Chapter 11 - Signal Transduction

8112019 Chapter 11 - Signal Transduction

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184

CONTENTS

bull Introduction 184

bull General Principles of Signal Transduction 184

bull Cell Structures 185

bull General Principles of Receptors and CellSignaling 186

bull Mechanisms of Intracellular Signaling 187

bull Select Signaling Systems Relevant to Allergy 197bull Additional Therapeutic Considerations 200

bull Conclusion 201

11Signal TransductionPAUL 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 collectivelyknown as signal transduction These events are critical for thecontrol of cell growth differentiation function movement andadaptability Cells can respond to diverse stimuli that range

from small molecules such as ions and various nutrients tolarger molecules including hormones cytokines growthfactors 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 specific signal transductionprocesses allowing the cell to respond appropriately to differingsituations and challenges Furthermore although many studieshave focused on the capacity of external factors to transduceintracellular signals and alter cell behavior a process termedoutside-in signaling there is an increasing appreciation for thereverse process inside-out signaling wherein intracellular eventscontrol how the cell interacts with its external environment Forexample certain intracellular signals can result in the activationor inhibition of cell-cell or cell-matrix adhesion molecules such

as the integrinsIn general signal transduction events are tightly regulated

and involve the coordinated action of numerous molecules toaffect the proper change in cell phenotype and functionHowever cells are exposed to numerous stimuli simultaneouslythat culminate in the ultimate behavioral change in the cell Theoutcome depends on a multitude of interactions that regulatethe appropriate response or because of an imbalance in media-tors can result in a disease state Modulation of specific cellular

signaling events cellular products or interactions is the basis ofmany pharmacologic interventions

The study of cell signaling is a diverse rapidly expandingfield driven by an understanding of how cells respond to variousstimuli in order to develop therapeutics that can selectivelytarget cell-specific behaviors Accordingly knowledge of themechanisms by which various hormones cytokines chemo-kines and allergens can modulate immune cell function isimportant to the field of allergy Although research into signaltransduction is extensive1 several major themes can be definedat the cellular level and with respect to immune cell functionThus this chapter first focuses on general principles in cellsignaling 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 inhibitoryreceptors their downstream signaling cascades and their mod-ulation Throughout therapeutic aspects associated with certainsignaling processes are examined

General Principles of SignalTransduction

This chapter focuses on the signal transduction mechanismsinitiated at the plasma membrane (outside-in signaling)

In memory of a great scientist mentor and teachermdashPaul John BerticsNovember 6 1956 to December 22 2011

SUMMARY OF IMPORTANT CONCEPTS

raquo Signal transduction is the process whereby various external andinternal stimuli (signals ligands) initiate a series of events thatmodulate cellular behavior

raquo Initiation of outside-in signaling begins with ligand binding of areceptor that is present on the cell membrane for nonpermeableligands or at intracellular sites for lipophiliccell-permeableligands

raquo Cell surface receptors often respond to ligand binding by modu-lating intracellular enzymes (kinases lipases phosphatases) Gprotein activity ion channels and gene transcriptionmRNA pro-cessing or by serving as scaffolding sites for other signaling orregulatory proteins

raquo Intracellular receptors generally act as DNAchromatin-bindingmolecules that promote or inhibit specific gene transcriptionalactivities

raquo Signal transduction is often amplified by the action of intermedi-ates (second messengers) including small molecules (egcAMP) proteins (G proteins) lipids (diacylglycerol) ions (Ca2+)and gases (nitric oxide)

raquo Receptor activation can regulate multiple cellular functions(secretiondegranulation migration replication differentiationapoptosis) offering numerous targets and tremendous possibili-ties for modulation of distinct intracellular pathways for the phar-macologic management of inflammation and specific diseases



11 Signal Transduction 18

Cell Structures

The cell is a sophisticated entity that can respond to a multitudof stimuli and changes from its microenvironment producin

a precise outcome that maintains the status quo of the bodyCellular anatomy has developed in such a manner as to isolatthe internal workings from nonspecific stimulation and to compartmentalize the machinery by grouping specific moleculethat can interact within and between their location Althougmany structures make up a typical cell the parts relevant tsignal transduction include the plasma membrane cytoskeleton and several organelles (See Alberts and associates2 for aextensive treatise on the biology of the cell)

PLASMA MEMBRANE

Eukaryotic cells are encapsulated by a plasma membrane thamakes the cell selectively permeable to many extracellula

factors including nutrients lipids proteins ions and pathogens The plasma membrane is a fluid lipid bilayer containina complex mixture of phospholipids glycolipids sterols anproteins This structure serves as an effective barrier to molecules that are poorly lipid soluble and also plays a critical roin the bidirectional flow of information This conduction oinformation includes the specific recognition of extracellulafactors including hormones toxins adhesion molecules anpathogens that function to modulate cellular responses As previously noted the recognition of these factors at the plasmmembrane is mediated by receptors and receptor engagemenelicits changes in cell behavior through the alteration o

Because cell signaling involves many effector molecules andcellular structures it is important first to define several basicconcepts (Fig 11-1)

For example external stimuli that cannot freely enter the

cell such as water-soluble factors (eg cytokines chemokines)or various externally tethered molecules (eg cell surface pro-teins extracellular matrix components) generally interact withspecific cellular recognition molecules (receptors) that possessan externally facing ligand-binding site These cell surfacereceptors then transduce information into the cell throughvarious conformational and enzymatic activities that allow forsignal amplification and regulation of specific intracellularenzyme activities ion fluxes cytoskeletal reorganization andsecretory events Also depending on the receptor cell surfacendashinitiated signaling may alter transcriptional activities chroma-tin structure messenger RNA (mRNA) stabilityprocessing(eg microRNAs) translational activity and posttranslationalprocessing Furthermore signaling through cell surface recep-

tors can result in receptor desensitization internalization andrecycling to achieve feedback control and to prevent excessivestimulation that may lead to pathology

With respect to lipid-soluble factors that can penetrate themembrane such as steroid hormones the receptors for thesemolecules are largely located within the cell often in the nucleuswherein ligand-receptor complex formation generally serves tomodulate gene transcription Once again feedback pathwaysexist to contain the magnitude and duration of the initiatedsignals Specific cellular structures receptors and processes arecommon to numerous signal transduction systems associatedwith 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 cognareceptor In general receptor activation leads to signal amplification and modification of cellular events including secretion cytoskeletal changeand certain enzymatic activities and alterations in gene expression (transcription mRNA stability protein induction) All these processes exhibcomplex feedback controls An understanding of these signaling systems is valuable in the development of therapeutics for allergic disorder

Intracellular

receptors

Lipid soluble(eg glucocorticoids)

Hormonesligands

Water soluble(eg IL-5)

Cell surface receptors

Feedback controls

Amplification cascades

Regulation of enzyme r eceptor activityCytoskeletal reorganizationIon fluxes secretion etc

Regulation of transcription

Protein stability mRNA stability and tr anscription control(eg microRNAs)

Modulation of processingsecretion

Nuclear receptorsregulatory proteins

Extracellular

Plasmamembrane

Cytoplasm

Nucleus



186 SECTION A Basic Sciences Underlying Allergy and Immunology

potential or permeability that occur with certain signalingevents can mediate programmed cell death (apoptosis)

General Principles of Receptorsand Cell Signaling

The initiation of outside-in signaling begins with a receptorgenerally a protein(s) that selectively binds the signal-initiating

factor (eg cytokine hormone) Receptors exist on the plasmamembrane for ligands that cannot readily enter the cell as wellas at intracellular sites (eg cytoplasm nucleus) for ligands thatare lipophilic or cell permeable (eg corticosteroids) Ligand-receptor (LR) interaction is selective and is required for initiat-ing the signaling response The biologic response is oftenproportional to LR complex formation which can be describedby the following equilibrium expression

[ ] [ ] [ ]L R LR +

where [L] [R] and [LR] are the concentrations of free ligandunoccupied receptor and ligand-receptor complex respectivelyThe dissociation constant (Kd) for LR breakdown follows

Kd L R LR = times[ ] [ ] [ ]

In general ligand-receptor interaction is of high affinity(Kd lt10minus9 M) and the receptor number per cell is small (satu-rable) which limits the signal that is produced Because [LR] isproportional to output the signal can be regulated by alteringligand or receptor concentration or by modulating receptoraffinity (Kd) This is a perfect therapeutic target primarilythrough the use of antibodies that can bind with higher affinityand block the binding of ligand to the receptor thus inhibitingactivation or sequestering the ligand before it binds to thereceptor For example the humanized antindashinterleukin-5 anti-bodies mepolizumab and reslizumab block the availability ofIL-5 to activate IL-5 receptors on eosinophils and are beingtested for the treatment of asthma4

In some cases multiple receptors for the same ligand permitseparate signaling behaviors ligand sensitivities and distinctcell type responses Receptor affinity and internalizationturnover are often controlled by ligand binding and theseevents can regulate or desensitize LR formation and cell respon-siveness Furthermore various end points may require differentdegrees of receptor occupancy for example some effects aredetected at low receptor occupancy because this induces enoughof one type of signal whereas other effects may need full recep-tor occupancy requiring a larger or different signal For exampleIL-5 or granulocyte-macrophage colony-stimulating factor(GM-CSF) at nanogrammilliliter (ngmL) levels can increaseeosinophil survival but higher cytokine levels are needed toenhance cell adhesion or degranulation

The following sections detail how certain cell surface recep-tors respond to ligand binding by activating enzymes such asprotein 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 intracellularreceptors often act as DNAchromatin-binding molecules thatpromote or inhibit specific gene transcriptional activities Theseevents can alter protein expression or activation and induce achange in cell behavior These cascades are not usually initiatedby a single hormone or receptor system in vivo but are ofteninfluenced by the concerted action of numerous factors that arepresented to the cell simultaneously or sequentially However

intracellular processes (signal transduction) These processesentail changes in the action of various enzymes structural pro-teins adapter molecules and transcription factors In additionthe regulated flow of ions (eg Ca2+) across the plasma mem-brane can modulate various signaling events

Another important feature of the plasma membrane is thepresence of specialized microdomains (ldquolipid raftsrdquo) that consistof a unique composition of sterols lipids and proteins These

localized differences in membrane structure promote therecruitment of certain receptors and associated molecules tothese regions facilitating the rapid activation of these signalingcomplexes in response to appropriate stimuli

CYTOSKELETON

The cytoskeleton consists of various fibers and filaments withspecialized functions that contribute to the cellrsquos shape mobil-ity and function (eg endocytosis) as well as to the intracellularmovement of proteins vesicles and chromosomes The majorcomponents include microfilaments (eg actin) intermediatefilaments (eg laminin vimentin) and microtubules (egβ-tubulin) Because of their nature (negatively charged) the

cytoskeletal elements present a favorable surface that allowstheir association with many signaling molecules (eg kinaseslow-molecular-weight guanine nucleotidendashbinding proteins [Gproteins] phospholipases) and these structures also providesupport for localized anchoring as well as directed transportwithin a cell3

