Principles of cell signaling - UT Southwestern

Ser Thr 

IRAK-M 

TRIF/ 

TICAM-1 

SOCS1 

/JAB 

ys63TRAF6 

0 

CAPK 

CAPK 

IRAK1 

Lys 

TBK-1 

P 

P 

TRAF2 TRAF1 

A20 

P Tyr STAT6 

K 

-2 

TNF 

TICAM-1 

P 

Ser 

IRAK1 

Thr 

Lys 

Ser 

IRAK1 

Thr Lys 

IKK 

Ser176 

Ser181 IKK 

IKK 

IKK 

P 

Ser176 

Ser181 IKK 

P 

IKK 

GM-CSFR 

GM-CSFR 

Uev1A 

Ubc13 

P Tyr STAT5 

PI3K 

MEKK 

Tyr STAT5 

GM-CSF 

TLR4 

PP2A 

TAB2 

P 

P 

P 

Thr184 

Ser192 TAK1 Thr187 P 

P P 

TBK-1 

P Ser TAB1 

Thr 

P 

P 

Tyr580 

P 

TRIF/ 

TICAM-1 

TRAM 

MD-2 

P P 

Tyr STAT5 

Tyr 

Ser386 

Ser385 

IRF-3 

Ser Thr IRAK1 

Lys 

Ub 

P 

P 

P 

P Tyr STAT3 

Tyr STAT3 

Thr38 Ser473 

Akt/PKB 

P 

P 

Thr38 Ser473 

Akt/PKB 

Ser Thr IRAK1 Lys 

Ub 

PKA 

PKA 

TRAM 

ASK 

Lys21 

Lys21 

I B 

Ser32 

Lys22 

Ser36 

NF- 

B 

p65+p50 

Ser276 

Ser529 

P 

Lys21 

I B 

Ser32 

Lys22 

Ser36 

P 

PKA 

NF- 

B 

p65+p50 

Ser276 

Ser529 

P 

Tyr701 

TAB2 

Tyr701 

Ser727 STAT1 

IKK 

Thr184 Ser192 TAK1 Thr187 

P 

P 

Ser 

TAB2 

TAB1 

Ser32 

Ser36 

IKK 

Ser Thr IRAK1 

Lys 

Ub 

Thr184 

Ser192 

TAK1 

Thr187 P 

SEK1/MKK4 

P 

Tyr701 

Ser727 

STAT1 

P 

P 

Tyr STAT2 

IRF-9 

Thr 

Ser386 

P 

P 

CK II 

Tyr JAK1 

IL-10R 

Tyr 

Tyr JAK1 

Ser 

IL-10R 

TAB1 

NF- B 

p65+p50 

Ser276 Ser529 

IL-10R 

Tyr 

NF- B 

p65+p50 

Ser276 Ser529 

P 

P 

NF- B 

p65+p50 

Ser276 Ser529 

P 

NF- 

B 

PKA 

p65+p50 

Ser276 

Ser529 

IRF-9 

P 

P 

Ser 

Thr 

glycerol 3P 

TAB2 

TAB1 

P 

IL-10R 

Tyr 

Tyk2 

Tyr 

IL-10R 

Thr184 

Ser192 

TAK1 

Thr187 

P 

Ser 

TAB2 

TAB1 

Thr 

Ser Thr IRAK1 

Lys 

Ub 

Thr184 

Ser192 

TAK1 

Thr187 

Thr 

SEK2/MKK7 

SEK1/MKK4 

Tyk2 Tyr 

Tyr STAT5 

p50 

p60 

P 

Thr183 ERK1 

Tyr185ERK2 

P 

TG 

PKA 

malic enzyme 

P 

Tyr 

P 

Tyr542 

Tyr580 SHP-2 

Tyr580 

P 

lipid droplet 

SOCS3 

SHP-1 

Tyr JAK1 

Tyr759 

Tyr915 

IL-6R 

Tyr767 

Tyr905 

Tyr814 

Tyr JAK1 

Tyr JAK1 

Tyr STAT3 P Tyr STAT3 

Thr 

Thr 

Ub 

Lys21 

Ub 

IL-1ra 

malate 

Tyr580 

Tyr542 SHP-2 

P Tyr STAT5 

JNK 

JNK 

Tyr 

Tyr 

P 

NF- B 

p65+p50 

Ser276 Ser529 

Ser276 Ser529 

P 

P 

P 

P 

Ser32 

Ser36 

P 

P 

pyruvate 

carrier 

SEK2/MKK7 

PAFR 

TNF 

TG 

y 

Tyr 

P 

malate 

dehydrogenase 

I B 

GM-CSF 

IL-6 

p50 

IL-1 

oxaloacetate 

PKA 

NADH+H+ 

Tyk2 

Tyr 

Tyr759 

Tyr915 

Tyr767 

IL-6R 

Tyr905 

Tyr814 

Tyk2 

Tyr 

Tyr759 Tyr915 

