and migration to sites of inammation, thus suppressing the
immune response. Also, corticosteroid therapy stimulates
immune cell apoptosis, perhaps through decreased cytokine
production necessary for cell survival. However, systemic
administration of these compounds results in many side effects,
making their long-term use difcult.
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