CELLULAR ORGANELLES

The endoplasmic reticulum (ER) nucleus and mitochondriaalso play key roles in intracellular signaling The ER is a mem-branous network of tubules and cisternae continuous with thenuclear envelope that participates in many cell functionsincluding protein synthesis ion sequestrationrelease and

processingtrafficking of membrane-associated and secretedproteins The ER similar to the plasma membrane containschannels that allow for ions especially calcium (Ca2+) toundergo regulated release into the cytoplasm where they mod-ulate various signaling processes

The nucleus is a membrane-enclosed organelle that containsmost of the cellrsquos genetic information (chromosomes) and is themajor site of gene regulation Cell responses to external andinternal stimuli can lead to changes in gene expression throughthe alteration of transcription factor activity and chromatinstructure In this regard DNA is tightly wrapped around pro-teins called histones thereby forming higher-order structuresknown as nucleosomes Modifications such as acetylation andmethylation in response to cellular environmental or develop-

mental cues can modulate histone function and affect geneexpression so-called epigenetics Following gene transcriptionthe mRNA is processed transported out of the nucleus andtranslated into protein at sites such as the ER These processesare subject to regulation by distinct signal transductionnetworks

Mitochondria are major sites of metabolic function includ-ing lipidcarbohydrate metabolism oxidative phosphorylationand adenosine triphosphate (ATP) synthesis This organelleparticipates in the metabolism (eg hydroxylation sulfation)of molecules for excretion and in the destruction of oxidativefree radicals Also alterations in mitochondrial membrane




dephosphorylates and inactivates extracellular signalndashregulate

kinases (ERKs) and p38 MAP kinases that are linked tthe control of gene transcription cell cycle progression anstress responses In contrast protein dephosphorylation casometimes lead to activation as with the tyrosine kinase Sr(see Fig 11-2)5

ASSEMBLY OF SIGNALING COMPLEXES

The orchestrated assembly of various proteins and lipids intdeliberate cascades is central to progression of signal transduction Many mechanisms are used to achieve this goal including the promotion of protein-protein andor protein-lipi

to some degree activation of a specific pathway leads to a well-documented series of choreographed steps that are comparableacross many cell types although cell-specific differences doexist

Mechanisms of Intracellular Signaling

Signals emanating from an activated receptor are often medi-

ated or amplified through effector molecules known as intra-cellular messengers or second messengers These intermediatesmay promote gene expression and protein synthesis but oftenthey regulate proteins and factors already present in the cyto-plasm initiating a rapid signaling response Common cell-signaling processes and posttranslational modifications includeprotein phosphorylation and dephosphorylation the assemblyof signaling complexes through protein-protein and protein-lipid interactions and protein modifications (eg ubiquity-lation sumoylation acetylation methylation) Other signalingprocesses promote the modification of membrane lipids andthe initiation of cytoplasmic ion fluxes (often Ca2+) Effectormolecules are often ldquoassembledrdquo into modules and compart-mentalized in cells and these signaling systems are frequently

similar between cell types and across species Interestingly theactivationdeactivation of these common signaling modulesdoes not necessarily result in the same response between celltypes (because of the differential expression of intracellulareffector molecules) or even within a cell type (because a celloften integrates multiple stimuli simultaneously) This dis-cussion introduces major signaling pathways that operate inmany cells

PHOSPHORYLATIONDEPHOSPHORYLATION

Activation or inhibition of signaling proteins can involve phos-photransfer from ATP to specific amino acids (generally serinethreonine andor tyrosine) by enzymes known as protein

kinases These enzymes exhibit unique substrate specificitiesand selectively regulate various pathways Kinase specificityarises from the recognition of certain amino acid sequencessurrounding the residue(s) to be phosphorylated and theserecognition sequences are one of the parameters used to classifykinases into different families In general the regulation ofprotein kinases leads to the control of their protein substratesthereby transmitting the signal to downstream signaling targetsIn certain cases phosphorylation can elicit a conformationalchange that removes an allosteric inhibitor allowing for proteinactivation (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 pseudosubstratedomain dissociates from the catalytic site This process leads

to kinase activation and allows substrate access to the catalyticsite Phosphorylation can also inhibit the function of certainenzymes such as myosin light chain kinase (MLCK) Phos-phorylation desensitizes MLCK to activation by Ca2+ and Ca2+-dependent kinases thereby preventing it from phosphorylatingmyosin which is necessary for force generation in musclecontraction

Protein phosphorylation is a transient modification andphosphoprotein phosphatases catalyze the removal of protein-associated phosphates Generally dephosphorylation haltsprotein activation and signal amplification for examplemitogen-activated protein (MAP) kinase phosphatase-1

Figure 11-2 Examples of intramolecular interactions that regulaprotein function A Intramolecular association of a pseudosubstratdomain in the amino terminal domain of protein kinase C zeta (PKC

sterically occludes the enzyme active site and blocks catalytic activitNearby adenosine triphosphate (ATP ) binding and phosphorylation this protein activates the enzyme by inducing a conformational changthat promotes pseudosubstrate dissociation and increased substraaccess to the catalytic site B Association of two domains within Sfamily kinases (eg Src Lyn) in the resting ldquoprimedrdquo and activstates In the inactive state (left ) the SH2 domain of these kinaseassociates with a phosphorylated tyrosine (P ) on the kinase domaC-terminal region On dephosphorylation of this tyrosine by specifiphosphotyrosine phosphatases (middle ) the protein ldquounfoldsrdquo and in a state suitable for activation which occurs when another tyrosinlocated in the kinase domain becomes phosphorylated (right )

ATP binding site

Cysteine r ich site

ATP binding amp

phosphorylation

ATP binding

COOH

COOH

Hinge

Protein kinase Czeta chain

C4

Pseudosubstrate

Pseudosubstrate

SubstrateBound to catalyticsite (kinase activation)

PP

Phosphoryl transf er site

A

B

C3

C3

C1

C1NH2

SH3

SH2

Kinase

NH2

P

SH3 SH3

Fully activeldquoPrimedrdquoInactive

SH2

Kinase

SH2

Kinase

P






Cyclic AMP and Protein Phosphorylation

The effects of cAMP are mediated by protein phosphorylatioevents catalyzed by the cAMP-dependent protein kinase (PKAPKA is composed of two catalytic (kinase) subunits (C) antwo regulatory subunits (R) that bind cAMP Inactive PKA iscomplex of R 2C2 but on binding two cAMPs to each R thcomplex dissociates and generates free C subunits that are catalytically active

Active PKA phosphorylates many enzymes transcriptiofactors and other proteins and this amplification allows forsmall amount of hormone to stimulate the production of manend products and effects The signal can be reversed by proteidephosphorylation of the target proteins via phosphatase

which are also regulated Additionally the degradation of cAMby phosphodiesterases which break the phospodiesterase bonalso limits the extent of the induced signal Conversely cAMaction can be prolonged by certain therapeutics that inhibspecific PDEs For example the PDE4 inhibitor roflumilast being tested for the treatment of chronic obstructive pulmonardisease and asthma7

Increases in cAMP can modulate gene transcriptiothrough PKA-mediated phosphorylation of transcriptiofactors including the cAMP regulatory (response) elementbinding (CREB) protein Phosphorylation of CREB regulateits interaction with DNA and other transcriptional contr

(GTP) for bound GDP The resulting Gα-GTP complex dissoci-ates from the GβGγ subunits The Gα-GTP complex depend-ing on the Gα isoform may either stimulate (Gαs) or inhibit(Gαi) adenylate cyclase The free GβGγ subunits can alsointeract with certain adenylate cyclases (as well as with othereffectors see later) G protein effects are rapidly but only tran-siently manifested because the Gα subunits possess intrinsicGTPase activity that slowly hydrolyzes GTP to GDP Thus GTPhydrolysis turns off the action of the Gα subunits and promotesthe reassociation of the Gα-GDP Gβ and Gγ subunits

Many receptors are coupled to G proteins (eg chemokinessee Chapter 7) and other classes of heterotrimeric G proteinisoforms besides Gs and Gi exhibit specific receptor coupling

profiles and regulate downstream effectors other than adenylatecyclases These other heterotrimeric G proteinndashcoupled systemsmodulate signaling molecules such as phospholipases nucleo-tide exchange factors or phosphodiesterases (see Fig 11-4)Additionally there are other G protein classes including thelow-molecular-weight (LMW about 21 kD) G proteins (RasRac Rho Cdc42) that exist as monomers and are regulated byother receptorsproteins that facilitate GDP-GTP exchange andGTPase activity As discussed later these LMW G proteins regu-late protein kinase cascades such as the MAP kinases and arelinked to cell growth control differentiation secretion geneexpression motility and cytoarchitecture

Figure 11-3 Heterotrimeric G proteins receptor association G protein cycling and target effectors Many signaling systems important fimmune cell function entail the extremely rapid activation of G proteinndashcoupled receptors (GPCRs) Each GPCR can regulate one or more heerotrimeric G protein complexes composed of α β and γ subunits In the resting state guanosine diphosphate (GDP ) is bound to Gα in threceptor-associated complex but on ligand-induced changes in GPCR conformation the GDP is replaced with guanosine triphosphate (GTPThe Gα-GTP dissociates from GβGγ and the subunits modulate specific effectors depending on the G protein isoform Gα contains intrinsGTPase activity that hydrolyzes the bound GTP to GDP and the Gα-GDP reassembles with GβGγ to return to the resting state GEF Guaninnucleotide exchange factor Lbc oncogene that encodes a Rho-GEF

No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP

Adenylate cyclase (cAMP increases)Protein kinase A (PKA)

Axin

Gαs (Gαs GαsXL Gαsolf )

Adenylate cyclase (cAMP decreases)PhospholipasesPhosphodiesterases

Gαiexcl (Gαiexcl1-3 Gαo Gαt Gαz Gαgust)

PLC β (intracellular Ca2+ increases)Phosphoinositide turnover Protein kinase C (PKC)Rho

Gαq (Gαq Gα14 Gα11 Gα15 Gα1516)

p115-Rho GEFLeukemia associated Rho-GEF (LARG)Post-synaptic diversity protein (PDZ)-Rho GEF

A-kinase anchoring protein (AKAP)-LbcRho

Gα12 (Gα12 Gα13)

P13 KinasePLCβIon channels

β

γ

βγ

Cytoplasm

Plasmamembrane

GPCR ligands (examples)

Peptides and proteins (chemokines)Biogenic amines (epinephrine histamine)Lipids (prostaglandins leukotrienes)

Amino acids (glutamate)Ions (calcium)Nucleotidesnucleosides (ATP)

Extracellular




Phospholipases

Phospholipid metabolites can act as intracellular and intercel-lular signaling molecules Many factors activate phospholipasesinvolved in the hydrolysis of either the head group or the fattyacids from the glycerol backbone of specific phospholipids (Fig 11-5 A) Different phospholipases hydrolyze distinctive por-tions of the phospholipid phospholipases A1 (PLA1) and A2 (PLA2) hydrolyze the ester bonds of the intact phospholipid atthe C-1 and C-2 positions of the glycerol backbone respectivelyPhospholipase C (PLC) hydrolyzes the phosphodiester bondbetween 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 groupMultiple isoforms of each phospholipase exist however certain

isoforms will act on only one type of phospholipid (eg phos-phatidylcholine or phosphatidylinositol) while others are lessspecific Additionally some isoforms are not tightly regulatedwhereas other isoforms are under strict control For exampleseveral PLA2 isoforms are activated by phosphorylation or byCa2+ or Ca2+-calmodulin binding