IL-6R 

Tyr767 Tyr905 

Tyr814 

Ub 

P 

Lys21 

I B 

Ser32 

Lys22 

Ser36 

Ub 

P 

PKA 

NF- 

B 

p65+p50 

Ser276 

Ser529 

IFN- 

IFN- 

IFNpyruvate 

pyruvate acetyl CoA 

A20 

P 

P 

Ser385 

Ser386 

P 

Ser386 

P 

NF- B 

p65+p50 

Ser276 Ser529 

Ser276 Ser529 

P 

P 

P 

P 

IL-6R 

gp130 

P 

P 

P 

Tyr759 Tyr915 Tyr759 Tyr915 

Tyr767 IL-6R IL-6R 

Tyr905 

Tyr767 

Tyr905 

P 

Tyr814 

P 

Tyr814 

P 

P 

P 

Tyr JAK1 

Tyk2 

Tyr 

P 

P 

SOCS3 

IL-1ra 

pyruvate 

carboxylase 

P 

Lys21 

I B 

Ser32 

Lys22 

Ser36 

P 

NF- 

B 

p65+p50 

Ser276 

Ser529 

IFN 

P 

P 

Ser385 

Ser386 

Ser386 

P 

P 

citrate 

liase 

PDH kinase 

PDH kinase 

P 

P 

pyruvate 

dehydrogenase 

dehydrogenase 

y 

Tyr JAK1 

IFN 

IFN 

Tyr440 

Tyr JAK1 

Tyk2 Tyr 

p38MAPK 

IFN- 

SCF 

R1 

IFN 

Tyr440 

R1 

Tyr701 

Ser727 STAT1 

P P 

Tyr STAT3 

Tyr 

P P 

Tyr Tyr STAT3 

P P 

Tyr Tyr STAT5 

TrCP 

UbcH5 

IFN 

acetyl CoA 

citrate 

Tyr1007 

R2 

Tyr 

P P 

Tyr Tyr 

STAT3 

SHP-1 

IFN 

R2 

Tyr 

JAK2 

Tyr1007 

PIAS3 

JAK2Tyr1007 

P 

P 

SOCS3 

P 

Tyr701 

Ser727 STAT1 

P 

P Tyr701 Tyr701 

P 

Ser727 STAT1 

Ser727 

P 

CoASH 

carnitine 

P 

P Tyr701 

Ser727 Tyr701 STAT1 

P 

P 

P Tyr701 Tyr701 

Ser727 STAT1 

P Ser727 

P 

carnitine 

acyl-CoA 

y 

P 

PIAS3 

P 

IL-4R 

Tyr 

IFN 

R1 

IFN 

R2 

Tyr440 

Tyr 

P 

P 

JAK2 

Tyr JAK1 

Tyr1007 

P 

SOCS1 

/JAB 

P 

P 

Thr38 Ser473 

Akt/PKB 

Thr38 Ser473 

Akt/PKB 

P 

IRF-9 

CoASH 

Tyr1007 

y 

P 

P 

acyl-CoA 

CoASH 

CPT I 

IL-4R 

Tyr580 

P 

P 

P 

P 

common 

Tyr 

chain 

JAK3Tyr 

SOCS1 

/JAB 

SOCS1 

/JAB 

SOCS1/JAB 

Tyr701 

Ser727 

STAT1 

Tyr STAT2 

IRF-9 

P 

Tyr STAT6 

IRF-2 

acylcarnitine 

acylcarnitine 

P 

J 3 y 

P 

IL-4R 

Tyr 

P Tyr STAT6 

Tyr 

TyrSTAT6 

MKP 

CACT 

CPT II 

P 

Tyr580 

Tyr542 SHP-2 SHP-1 

P 

SHIP 

PI3K 

PI3K 

PIAS1 

IRF-2 

IRF-2 

P P 

Tyr Tyr STAT6 

P P 

Tyr Tyr STAT6 

ATP 

ADP 

Tyr Fyn 

IRF-1 

y 

IL-4R 

Tyr 

Tyr JAK1 

P Tyr Fyn 

Tyr JAK1 

Tyr701 

Tyr701 

Ser727 STAT1 

Ser727 

P P Tyr701 

Ser727 

Tyr701 

STAT1 

P 

Ser727 

P 

PIAS1 

IRF-1 

IRF-1 

malonyl CoA 

fatty acid 

ATP 

synthetase 

IL-4 

JAK3Tyr 

IL-4 

y 

common 

Tyr 

chain 

JAK3Tyr 

Tyr 

IRS 

Ser312(307:R) 

p53 

NOSII/iNOS 

NOSII/iNOS 

common 

Tyr 

chain 

P 

SOCS3 

IRF-7 

Ser484 

Ser485 IRF-7 

Tyr 

Gab2 

P Tyr Gab2 

P 

Tyr 

IRS 

Ser312(307:R) 