Phospholipase A2 Activation

Many systems that induce Ca2+ mobilization result in enhancedPLA2 activity and the release of arachidonic acid a 20-carbonfatty 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 specific cAMP-regulated enhancer (CRE) regions in the IL-6 and induciblenitric oxide synthase genes and induces their expression

Ions in Cell Signaling

Fluxes in intracellular ion concentrations can affect many cellprocesses8 including membrane depolarization protease acti-vation and the activity of numerous phospholipases andprotein and lipid kinases As such ion fluxes are integral to thedissemination of signals from the plasma membrane and cancontrol events such as secretiondegranulation gene transcrip-tion and cytoskeletal reorganization These events can affectprocesses associated with immune function including chemo-taxis survival and the degranulation of cytotoxic proteins that

contribute to inflammatory responsesCytoplasmic free [Ca2+] is normally about 10 to 100 nmolL

but can be increased rapidly in response to stimuli such asepinephrine and various chemoattractants (see Fig 11-4) Thischange in cytoplasmic free [Ca2+] can be achieved by two majormechanisms the release of intracellularly stored Ca2+ such asfrom the ER and the influx of extracellular Ca2+ (generallyabout 1 mmolL) These events can elevate intracellular free[Ca2+] to 1 to 10 983221molL which is enough to activate proteinssuch as the Ca2+-dependent protein kinases and certain phos-pholipases In some cases these Ca2+ effects are mediated by theCa2+-binding protein calmodulin

Figure 11-4 Common signaling pathways Several widespread systems involved in outside-in signaling are linked to the generation of secondmessengers such as cAMP cytoplasmic free calcium ion (Ca2 +) and phospholipid metabolites Left Activation of single transmembrane-spanningreceptors that possess intrinsic tyrosine kinase activity andor activaterecruit nonreceptor kinases that regulate downstream kinases and phos-pholipases such as PLC-γ isoforms These PLCs catalyze the breakdown of PIP2 to IP3 and DAG which promote Ca2+ release from intracellularstores (enoplasmic reticulum) and protein kinase C (PKC) activation respectively Middle GPCRs are seven transmembrane receptors whoseintracellular domains interact with specific heterotrimeric G proteins Multiple G protein subfamilies exist (see Fig 11-3) that regulate adenylatecyclase (with changes in cAMP levels and PKA activation) andor phospholipases (PLCs or PLA2) that modulate Ca2+ fluxes or the production ofproinflammatory eicosanoids Right Ligand-gated ion channels can permit ions (eg Ca2+) to traverse the plasma membrane into the cell downtheir concentration gradient which activates signaling networks that can involve Ca2+-binding proteins (calmodulin) and various phospholipases(eg PLA2) and protein kinases (eg PKCs)

PLC-12 PLC

P

P

PIP2 IP3 + DAG

Intracellular Ca2+ mobilization

Ca2+

+calmodulin

Ca2+

Cytoplasm

ERPKC activation(gt12 isoforms)

Regulation of various downstream enzyme activities ion channels and transcriptional processes

Regulation of adenylate cyclase

PLA2

Ion channelactivation

G proteinndashcoupledreceptor

Cytokinegrowth factor receptor

Receptornon-receptor Tyrosine kinases

Ion channel

HeterotimericLigand-gated

G proteins

Eicosanoids

PKA Protein kinaseactivation

Alterationsof cAMP levels

Plasmamembrane

Extracellular




family (protein kinase C PKC) that can bind tightly to plasmand nuclear membranes PKC phosphorylates serine-threonin

residues of specific proteins (eg receptors transcriptiofactors) thus modulating their activity

Phosphoinositide 3-Kinase

Besides serving as a precursor for DAG and IP3 PIP2 caalso be phosphorylated by phosphoinositide 3-kinase (PI3Kat the 3 position of the inositol ring to form PIP3 PI3possesses phosphotyrosine-binding sites (SH2 domains) anis recruited to certain plasma membrane receptors aftetheir ligand-induced phosphorylation At the membranPIP2 is converted to PIP3 by PI3K and PIP3 activat3-phosphoinositide-dependent kinases (PDKs) In turn thPDKs can phosphorylateactivate other protein kinases such aAkt (Table 11-2 and Fig 11-5 C ) Activated PI3K and Ak

appear essential for mediating many hormone and cytokineffects including nutrient uptake gene expression and cell suvival Subsequently active Akt can be regulated by dephospho

ylation via protein phosphatase 2A

Low-molecular-weight G Proteins

The Ras and Rho families of LMW G proteins function amolecular switches cycling between an inactive GDP-bounstate and an active GTP-bound state and serve to regulate thactivation of various protein kinase cascades (eg MAP kinascascades Fig 11-6) The cycling between the active and inactivforms of these G proteins is controlled by (1) a group o

water-soluble factors known as the eicosanoids These factorsinclude the prostaglandins prostacyclins thromboxanes and

leukotrienes such as LTC4 which can induce many effectsincluding bronchospasm mucus secretion and eosinophilrecruitment These lipid derivatives act locally as intercellularmediators that can affect inflammation smooth muscle con-traction and platelet aggregation In turn the therapeuticeffects of decreased PLA2 expression and the attenuated actionproduction of various eicosanoids are important in treatingcertain inflammatory responses associated with allergy9

Phospholipase C Activation and PhosphoinositideHydrolysis

Phosphatidylinositol (PI) metabolites are often important forcell responses to stimuli that mobilize intracellular Ca2+ In thisregard a small pool of PI in the plasma membrane is sequen-

tially phosphorylated to phosphatidylinositol-4-phosphate(PIP) and phosphatidylinositol-45-bisphosphate (PIP2) byseveral PI kinases (Fig 11-5 B) PIP2 can be hydrolyzed todiacylglycerol (DAG) and 145-trisphosphoinositol (IP3)by phosphoinositide-specific PLC isoforms (PI-PLCs) Withappropriate stimuli ligand-receptor complex formation canactivate PI-PLCs by either tyrosine phosphorylation (eg PLC-γ 1PLC-γ 2) or heterotrimeric G proteins (Gq) that stimulatea PIP2-specific PLC (eg PLC-β) Both DAG and IP3 serve asintracellular messengers IP3 interacts with Ca2+ channels in theER and rapidly promotes Ca2+ release whereas DAG activatesmembers of a Ca2+phospholipid-dependent protein kinase

Figure 11-5 Examples of phospholipid metabolism in signtransduction A Site-specific cleavage of membrane phopholipids by phospholipases modulates the production ometabolites that serve either as second messengers (DAG IPor as lipid mediators in inflammation (eg arachidonic ac

metabolites released via PLA2) B Modification of phospholiids by lipid kinases (eg phosphoinositide [PI] kinases) anphospholipases (PI-PLCs) can also generate second messegers (IP3 DAG) C Phosphorylation of PIP2 by PI3 kinasresults in PIP3 a lipid product that activates phosphoinositiddependent kinases (PDKs) In turn the PDKs phosphorylateactivate Akt which is a protein kinase that induces many celular effects including cell survival This effect can be inhibiteby protein phosphatase 2A (PP2A) which would result in thdephosphorylation and inactivation of Akt

Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2

Arachidonic acid(Eicosanoids)

(Head gr oup)

Phospholipase DPhospholipase C

TyrosinePhosphorylation

R2

X

CH2

CH2

CH

O

O

O

Pl-PLC

Pl-4-kinase Pl-5-kinase

Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival

Gq11Mobilize Ca2+ fr omintracellular stores

IP3 + DAG

Activate PKC isoforms




activated in response to cell stimulation by agents such asgrowth factors cytokines chemotactic factors and phorbol

esters10

The MAP kinase family includes the ERKs the c-JunNH2-terminal kinases (JNKs) and the p38 stress-activatedprotein kinases These kinases are regulated by members of theLMW G protein family such as Ras and Rac (see Fig 11-6) Forexample active Ras can interact with effector molecules includ-ing the serinethreonine kinase Raf-1 Active Ras recruits Raf-1to the membrane whereon it phosphorylates and activatescertain dual-specificity kinases (MAPKERK kinases or MEKs)which phosphorylate and activate specific members of theMAP kinase family (eg ERK1 and ERK2) Substrates for ERKsinclude cytoplasmic PLA2 the p90 ribosomal S6 kinase (p90Rsk) cytoskeletal proteins membrane-localized receptors andcertain transcription factors As such the accumulation ofactive Ras and the consequent stimulation of ERK1 and ERK2

lead to the control of many cellular processes including theproduction of lipid mediators cytoskeletal changes and tran-scriptional events In fact the ERKs together with the otherMAP kinase family members can trigger the activation ofnumerous transcription factors (eg Elk-1 CREB ATF2 CEBP-β NFAT c-Junc-Fos) which can modulate cytokine andinflammatory mediator expression

Cytoplasmic Tyrosine Kinases

Cytokine and chemoattractant signaling in immune cellshas been shown to be critically dependent on receptor inter-action and activation of multiple cytoplasmic tyrosine kinases

accessorynucleotide exchange factors (eg Sos or Vav) that areoften recruited to tyrosine phosphorylated receptors by adaptermolecules such as Shc and Grb2 at which point they promotethe accumulation of the active GTP-loaded form of the Gprotein and (2) proteins that induce G protein inactivationthrough 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 tyrosinekinasendashdependent pathways and are localized in proximity tothese G proteins through association with adapter proteins Inturn 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 andcytoskeletal reorganization

For example active Ras can interact with multiple effectormolecules such as the protein kinase Raf-1 and PI3 kinase Therecruitment of Raf-1 to the plasma membrane by active Ras andthe initiation of MAP kinase cascades (see Fig 11-6) comprisea well-characterized signaling cascade crucial for transcriptionfactor regulation

MAP Kinase Cascades

The MAP kinase family is composed of serinethreonine proteinkinases that are highly conserved throughout evolution and are

Figure 11-6 Examples of low-molecular-weight (LMW) G proteinndashMAP kinase signaling cascades frequently used by immune stimuliMany 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 occursvia cell surface receptors that activate low MW G proteins (Ras Rho)through receptor recruitment of adapter molecules (Grb2 Shc) andnucleotide exchange factors (Sos Vav) resulting in the cascade ofMAPKKKs MAPKKs and MAPKs In the case of ERK activation Rasactivation leads to several steps of protein phosphorylationactivationresulting in specific substrate activation such as transcription factorsSimilar cascades of LMW G proteins are involved in regulating theJNK and p38 MAP kinases which control the expression of manyinflammatory gene products GPCRs G proteinndashcoupled receptorsSer serine Thr threonine Tyr tyrosine

MAPKKK

Low Mol Weight G-Protein Ras

Raf PI3K

GDP-GTP exchange

Activationphosphorylation

Ser phosphorylation

ThrSer phosphorylation

MAPKK MEK1 MEK2

Tyrosine Kinases GPCRs Cytokine ReceptorsReceptors

ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36

Substrates in the cytoplasm and nucleus

TyrThr phosphorylation

ExamplesShared Intracellular Cascade Pathways withFunctions in Signal Transduction

ModulesComponents Select Functions

RasndashRaf-1ndashMEK-ERK(MAP kinase)

Receptor regulation cytoskeletalchangers cPLA2 activationtranscription factor activation

RacMLKMKKp38

(MAP kinase)

Transcription factor activation

RacASK1MKKJNK(MAP kinase)


PLCIP3-Ca2+DAG-PKC Secretion contraction motilitychanges regulation of wide varietyof enzymes (eg protein kinasesphospholipases proteases)

cAMPPKA Response to many receptors formodulation of signaling

ShcGrb2Sos Dynamically associated Ras-activatingmodule

CalcineurinNFAT Ca2+-dependent phosphatase in NFATactivation

mTORS6 kinase Mammalian target of rapamycin incell proliferation and growth

IP3AktPDK Activation of transcription factors forcell survival

Pyk2FAK Cytoskeletal regulation adhesionmoleculendashimmunoreceptor crosstalk

JAKSTAT JAK2STAT5 (example of Src sharedcytokine-signaling pathway)

NF-κ BRelAIκ Bα DNA-binding factorstranscriptionalregulators

ITIMSHIPSHP-1 Immunoreceptor inhibitory pathway

ITAMSrc familykinasesSYKZAP70

Immunoreceptor stimulatory pathways

SMADs 1 to 7 TGF-β superfamily intracellularsignaling

TABLE 11-2






SMAD signaling and subsequent propagation of profibroticmediators have been linked to airway remodeling seen in asthmapatients Similar to STATs the phosphorylation of receptorSMADs (R-SMADs) leads to their dimerization and transportinto the nucleus to regulate gene transcription Interestinglysome SMADs facilitate nuclear import of activated R-SMADsand others inhibit SMAD-dependent transcription17

Glucocorticoid Signaling

Glucocorticoids (GCs eg prednisone dexamethasone hydro-cortisone) are some of the most effective antiinflammatorytherapeutics used for controlling a wide variety of inflamma-tory diseases such as asthma and allergies Because of theirlipophilic nature GCs are able to traverse through the plasmamembrane and bind glucocorticoid receptor α (GR α) inthe cytoplasm After binding the GCGR α complex trans-locates into the nucleus and homodimerizes to bind to gluco-corticoid response elements (GREs) on the promoters of anumber of antiinflammatory genes to elicit transcription9 (seeChapter 99)

Histone AcetylationMethylationand Gene Transcription

DNA-histone interactions are important for chromatin struc-ture and gene regulation Histones are subject to modificationsthat influence their activities For example histone acetylationcan loosen the tightly wound DNA structure and allow forincreased DNA access to transcription factors thus allowinggene transcription to occur18 Histone acetyltransferases (HATs)catalyze this process and act as transcriptional coactivatorsHistone 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 modification that can either repress oractivate gene expression and is regulated by signaling pathwaysthat 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 highlocal concentrations of cholesterol and sphingolipids allowmany eukaryotic cells to organize a subset of their receptor-signaling systems into these compartments19 (Fig 11-10) Thisarrangement facilitates the temporal and spatial regulationof cellular functions For example studies using neutrophilshave revealed that IL-8 priming promotes the recruitmentof NADPH oxidase components to lipid rafts and on stimula-tion with chemoattractants superoxide production is greatly

enhanced20

Many signaling molecules are localized to membrane micro-domains including Ras Src family kinases β-arrestins GPCRsand glycosyl-phosphatidylinositolndashanchored proteins (eguPAR CD16) The movement of molecules in and out of thesemicrodomains together with interactions between micro-domains appears important in the control of cell signaling Inmany 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 ofcertain signaling events such as ERK activation and PLD activ-ity respectively21

importance in allergic diseases because its activation is criticalfor IL-4ndash and IL-13ndashmediated events such as helper T cell type2 (Th2) chemokine production and has been targeted forasthma therapeutics15

Nuclear Factor-κ B

One signaling event initiated by many immune stimuli (eg

TNF-α) is the activation of the NF-κ B family of transcriptionalregulators (Fig 11-8) In fact the expression of many survivalfactors cytokines chemokines and enzymes involved in inflam-matory mediator production is associated with NF-κ B activa-tion Briefly the NF-κ B or Rel family of transcription factorsexists in the cytoplasm basally and consists of various homodi-meric or heterodimeric pairings between family members Theactivity of these dimers in the cytoplasm is suppressed by thebinding of members of an inhibitory protein family Iκ B suchthat one Iκ B molecule binds each dimer On the initiation ofsignaling events that activate cytoplasmic Iκ B kinases (IKKs)the serine phosphorylation of Iκ B isoforms by the IKKs targetsIκ B for proteasome-mediated degradation Iκ B removal allowsthe NF-κ B complex to translocate into the nucleus where it

binds to regulatory elements present in various gene promoterregions NF-κ B activation and nuclear import are also associ-ated with the subsequent induction of Iκ B isoforms such thatthe system exhibits a self-regulatory behavior Interestinglyactivated NF-κ B can also bind to CBP When both CREB andNF-κ B are present CREB will act as an inhibitor of NF-κ B bycompeting for a restricted pool of CBP16

SMADs

Activation of the SMAD family of transcription factors is theprimary signaling cascade initiated by the transforming growthfactor-β (TGF-β) superfamily (Fig 11-9) TGF-βndashinduced

Figure 11-8 General signaling mechanisms associated with thenuclear factor-κ B (NF-κ B ) module In the inactive state cytoplasmicNF-κ BRelA dimers are associated with an Iκ B isoform that maintainsNF-κ B Upon cell stimulation Iκ B is phosphorylated by the IKKs isubiquitylated and undergoes proteosomal degradation allowing the

NF-κ BRelA complex to translocate into the nucleus NF-κ B is furtherprocessed in the nucleus (eg phosphorylation acetylation) and asso-ciates with coactivators to initiate transcription or corepressors toinhibit transcription

Tyrosine kinases

Ubiquitylationand proteosomaldegradation

Nuclear membrane

Modified byPhosphorylation

AcetylationDephosphorylation

Inhibition of transcr iption

Activation of transcr iption

RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα

Inactive complexin cytoplasm

P P

Iκ

Bα

P P




Caveolins

In some cells there exists Triton Xndashresistant plasma membranmicrodomains termed caveolae that are rich in cholesterol anproteins (caveolins) critical for transport processes such as cholesterol and receptor trafficking23 Caveolae are enriched fovarious receptors effector proteins and lipids important for cesignaling The caveolins are integral membrane proteins of 2to 24 kD that act as the coat protein of caveolae Interestingla cytosolic N-terminal juxtamembrane region the caveolin-

scaffolding domain can interact with certain lipid-modifiesignaling molecules such as heterotrimeric G proteins Ras anSrc family tyrosine kinases These interactions appear to sequeter the proteins within caveolae and to modulate or suppretheir activities until proper ligand stimulation leads to signalincomplex formation

MECHANISMS FOR SIGNAL DOWNREGULATION

Receptor Internalization

Ligand-induced receptor internalization through clathrincoated pits is one mechanism by which cells downregulat

SELECT SCAFFOLDING MOLECULES

β -Arrestins

The GPCRs are a large family of signaling molecules that areknown 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 ofmolecules known as the β-arrestins The arrestins desensitize

GPCRs and attenuate G protein binding but can also redirectGPCR signaling to pathways involving Src family kinases andthe ERKs Arrestin interaction with a GPCR is mediated by itsselectivity for the phosphorylated and activated (ligand-bound)form of the receptor The ldquophosphate-sensingrdquo core of arrestininteracts with phosphorylated GPCRs and facilitates their traf-ficking into clathrin-coated pits for internalization and degra-dation Arrestin-GPCR interaction also involves a conformationalchange of the arrestin molecule that allows for the binding ofSrc MAP kinases (ERKs JNKs p38) and associated moleculessuch as Raf-1 Ask1 MEKs and MKKs leading to MAP kinaseactivation22

Figure 11-9 Example of SMAD signaling Dimeric ligands of the transforming growth factor-β superfamily (eg TGF-β activins) bind to typII membrane receptor serine-threonine kinases and transphosphorylate the Gs domains of type I receptors to promote docking of receptoassociated SMADs (R-SMADs) Activated type I receptors subsequently phosphorylate recruited R-SMADs on C-terminal serines and this assciation is stabilized by adapter proteins (eg SARA Dab2 ) Upon phosphorylation R-SMADs dissociate from type I receptors dimerize anform a complex with a co-SMAD (eg SMAD4) translocate into the nucleus and initiate transcription binding to SMAD-binding elements (SBEin the promoters of target genes The R-SMADs are eventually dephosphorylated which results in the dissociation and export of inactive SMADto the cytoplasm SMAD signaling is also regulated by inhibitory SMADs (I-SMADs) and SMAD ubiquitylation-regulatory factors (SMURF ) 1 an2 target R-SMADs for degradation

P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF

SMAD-binding elements

SMURF 12

SAR A

Dab2

SARA

I

I




Sumoylation has been associated with various cellular eventsincluding signal transduction nuclear transport cell cycle pro-gression and stress responses Unlike ubiquitylation sumoyla-tion does not promote protein degradation but may augmentprotein stability and control protein localization AlthoughSUMO 1 to 3 exhibit a wide tissue distribution SUMO4 expres-sion appears restricted to the kidney and immune cells Interest-

ingly sumoylation of Iκ Bα with SUMO1 and SUMO4 makesIκ Bα more resistant to proteasome-mediated ubiquitylationand degradation leading to attenuated NF-κ B activity

Attenuation of JAK-STAT Signaling

Signaling through the JAK-STAT pathway by hematopoieticreceptors such as various interferon and interleukin receptorsis also subject to feedback control (see Fig 11-7) For examplecertain STAT-induced genes serve as JAK-STAT inhibitorsincluding a family of proteins known as the suppressors of cyto-kine signaling (SOCS) that interacts either with the receptor toinhibit JAK or STAT binding or with JAK family members toinhibit their catalytic activity27 SOCS-mediated inhibitioncan also involve the recruitment of the ubiquitin-transferase

system that targets proteins for degradation28

Eight membersof the SOCS family have been identified including SOCS 1to 7 and CIS Interestingly the SOCS1 knockout mouseexhibits a phenotype consistent with augmented signaling byinterferon (IFN)-γ and administration of IFN-γ ndashneutralizingantibodies corrected the phenotype suggesting that SOCS1serves as a critical modulator of cytokine signaling Besides theSOCS proteins members of the protein inhibitor of activatedSTAT (PIAS) family also appear to regulate the STATs as wellas NF-κ B and the tumor suppressor protein p53 The PIASproteins can modulate transcription through multiple mecha-nisms including antagonizing the DNA-binding activity of

signaling pathways24 Other mechanisms include protease-mediated shedding of cell surface receptors and the desensitiza-tion of receptors through complex formation with certaineffector molecules As discussed for β-arrestins the latter mech-anism can be seen with the β-adrenergic receptors which areGPCRs that exhibit ligand-stimulated phosphorylation (viaGRKs) followed by interaction with the β-arrestins and recruit-

ment into endosomal vesicles where they are either recycled tothe cell surface or degraded (resulting in decreased surfaceexpression)22

Ubiquitylation

Ubiquitin is a 76ndashamino acid peptide that can be conjugated toselect proteins to modulate their turnover and signaling25 Ubiquitylation involves ubiquitin conjugation to a lysine residueof a target protein or to an already-bound ubiquitin moleculethereby forming a branching structure This modification canserve to target the protein for degradation by the 26S protea-some if the multiple ubiquitin molecules are conjugated bylysine-48 Conversely protein conjugation to ubiquitin lysine-63 can promote protein-protein interaction between the

monoubiquitinated substrate and proteins with ubiquitin-binding domains This modification can also be transientbecause there are enzymes that remove the ubiquitin modifica-tion such as ubiquitin-specific proteases

Sumoylation

Another posttranslational event relevant to cell signaling issumoylation which is a protein modification involving theaddition of a member of the small ubiquitin-related modifierfamily (SUMO 1 to 4)26 These proteins are approximately 100amino acids in length but unlike ubiquitin which can formbranching 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 tocell stimuli and are mediated through protein-protein and protein-lipid interactions Depicted here are several common groupings for cytokinereceptors (left ) and the G proteinndashcoupled receptors (GPCRs right ) These complexes can form around the seed of a specific receptor type or

may inherently be associated with membrane microdomains (lipid rafts) which often contain a diverse array of receptor types This spatialassociation promotes not only rapid responses but also the capacity for crosstalk between receptor systems This includes the recruitment andactivation of scaffolding molecules (eg β-arrestins) to GPCRs that can serve to link separate systems together to coregulate specific pathways(eg ERKs and Src family kinases) and thus allow for one system to ldquoprimerdquo the other

β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival

Cytoskeleton reorganization

Mediatorgranule release

Motility

Lipid rafts

(cholesterol rich) GPCR

γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc




increased cytoplasmic IP3Ca2+ levels respectively SimilarlCysLT1 promotes bronchial smooth muscle contraction througincreased Ca2+ fluxes and kinase activation (PKC MAP kinasesAntagonists of CysLT1 include montelukast pranlukast anzafirlukast and these agents can reduce IL-4IL-13 productionattenuate eosinophil numbers and decrease airway remodelin(see Chapters 9 and 100)

Histamine Receptors

Histamine can induce symptoms associated with rhinitis bronchospasm or cutaneous wheal and flare responses31 Histaminreleased from mast cells and basophils acts on vascular endothelium and bronchial smooth muscle cells Histamine receptors (HR1 to HR4) are GPCRs widely distributed across tissutypes The role of HR2 to HR4 in inflammatoryallergic processes is less clear than HR1 which is coupled to Gαq11 anstimulates IP3 production and Ca2+ fluxesPKC activation Theevents increase nitric oxide generation and leukotriene production HR1 also promotes NF-κ B activation and inflammatorcytokinechemokine production31 Many therapeutic antihistamines including loratadine fexofenadine and cetirizine arinverse agonists of HR1 and potently inhibit allergen-induce

responses in the skin nose and airways

32

(see Chapter 94)Adrenergic Receptors

Widely distributed β-adrenergic receptors (BAR 1 to 3) alinked to Gs and the activation of adenylate cyclase BAR2 found in the respiratory tract including airway smooth musclepithelial and endothelial cells and mast cells Interestingly ainverse relationship exists between forced expiratory volume i1 second (FEV1) and BAR2 density in the lungs33 When stimulated these receptors allow for bronchiole smooth muscle relaxation Common BAR2 agonists include the short-acting agonisalbuterol and procaterol and the long-acting agonists fomoterol and salmeterol

Proposed mechanisms for BAR2-induced smooth musc

relaxation and bronchospasm alleviation include PKAdependent phosphorylation of proteins that control musctone and inhibiton of Ca2+ release from intracellular stores oreduced Ca2+ entry into the cell Other studies suggest BARagonists relax airway smooth muscle by modulating potassiumchannels through Gαs to counteract the excitatory response oCa2+ currents as well as altering MLCK phosphorylation profiles thus reducing smooth muscle contractile force3435

The use of BAR2 agonists is beneficial despite possibadverse side effects of increased asthma exacerbations andecreased agonist ability to produce bronchodilation Patienbecome refractory to prolonged or repeated agonist exposurpossibly because of receptor desensitization and internalizationIn addition BAR2 polymorphisms may alter receptor behavio

resulting in greater receptor downregulation36

and airwahyperreactivity33 Adverse effects may also be linked to agoniaction on cells other than airway smooth muscle BAR2 agoniscan suppress eosinophil apoptosis37 and upregulate HR1 expresion38 thus aggravating this condition BAR2 agonists can alspromote positive effects such as the inhibition of histamine anleukotriene release from mast cells9 (See Chapter 95 for morinformation on β-adrenergic therapies)

NucleosideNucleotide Receptors

Adenine nucleotides can be released from cells in a regulatemanner (platelet degranulation) or as a consequence of ce

transcription factors promoting protein sumoylation andrecruiting transcriptional corepressors or coactivators Target-ing the JAK-STAT pathway with tofacitinib has been effective intreating rheumatoid arthritis29

Select Signaling Systems Relevantto Allergy

For the proper organization and effective mediation of a spe-cific immune response multiple signaling events must be inte-grated The examples discussed here address select receptortypes and downstream signaling pathways important in cellularand physiologic responses to allergens as well as current thera-pies used to modulate these responses

G PROTEINndashCOUPLED RECEPTORS

G proteinndashcoupled receptors are plasma membrane localizedand composed of seven transmembrane segments The specificG proteins associated with these receptors can either activate orinhibit various signaling pathways (see Fig 11-3) Besides theirlinkage to heterotrimeric G proteins GPCRs may associate with

other effector molecules such as JAK2 and β-arrestins

22

Theseeffectors can then facilitate receptor signaling or desensitizationand downregulation Because of their role in initiating signaltransduction pathways many therapeutics target the GPCRs asdiscussed next

Chemokine Receptors

Chemokine receptors are a superfamily of GPCRs that controlimmune cell behavior they promote chemotaxis cell adhesionand mediator release (see also Chapter 7) The chemokinereceptor superfamily is divided into four classes based on thechemokineligand interaction motif to which they bind (CCCXC CX3C or XC) Many chemokine-induced events areinhibited by cell treatment with Bordetella pertussis toxin which

ADP ribosylates and inactivates the α subunit of GiGo sug-gesting that certain chemokine receptors are coupled to these Gproteins However some chemokine-induced events are pertus-sis toxin insensitive IL-8ndashinduced chemotaxis does not requireGαi but is mediated by GβGγ after receptor activation Che-mokine receptors can also activate PLC isoforms leading to IP3 production and increased cytoplasmic Ca2+ which can regulatevarious kinases and promote cytoarchitectural changes Incertain cases chemokine receptor signaling also activates LMWG proteins (RasRho) which mediate many actin-dependentprocesses such as degranulation and chemotaxis For examplethe chemokines RANTES eotaxin and MCP-1 have been linkedto eosinophil activation and recruitment to the lungs of allergicasthmatic patients30

Leukotriene Receptors

Leukotrienes are lipid-based mediators derived from arachi-donic acid liberated from membrane phospholipids afterPLA2 activation and are separated into two groups LTB4 andcysteinyl leukotrienes including LTC4 LTD4 and LTE4 Theseagents are implicated in inflammatory diseases includingasthma because of their potent action as immune cell chemoat-tractants and bronchospasm stimulatorsinducers LTB4 hastwo identified receptors BLT1 and BLT2 the cysteinyl leukot-rienes also have two CysLT1 and CysLT2 LTB4-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 cells4344 Therapy in subsets of asthmatic patients with humanized antindashIL-5 antibody has been promising45

Interleukin-1

The IL-1 family ligands and receptors (IL-1Rs) are primarilyassociated with autoinflammatory conditions and are viable

therapeutic targets in diseases such as rheumatoid arthritisIL-1 promotes secretion of additional IL-1 and leads to a pro-inflammatory response The IL-1Rs contain a cytoplasmic TollIL-1 receptor domain that leads to downstream signalingthrough association of MyD88 an adapter protein and activa-tion of IRAKs (IL-1Rndashassociated kinases) and IKKβndashNF-κ Bsimilar to Toll-like receptors These signaling cascades activatecaspase 1 a cysteine protease responsible for the final processingof pro-IL-1β into its secretableactive form This proinflamma-tory response is associated with diseases such as rheumatoidarthritis and can be treated by blocking access of IL-1β to thereceptor (eg anakira) or by using neutralizing antibodies (egcanakinumab) against IL-1β Rilonacept a fusion of the extra-cellular portions of IL-1R1IL-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-specific α chains and acommon γ subunit important for cell signaling47 The IL-2 andIL-15 receptors also contain a β subunit that contributes tosignaling48 Stimulation of receptors sharing the common γ subunit promotes JAK13 phosphorylation49 whereas the Srcfamily kinases Lck50 Lyn and Fyn51 are associated with the β subunit The IL-2 receptor also activates other signaling mole-cules including PI3 kinase PLC-γ Ras Raf-1 and MAP

kinases47

The IL-13R shares the IL-4 α subunit and both recep-tors signal through JAK1 and Tyk2 activating STAT3652 IL-13has been shown to be a viable target in the treatment of asthmapatients with lebrikizumab a neutralizing antibody to IL-13which resulted in an increase in lung function53 In mice neu-tralizing antibodies against IL-13 suppress ovalbumin-inducedairway hyperresponsiveness eosinophil infiltration inflamma-tory cytokine production serum IgE levels and airway remod-eling Similarly neutralizating IL-9 in mice reduces airwayinflammation hyperresponsiveness and bone marrow eosino-philia suggesting that both IL-13 and IL-9 may be therapeutictargets in asthma (see Chapter 51)

The IL-6 receptor family includes receptors for IL-6 IL-11leukemia 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 JAK2and TYK254 Stimulation with IL-6 promotes STAT1 and STAT3activation and heterodimerization as well as activation ofprotein tyrosine phosphatase 2 (SHP2) which links this systemto Ras-ERK signaling and signal attenuation

Interleukin-12 signals through two receptors (IL-12R β1IL-12R β2) that regulate the activation of Lyk Tyk2 JAK2and STAT4 Although administered to alleviate asthma symp-toms IL-12 induces several toxic effects including malaise andcardiac arrhythmia suggesting that this treatment may not bepractical55

damage or death EctoATPases and nucleotidases modulate thebioavailability of extracellular nucleotides and generate metab-olites that are ligands for receptors on many cells Nucleotidereceptors are divided into P1 and P2 classes P1 receptors bindadenosineAMP whereas P2 receptors bind ATP adenosinediphosphate (ADP) and the pyrimidine nucleotides uridinetriphosphate (UTP) and uridine diphosphate (UDP) The P2family is divided into P2Y and P2X subclasses based on their

predicted structures The P1 and P2Y receptors are GPCRswhereas P2X receptors are ligand-gated ion channels39

Mast cells eosinophils lymphocytes neutrophils andmacrophages express P1 (adenosine) receptors and exhibitinflammatory mediator elaboration on stimulation39 Inhaledadenosine elicits bronchoconstriction in asthma and inducesmast cell release of histamine leukotrienes and inflammatorycytokineschemokines Adenosine receptor subtypes includeA1 A2AA2B and A3 A1 receptors are ubiquitously expressedwhereas A2 receptors are expressed in mast cells neutrophilsplatelets pancreas smooth muscle and the vasculature Activa-tion of A2 receptors promotes Gs-induced elevations in cAMPand IL-8 release A3 receptors are expressed in many tissuesespecially the lung and liver Its activation results in decreased

cAMP increased Ca

2+

fluxes and histamine release Elevatedadenosine levels activate A3 and promote eosinophil apoptosisand inhibit eosinophil and neutrophil degranulation reactiveoxygen species (ROS) release and chemotaxis

Widely expressed in immune cells the P2Y receptors arecoupled to Gq11 Gs (stimulator) or Gi (inhibitor) and regu-late IP3 generation Ca2+ fluxes and cAMP formation Notablythe leukotriene receptor antagonists montelukast and pranlu-kast can attenuate P2Y-mediated activation of PLC and intra-cellular Ca2+ mobilization thus modulating P2Y-mediatedinflammatory processes in allergy

The P2X receptors widely expressed on immune cells areATP-gated ion channels that elevate intracellular Ca2+ andmediate fast permeability to cations including Na+ and K+

Interestingly P2X 7 has been implicated in asthma-associatedairway inflammation40 P2X 7 activation can enhance endotoxin-induced responses in macrophages promote cytoskeletal reor-ganization and membrane blebbing (via Rho and p38) activatetranscription factors (eg NF-κ B CREB AP-1) and lead to theelaboration of mediators (eg IL-1β VEGF ROS)41 Thusmodulating P2X and P2Y receptors may effectively reduceinflammatory responses associated with allergy42

HEMATOPOIETIC RECEPTORS

Hematopoietic cytokines and their respective receptors controlthe differentiation maturation priming and activation ofmany 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-StimulatingFactorInterleukin-3Interleukin-5

The GM-CSFIL-3IL-5 cytokine receptor family shares acommon β chain necessary for signaling and forms a high-affinity receptor when associated or dimerized with a ligand-specific α chain Signaling through these receptors includes theactivation of cytoplasmic tyrosine kinases (eg Lyn Syk) Ras-ERK and JAK2-STAT35 IL-5 primarily targets eosinophilsand contributes to eosinophilic differentiation and survival in




these receptors initiates formation of TLR homodimers anheterodimers resulting in production of proinflammatorcytokineschemokines and other mediators TLRs are expressein monocytesmacrophages dendritic cells B cells and macells but some are found in certain nonimmune cells includincells lining mucosal surfaces (eg intestinal epithelial cellsAlso receptor expression is modulated by microbial invasionmicrobial components and cytokines58 (see Chapter 1)

INHIBITORY AND STIMULATORY RECEPTORS

Balancing stimulatory and inhibitory networks is important iproviding appropriate activation thresholds and sensitivtuning mechanisms for cell responses Many cell surface receptors and signaling molecules are critical for this balance including Fc receptors (FcRs) and their homologs leukocytimmunoglobulin-like receptors (LIRs) and paired Ig-likreceptors and signal-regulatory proteins (SIRPs) These proteins belong to a large class of receptors characterized bcommon cytoplasmic tyrosine-based signaling motifs immunoreceptor tyrosine-based activating motifs (ITAMs) thcontain two repeats of the consensus sequence Y-X-X-LI space

by six to eight amino acids or the immunoreceptor tyrosinebased inhibitory motifs (ITIMs) with a sixndashamino acid consensus sequence (IVLS)-X-Y-X-X-(LV) (see Table 11-1) Liganbinding to activating receptors leads to rapid phosphorylatioof ITAM tyrosines by Src family kinases which then initiat

For more information on cytokines and their receptors seeChapter 5

TUMOR NECROSIS FACTOR

Tumor necrosis factor (TNF) a major cytokine released pri-marily from macrophages but also from many other cells (eglymphoid cells mast cells fibroblasts) and involved in proin-

flammatory responses is associated with several diseasesincluding Crohn disease and rheumatoid arthritis TNF signalsthrough homotrimeric receptors 1 and 2 (TNF-R1 TNF-R2)which activate caspases (involved in apoptosis) NF-κ B andactivator protein 1 (AP-1) signaling cascades TNF is a potentialtherapeutic target implicated in airway inflammation and bron-chial hyperresponsiveness but late stage clinical trials weredisappointing56

TOLL-LIKE RECEPTORS

Toll-like receptors (TLRs) a superfamily of transmembraneIL-1Rndashlike molecules function in the innate immune system bydistinguishing different pathogens based on molecular signa-

tures TLRs are pattern recognition receptors (Fig 11-11) Theycontain leucine-rich motifs and a cytoplasmic region TollIL-1R (TIR) domain that is necessary for cell signaling57 Thesereceptors are largely expressed on the cell surface althoughTLRs 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 interactioresponses and modulation of inhibitory and stimulatory cell signals Toll-like receptors (TLRs) are a superfamily of transmembrane interleukinreceptor (IL-1R)ndashlike molecules that function in the innate immune system by distinguishing different pathogens based on their molecular signatures Many other immune cell surface receptors and signaling molecules are critical in mediating a balance between inhibitory and stimulatocell activities including the Fc receptors (FcRs) and their homologs the leukocyte immunoglobulin-like receptors (LIRs) and signal-regulatorproteins (SIRPs) These proteins belong to a large class of receptors (eg T cell receptors Siglecs paired Ig-like receptors) and are characteized by the possession of common cytoplasmic tyrosine-based signaling motifs the ITAMs for stimulatory systems and ITIMs for inhibitosystems Ligand binding to activating receptor complexes promotes ITAM tyrosine phosphorylation and the recruitment of Src family kinasethat induce positive cell responses Ligand binding to inhibitory receptor complexes promotes ITIM tyrosine phosphorylation and the recruiment of SH2 domainndashcontaining 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 familykinasesZAP70

TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P




the these receptors ITIM phosphorylation creates docking sitesfor SH2 domainndashcontaining phosphatases such as SHP-1 andthe inositol phosphatase SHIP which then deliver an inhibitorysignal to receptor systems such as FcRs or cytokine receptorsthat use tyrosine kinases (eg Src family kinases Syk JAKs) orinositol phosphates (IP3 PIP3) as stimulatory signals The utilityof LIRs containing multiple cytoplasmic ITIMs is undefined butmay allow for signal amplification or recruitment of signaling

moleculesStimulatory LIRs possess short cytoplasmic domains devoidof ITIMs and a positively charged arginine within their trans-membrane domains This arginine may direct LIR associationwith other surface receptors such as FcR common γ chainwhich interacts with FcRs for IgA IgE and IgG and has a nega-tively charged aspartate within its transmembrane region Theinteraction of these stimulatory LIRs with FcR-γ may berequired for cell surface expression and assembly of a signaling-capable multimeric receptor FcR-γ 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 proteinkinases that phosphorylate tyrosine residues within the ITAMcreating binding sites for SH2 domainndashcontaining kinases Syk

and ZAP-70 thus promoting initiation of stimulatory signalsSignal-regulatory Proteins

The regulation of immune responses also involves SIRP familymembers expressed by myeloid cells that can interact with cellsurfacendashtethered proteins either amplifying or attenuating cellsignaling60 The three SIRP members each contain three highlyhomologous Ig-like domains in their extracellular regions butdiffer in their cytoplasmic regions by the presence or absenceof ITIMs

The SIRP family comprises α β and γ types SIRPα foundin myeloid cells and neurons utilizes CD47 and surfactant pro-teins associated with lung inflammation (eg SP-A) as ligandsSIRPβ also known as CD172b and found in macrophages and

neutrophils has no known ligand SIRPγ also known as CD172gor SIRPβ2 binds CD47 and is expressed in lymphocytes andnatural killer (NK) cells SIRPα initiates inhibitory signals suchas reduced macrophage phagocytosis and TNF productionthrough cytoplasmic ITIMs Conversely SIRPβ has a basicamino acid side chain in its transmembrane region essential forbinding the activating adapter protein DAP12 which containsa single ITAM that provides stimulatory signals SIRPγ has ashort cytoplasmic domain with no known motifs for recruitingsignaling molecules and no charged residue to mediate associa-tion with DAP12 or other adapter proteins thus its signalingpotential is unclear

Because of their interaction with cell surfacendashtethered pro-teins SIRPs are sometimes termed paired receptors Additional

examples of inhibitorystimulatory receptor systems includeCD40CD40L CD200 receptor cytotoxic T lymphocyte antigen4 intercellular adhesion molecules (ICAMs) sialic acidndashbindingIg-like lectins (Siglecs)61 and the T cell receptors (TCRs)

Additional TherapeuticConsiderations

Therapeutics affecting protein kinases and transcription factorsare used to treat asthma airway inflammation and otherinflammatory diseases Many kinase inhibitors including thosetargeting Src kinases JAKs and PI3 kinase isoforms have been

signaling cascades leading to cellular activation In contrastbinding to the inhibitory receptors results in tyrosine phos-phorylation of ITIMs which generates docking sites for SH2domainndashcontaining phosphatases that can attenuate cellularactivation The balance between these activating and inhibitoryreceptors 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 andantibodies Accordingly receptors exist for each antibody classFcγ R binds IgG FcαR binds IgA FcεR binds IgE Fcα983221R bindsIgM and FcδR binds IgD Various leukocytes and other immunecells express subsets of these receptors There are two major FcRtypes one triggers cell activation and one leads to inhibitorysignals Activating FcR subtypes possess cytoplasmic ITAMs(see Table 11-1 and Fig 11-11) and can be divided into twosubclasses one is composed of a ligand-binding α subunit asso-ciated with one or two ITAMs containing intracellular signalingmolecules and the second consists of two related single-chainIgG receptors that have a single cytoplasmic ITAM InhibitoryFcRs contain cytoplasmic ITIMs and after phosphorylation

recruit phosphatases that inhibit cell activation through proteindephosphorylation Thus negative regulation of FcRs resultsfrom coaggregation with receptors containing ITIMs

Of relevance to allergy the high-affinity IgE receptor (FcεRI)is primarily expressed by mast cells and basophils its activationleads to the release of histamine eicosanoids and proteases thatresult in airway obstruction smooth muscle contraction vas-cular leakage and mucus secretion Upon IgE binding FcεRIaggregates promoting rapid ITAM phosphorylation of thereceptor γ portion and subsequent activation of Syk and Srcfamily kinases (eg Lyn Fyn) With the aid of adapter proteins(eg LAT and NATL) these kinases activate multiple signalingcascades including PLCγ PKC PI3KPLD and MAPKs as wellas the transcription factors NF-κ B AP-1 and nuclear factor of

activated T cells (NFAT) Many patients with allergic diseasepresent with an elevated IgE level that correlates with diseasesymptoms and therefore makes inhibiting FcεRI signaling animportant therapeutic target For example omalizumab ahumanized monoclonal antibody binds circulating IgE pre-venting activation of the FcεRI and is used to treat moderateto severe allergic asthma In general FcRs are key targets intreating allergic inflammation (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 Althoughtheir ligands are not completely defined some may recognizemajor histocompatibility complex (MHC) class I molecules59

LIRs contain two or four Ig-like domains in their extracellularregions and are either inhibitory cell surface transmembranereceptors with larger intracellular domains (LIRs 1 2 3 5 8)stimulatory cell surfacendashassociated receptors with short cyto-plasmic sequences (LIRs 6a 6b 7) or soluble molecules (LIR4)Although the role of the first two LIR classes have been charac-terized the function of LIR4 is unclear but it may act as asoluble ligand triggering other cell-surface receptors or as anantagonist of membrane-bound LIRs by competing for ligands

It is thought that when inhibitory ITIM-containing LIRs arebrought into close proximity with activating receptors theITIMs become tyrosine phosphorylated by kinases recruited to




Conclusion

Targeting various components of signaling pathways shoulallow for potent treatments of allergic disease Because of thubiquitous nature of these molecules however these therapieface greater challenges in terms of safety and selectivity thatherapies targeted toward individual receptors that are morrestricted in their expression

AcknowledgmentsPaul J Bertics who held the endowed Robert Turell Profesorship in the Department of Biomolecular Chemistry athe University of Wisconsin (UW) School of Medicine anPublic Health died unexpectedly on Dec 22 2011 at the agof 55

Paul was regarded across multiple disciplines in the scientific community as a person with outstanding insight anintelligence His scientific interests focused on inter- and intramolecular mechanisms that translate ligandreceptor interactions into physiologic outcomes Paulrsquos research career begawith examining signal transduction events through EGF receptors on cancer cells and expanded to purinergic and IL-5 fami

receptors on innate immune cells as well as state-of-the-aresearch utilizing liquid crystal and nanostructured surfaces tdevelop innovative detection methods for the rapid characterization of disease states Although the extent of his academiand scientific contributions was immense he was and alwayremained an unassuming and humble man Paul was a naturaleader who brought out the best in people by sharing hwisdom empathy compassion concern and most importantlhis humor and infectious laugh

As a scientist teacher colleague friend and above ala loving father and husband Paul will be greatly missedbut his legacy will continue in those who knew and learnefrom him

examined in inflammation models and are in development forpossible human use In terms of kinases the p38 inhibitorsVX-702 and HEP 689 have been evaluated in clinical trials forpsoriasis and other chronic inflammatory disorders BIRB796another p38 inhibitor is being evaluated in Crohn diseasebecause CNI-1493 a dual inhibitor of JNK and p38 was effec-tive in a small study of patients with Crohn disease62 A Sykinhibitor R-112 (Rigel) has been effective against allergic rhi-

nitis symptoms

63

Another Syk antagonist fostamatinib hasentered a phase III study for rheumatoid arthritis Because Sykis involved in many processes and cell types the selectivitysafety and mode of delivery of these drugs are areas of intenseanalysis Furthermore inhibitors targeting Brutonrsquos tyrosinekinase (BTK) are in clinical trials for B cell malignancies andalso inhibit degranulation from basophils12

In terms of transcription factors in mouse models an oralsmall-molecule inhibitor (BMS345541) that blocks IKK2-dependent phosphorylation of Iκ B and attenuates NF-κ B-dependent transcription reduces the incidence and severity ofarthritis However NF-κ B is a ubiquitous transcription factorgiving rise to concerns that its inhibition may cause increasedsusceptibility to infections Similarly one of the most common

therapies prescribed for asthmatic patients to control symp-toms is inhaled or systemic glucocorticoids which signalthrough intracellular receptors to regulate gene transcription64 For example corticosteroid-induced upregulation of Iκ Battenuates NF-κ B action and inhibits mediator production65 This subsequent decrease in cytokine and chemokine produc-tion attenuates lymphocyte and macrophages proliferationand migration to sites of inflammation thus suppressing theimmune response Also corticosteroid therapy stimulatesimmune cell apoptosis perhaps through decreased cytokineproduction necessary for cell survival However systemicadministration of these compounds results in many side effectsmaking their long-term use difficult

REFERENCES

Introduction

1 Bradshaw R Dennis E editors Handbook ofcell signaling London Elsevier 2003

Cell Structures

2 Alberts B Bray D Hopkin K et al Molecularbiology of the cell 3rd ed New York GarlandSciences 2009

3 Janmey P The cytoskeleton and cell signalingcomponent localization and mechanical cou-pling Physiol Rev 199878763-81

General Principles of Receptors and CellSignaling

4 Molfino NA Gossage D Kolbeck R Parker JMGeba GP Molecular and clinical rationale fortherapeutic targeting of interleukin-5 and itsreceptor Clin Exp Allergy 201242712-37


5 Mustelin T Vang T Bottini N Protein tyrosinephosphatases and the immune response NatRev Immunol 2005543-57

6 Pierce KL Premont RT Lefkowitz RJ Seven-transmembrane receptors Nat Rev Mol CellBiol 20023639-50

7 Page C Spina D Phosphodiesterases as drugtargets Handb Exp Pharmacol 2011204391-414

8 Berridge MJ Bootman MD Roderick HLCalcium signalling dynamics homeostasis andremodelling Nat Rev Mol Cell Biol 20034517-29

9 Barnes PJ Biochemical basis of asthma therapyJ Biochem Chem 201128632899-905

10 Kolch W Coordinating ERKMAPK signallingthrough scaffolds and inhibitors Nat Rev MolCell Biol 20056827-37

11 Wong WSF Leong KP Tyrosine kinase inhibi-tors a new approach for asthma BiochimBiophys Acta 2004169753-69

12 MacGlashan Jr D Honigberg LA Smith ABuggy J Schroeder JT Inhibition of IgE-mediated secretion from human basophils witha highly selective Brutonrsquos tyrosine kinaseBtk inhibitor Int Immunopharmacol 201111475-9

13 Wen AY Sakamoto KM Mil ler LS Therole of the transcription factor CREB inimmune function J Immunol 20101856413-9

14 Shuai K Liu B Regulation of JAK-STAT signal-ling in the immune system Nat Rev Immunol20033900-11

15 Oh C Geba G Molfino N Investigational thera-peutics targeting the IL-4IL-13STAT-6 pathwayfor the treatment of asthma Eur Respir Rev20101946-54

16 Perkins ND Integrating cell-signalling pathwawith NF-κ B and IKK function Nat Rev Mol CBiol 2007849-62

17 Massagueacute J Seoane J Wotton D Smad trascription factors Genes Dev 2005192783-81

18 Grant PA A tale of histone modificationGenome Biol 20012Reviews00031-reviews6

19 Lingwood D Simons K Lipid rafts as membrane-organizing principle Science 201327(5961)46-50

20 Guichard C Pedruzzi E Dewas C et Interleukin-8-induced priming of neutrophoxidative burst requires sequential recruitmeof NADPH oxidase components into lipid rafJ Biol Chem 200528037021-32

21 Diaz O Mebarek-Azzam S Benzaria A et aDisruption of lipid rafts stimulates phosphopase D activity in human lymphocytes impliction in the regulation of immune functioJ Immunol 20051758077-86

22 Whalen EJ Rajagopal S Lefkowitz RJ Therpeutic potential of β-arrestin- and G proteinbiased agonists Trends Mol Med 20111126-39

23 Parton RG Simons K The multiple facof caveolae Nat Rev Mol Cell Biol 2007185-94

24 Scita G Di Fiore PP The endocytic matriNature 2010463(7280)464-73




25 Weissman AM Themes and variations onubiquitylation Nat Rev Mol Cell Biol 20012169-78

26 Muller S Hoege C Pyrowolakis G Jentsch SSUMO ubiquitinrsquos mysterious cousin Nat RevMol Cell Biol 20012202-10

27 Krebs DL Hilton DJ SOCS physiological sup-pressors of cytokine signaling J Cell Sci 2000113(Pt 16)2813-9

28 Yoshimura A Negative regulation of cytokine

signaling Clin Rev Allergy Immunol 200528205-2029 Yazici Y Regens A Promising new treatments

for rheumatoid arthritis the kinase inhibitorsBull NYU Hosp Joint Dis 201169233-7

Select Signaling Systems Relavantto Allergy

30 Viola A Luster AD Chemokines and theirreceptors drug targets in immunity andinflammation Annu Rev Pharmacol Toxicol200848171-97

31 Jutel M Blaser K Akdis CA Histamine in aller-gic inflammation and immune modulation IntArch Allergy Immunol 200513782-92

32 Simons FER Simons KJ Histamine andH1-antihistamines celebrating a century of

progress J Allergy Clin Immunol 20111281139-50e433 Johnson M The beta-adrenoceptor Am J Respir

Crit Care Med 1998158(5 Pt 3)S146-5334 Price DM Chik CL Ho AK Norepinephrine

induction of mitogen-activated protein kinasephosphatase-1 expression in rat pinealocytesdistinct roles of alpha- and beta-adrenergicreceptors Endocrinology 20041455723-33

35 Kotlikoff MI Kamm KE Molecular mecha-nisms of beta-adrenergic relaxation of airwaysmooth muscle Annu Rev Physiol [Review]199658115-41

36 Reihsaus E Innis M MacIntyre N Liggett SBMutations in the gene encoding for the β2-adrenergic receptor in normal and asthmaticsubjects Am J Respir Cell Mol Biol 19938

334-937 Kankaanranta H Lindsay MA Giembycz MAet al Delayed eosinophil apoptosis in asthmaJ Allergy Clin Immunol 2000106(1 Pt 1)77-83

38 Mak JC Roffel AF Katsunuma T et al Up-regulation of airway smooth muscle hista-mine H(1) receptor mRNA protein and func-tion by beta(2)-adrenoceptor activation MolPharmacol 200057857-64

39 Polosa R Adenosine-receptor subtypes theirrelevance to adenosine-mediated responses inasthma and chronic obstructive pulmonarydisease Eur Respir J 200220488-96

40 Denlinger L Sorkness R Lee WM et al Lowerairway rhinovirus burden and the seasonal riskof asthma exacerbation Am J Respir Crit CareMed 20111841007-14

41 Lenertz L Gavala M Zhu Y Bertics P Transcrip-tional control mechanisms associated with the

nucleotide receptor P2X7 a critical regulator ofimmunologic osteogenic and neurologic func-tions Immunol Res 20115022-38

42 Muumlller T Vieira RP Grimm M et al A potentialrole for P2X7R in allergic airway inflammationin mice and humans Am J Respir Cell Mol Biol201144456-64

43 Lopez AF Hercus TR Ekert P et al Molecularbasis of cytokine receptor activation IUBMBLife 201062509-18

44 Pouliot P Olivier M Opposing forces in asthmaregulation of signaling pathways by kinasesand phosphatases Crit Rev Immunol 200929419-42

45 Haldar P Brightling CE Hargadon B et alMepolizumab and exacerbations of refractoryeosinophilic asthma N Engl J Med 2009360

973-8446 Dinarello CA Interleukin-1 in the pathogenesisand treatment of inflammatory diseases Blood20111173720-32

47 Sugamura K Asao H Kondo M et al Theinterleukin-2 receptor γ chain its role in themultiple cytokine receptor complexes and T celldevelopment in XSCID Annu Rev Immunol199614179-205

48 Giri JG Ahdieh M Eisenman J et al Utilizationof the β and γ chains of the IL-2 receptor bythe novel cytokine IL-15 Embo J 1994132822-30

49 Taga T Kishimoto T Signaling mechanismsthrough cytokine receptors that share signaltransducing receptor components Curr OpinImmunol 1995717-23

50 Hatakeyama M Kono T Kobayashi N et alInteraction of the IL-2 receptor with the src-family kinase p56lck identification of novelintermolecular association Science 1991252(5012)1523-8

51 Kobayashi N Kono T Hatakeyama M et alFunctional coupling of the src-family proteintyrosine kinases p59fyn and p5356lyn withthe interleukin-2 receptor implications forredundancy and pleiotropism in cytokine signal

transduction Proc Natl Acad Sci USA 1993904201-5

52 Izuhara K Arima K Signal transduction ofIL-13 and its role in the pathogenesis of bron-chial asthma Drug News Perspect 20041791-8

53 Corren J Lemanske RF Hanania NA et alLebrikizumab treatment in adults with asthmaN Engl J Med 20113651088-98

54 Ihle JN Witthuhn BA Quelle FW Yamamoto K

Silvennoinen O Signaling through the hemato-poietic cytokine receptors Annu Rev Immunol199513369-98

55 Yamagata T Ichinose M Agents against cytokinesynthesis or receptors Eur J Pharmacol 2006533289-301

56 Wenzel SE Barnes PJ Bleecker ER et al ARandomized double-blind placebo-controlledstudy of tumor necrosis factor-α blockade insevere persistent asthma Am J Respir Crit CareMed 2009179549-58

57 Akira S Toll-like receptor signaling J Biol Chem200327838105-8

58 Takeda K Kaisho T Akira S Toll-like receptorsAnnu Rev Immunol 200321335-76

59 Borges L Cosman D LIRsILTsMIRs inhibi-tory and stimulatory Ig-superfamily receptors

expressed in myeloid and lymphoid cells Cyto-kine Growth Factor Rev 200011209-1760 Barclay AN Brown MH The SIRP family of

receptors and immune regulation Nat RevImmunol 20066457-64

61 Von Gunten S Bochner BS Basic and clinicalimmunology of Siglecs Ann NY Acad Sci2008114361-82

Additional Therapeutic Considerations

62 Adcock IM Chung KF Caramori G Ito KKinase inhibitors and airway inflammation EurJ Pharmacol 2006533118-32

63 Patou J Holtappels G Affleck K van Cauwen-berge P Bachert C Syk-kinase inhibition pre-vents mast cell actiavtion in nasal polypsRhinology 201149100-6

64 Goodman amp Gilmanrsquos the pharmacologicalbasis of therapeutics 11th ed New YorkMcGraw-Hill 2006

65 McKay LI Cidlowski JA Molecular control ofimmuneinflammatory responses interactionsbetween nuclear factor-κ B and steroid receptorndashsignaling pathways Endocr Rev 199920435-59




Cell Structures

The cell is a sophisticated entity that can respond to a multitudof stimuli and changes from its microenvironment producin

a precise outcome that maintains the status quo of the bodyCellular anatomy has developed in such a manner as to isolatthe internal workings from nonspecific stimulation and to compartmentalize the machinery by grouping specific moleculethat can interact within and between their location Althougmany structures make up a typical cell the parts relevant tsignal transduction include the plasma membrane cytoskeleton and several organelles (See Alberts and associates2 for aextensive treatise on the biology of the cell)

PLASMA MEMBRANE

Eukaryotic cells are encapsulated by a plasma membrane thamakes the cell selectively permeable to many extracellula

factors including nutrients lipids proteins ions and pathogens The plasma membrane is a fluid lipid bilayer containina complex mixture of phospholipids glycolipids sterols anproteins This structure serves as an effective barrier to molecules that are poorly lipid soluble and also plays a critical roin the bidirectional flow of information This conduction oinformation includes the specific recognition of extracellulafactors including hormones toxins adhesion molecules anpathogens that function to modulate cellular responses As previously noted the recognition of these factors at the plasmmembrane is mediated by receptors and receptor engagemenelicits changes in cell behavior through the alteration o

Because cell signaling involves many effector molecules andcellular structures it is important first to define several basicconcepts (Fig 11-1)

For example external stimuli that cannot freely enter the

cell such as water-soluble factors (eg cytokines chemokines)or various externally tethered molecules (eg cell surface pro-teins extracellular matrix components) generally interact withspecific cellular recognition molecules (receptors) that possessan externally facing ligand-binding site These cell surfacereceptors then transduce information into the cell throughvarious conformational and enzymatic activities that allow forsignal amplification and regulation of specific intracellularenzyme activities ion fluxes cytoskeletal reorganization andsecretory events Also depending on the receptor cell surfacendashinitiated signaling may alter transcriptional activities chroma-tin structure messenger RNA (mRNA) stabilityprocessing(eg microRNAs) translational activity and posttranslationalprocessing Furthermore signaling through cell surface recep-

tors can result in receptor desensitization internalization andrecycling to achieve feedback control and to prevent excessivestimulation that may lead to pathology

With respect to lipid-soluble factors that can penetrate themembrane such as steroid hormones the receptors for thesemolecules are largely located within the cell often in the nucleuswherein ligand-receptor complex formation generally serves tomodulate gene transcription Once again feedback pathwaysexist to contain the magnitude and duration of the initiatedsignals Specific cellular structures receptors and processes arecommon to numerous signal transduction systems associatedwith 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 cognareceptor In general receptor activation leads to signal amplification and modification of cellular events including secretion cytoskeletal changeand certain enzymatic activities and alterations in gene expression (transcription mRNA stability protein induction) All these processes exhibcomplex feedback controls An understanding of these signaling systems is valuable in the development of therapeutics for allergic disorder

Intracellular

receptors

Lipid soluble(eg glucocorticoids)

Hormonesligands

Water soluble(eg IL-5)

Cell surface receptors

Feedback controls

Amplification cascades

Regulation of enzyme r eceptor activityCytoskeletal reorganizationIon fluxes secretion etc

Regulation of transcription

Protein stability mRNA stability and tr anscription control(eg microRNAs)

Modulation of processingsecretion

Nuclear receptorsregulatory proteins

Extracellular

Plasmamembrane

Cytoplasm

Nucleus








[ ] [ ] [ ]L R LR +


Kd L R LR = times[ ] [ ] [ ]







CYTOSKELETON



CELLULAR ORGANELLES

























ATP binding site

Cysteine r ich site

ATP binding amp

phosphorylation

ATP binding

COOH

COOH

Hinge


C4

Pseudosubstrate

Pseudosubstrate


PP


A

B

C3

C3

C1

C1NH2

SH3

SH2

Kinase

NH2

P

SH3 SH3


SH2

Kinase

SH2

Kinase

P















No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP


Axin








Gα12 (Gα12 Gα13)


β

γ

βγ

Cytoplasm

Plasmamembrane




Extracellular




Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures





















































































[ ] [ ] [ ]L R LR +


Kd L R LR = times[ ] [ ] [ ]







CYTOSKELETON



CELLULAR ORGANELLES

























ATP binding site

Cysteine r ich site

ATP binding amp

phosphorylation

ATP binding

COOH

COOH

Hinge


C4

Pseudosubstrate

Pseudosubstrate


PP


A

B

C3

C3

C1

C1NH2

SH3

SH2

Kinase

NH2

P

SH3 SH3


SH2

Kinase

SH2

Kinase

P















No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP


Axin








Gα12 (Gα12 Gα13)


β

γ

βγ

Cytoplasm

Plasmamembrane




Extracellular




Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

































































































ATP binding site

Cysteine r ich site

ATP binding amp

phosphorylation

ATP binding

COOH

COOH

Hinge


C4

Pseudosubstrate

Pseudosubstrate


PP


A

B

C3

C3

C1

C1NH2

SH3

SH2

Kinase

NH2

P

SH3 SH3


SH2

Kinase

SH2

Kinase

P















No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP


Axin








Gα12 (Gα12 Gα13)


β

γ

βγ

Cytoplasm

Plasmamembrane




Extracellular




Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures




























































































No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP


Axin








Gα12 (Gα12 Gα13)


β

γ

βγ

Cytoplasm

Plasmamembrane




Extracellular




Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


























































































No ligand

GPCR

Ligand

α-GTP

GTP GDP

α-GDP

GTPase

Pi

α-GDP α-GTP


Axin








Gα12 (Gα12 Gα13)


β

γ

βγ

Cytoplasm

Plasmamembrane




Extracellular




Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

















































































Phospholipases











PLC-12 PLC

P

P

PIP2 IP3 + DAG


Ca2+

+calmodulin

Ca2+

Cytoplasm




PLA2





Ion channel


G proteins

Eicosanoids



Plasmamembrane

Extracellular



















Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures
































































































Phospholipase A1 O

O

O

C

C

P

O ndash

R1Phospholipase A2


(Head gr oup)



R2

X

CH2

CH2

CH

O

O

O

Pl-PLC


Pl3 kinase

Pl

A

B

C

PlP PlP2

PlP3 PDK 12 PP2 A

Akt

Phosphorylated Akt

Cell sur vival


IP3 + DAG






esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


















































































esters10








MAP Kinase Cascades




MAPKKK


Raf PI3K

GDP-GTP exchange


Ser phosphorylation


MAPKK MEK1 MEK2


ERK1 ERK2MAPK

Rac

ASK1

MKK36

JNKp38

MKK47

MLK36







RacMLKMKKp38

(MAP kinase)

















TABLE 11-2













enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


























































































enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures
























































































enhanced20



Nuclear Factor-κ B




SMADs




Tyrosine kinases


Nuclear membrane





RelA

RelA

Hormonalactivation

IKKs

NF-κ B

NF-κ B

Iκ Bα

Iκ Bα


P P

Iκ

Bα

P P




Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

















































































Caveolins







β -Arrestins




P

P

II

III

II

II

P

P

Dab2R-SMAD

R-SMAD

R-SMAD

PR-SMAD

R-SMAD

R-SMAD

co-SMAD

P P

R - S M

A D

c o - S M A D

co-SMAD

D i m e r i z

a t i o n

I-SMAD

eg Type 1 collagen

I-SMAD

FKBP12

Gs domain

Kinasedomains

Adapter proteins

SMURF


SMURF 12

SAR A

Dab2

SARA

I

I












Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

























































































Ubiquitylation



Sumoylation




β

RasRaf

MEK

ERK

1 amp 2

α

β-arrestins

β-arrestins

Cytokine receptor

Mediator production

Gene expression

Survival



Motility

Lipid rafts


γ

JAKs

STATs

Lyn Lyn

RasGRK

SrcSyk

Grb2Sos

Shc





Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


















































































Histamine Receptors



32
















22


Chemokine Receptors









Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


















































































Interleukin-1






kinases47








cAMP increased Ca

2+





















TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

























































































TOLL-LIKE RECEPTORS




P

+

P

P

+

S H P - 1 2

S H P - 1

S H I P

Z a p 7 0


TRAFs

TAK TABs

TRIF TRAM

TLRsIL-1R

SIRPβ(stimulatory)

β

LIR(stimulatory)

FcRsγ chains

γ γ

SIRPα(inhibitory)

α

LIR(inhibitory)

IRAK

IKK

NF-κ B

NF-κ B

Iκ Bα

My D88 D A P 1 2

Syk

ITIMs

ITAMs

P

P

P















Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures




























































































Fc Receptors












Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures

















































































Conclusion







nitis symptoms

63




REFERENCES

Introduction


Cell Structures


































































































































Documents

Chapter 11 - Signal Transduction