P 

P Ser21 P 

Ser32 Ser374 

c-Fos 

Ser42 Ser113 

P Ser70 P 

P 

P 

P 

Ser63 Ser73 

c-Jun 

P 

P Ser21 P 

Ser32 Ser374 

c-Fos 

Ser42 Ser113 

P Ser70 P 

P 

P 

Tyr 

P 

P 

Ser63 Ser73 

c-Jun 

CPT1 

acyl CoA 

synthetase 

Tyr 

IRS 

Ser312(307:R) 

P 

P 

Ser484 

IRF-7 

Ser485 

P 

P 

Ser484 

IRF-7 

Ser485 

P 

calpain 

Tyr706 

Tyr544 Tyr974Tyr921 

M-CSFR 

Tyr559 Tyr807 

Tyr721 

P Tyr Gab2 

AP-1 

c-Fos+c-Jun 

Tyr580 Tyr542SHP-2 

P 

P 

Thr 

Tyr 

JNK 

Ser133 

CREB 

Tyr580 

nucleus 

acetyl CoA 

carboxylase 

NADPH 

Grb2 

acetyl CoA carboxylase 

xanthine 

oxidase 

Tyr771 

PLC 

Tyr783 Tyr1254 

PP2B 

P 

Thr183 ERK1 

Tyr185 

ERK2 

P 

P 

P 

P 

P 

Ser63 Ser73 

Ser63 Ser73 

c-Jun 

P Tyr Gab2 

P 

Tyr580 Tyr542SHP-2 

P 

P 

P 

Ser369 Thr577 

RSK 

Ser386 Ser227 

P 

xanthine 

P 

Ser 

SOS 

Thr 

Ser63 Ser73 

c-Jun 

c-jun 

P 

Ser133 

CREB 

fatty acid 

synthetase 

Tyr706 

P 

P 

fatty acid synthetase 

NADPH 

oxidase 

PP2A 

c-fos 

P 

RasGAP 

GTP 

GDP 

Ras 

Grb2 

Ser21 

Ser32 Ser374 

c-Fos 

Ser42 Ser113 

Ser70 

Grb2 

PI3K 

PP2B 

Ser 

SOS 

Thr 

acyl CoA synthetase 

Fc RIa 

chain 

? 

Ser 

SOS 

Thr 

proteasome 

MKP 

P 

Tyr771 

Tyr783 

P 

P 

Ser383 

Elk-1 

Ser389 

P 

Site-2 protease 

PLC 

Pi 

Ser383 

Ser389 Elk-1 

Thr183 

Thr183 ERK1 

Tyr185ERK2 

Tyr185 

. O2 - 

RXR 

RasGAP 

SOD 

chain 

Tyr 

Tyr 

Tyr1254 

P 

chain 

Tyr 

P 

Tyr518 

GDP 

SREBP1c 

/bHLH 

SREBP1c 

/bHLH 

GTP 

Ras 

P 

P 

Thr183 

Thr183 ERK1 

Tyr185ERK2 

Tyr185 

P 

P 

MPO 

LOOH 

Golgi 

LXR 

LXR 

Tyr341 Ser4 

Raf 

Ser338 Ser62 

Tyr341 Ser4 

Raf 

Ser338 Ser62 

LXR 

27-hydroxyChol 

SREBP1c 

/bHLH 

Tyr518 

Tyr518 

Syk 

Tyr519 

Src 

P 

Ser 

Grb2 

SOS 

Thr 

P 

P 

Ser369 

P 

P 

Ser369 

Ser386 

P 

LXR 

SCAP 

HOCl 

P 

P 

Thr577 

P 

R 

9 

r 

Site 

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 589 

14 

Principles of cell 

signaling 

Melanie H. Cobb and Elliott M. Ross 

The University of Texas Southwestern Medical Center at Dallas 

This image represents about 10% of the map of the known signaling interactions and 

reactions in the mouse macrophage. Preparing such a map in a computable format is 

the first step in analyzing a large signaling network. This map was prepared by the group 

led by Hiroaki Kitano at the Systems Biology Institute, Tokyo, using their CellDesigner 

program. Map courtesy of Kanae Oda, Yukiko Matsuoka, and Hiroaki Kitano (The Systems 

Biology Institute). 

Ub Lys63 TRAF6 

Lys63TRAF6 

Ub 

Lys63TRAF6 

Ub 

P 

Ser385 

IRF-3 

Ub Lys63 TRAF6 

Lys22 I B P 

P 

Tyr542 SHP-2 

Lys22 I B IL-10 

Ser385 

IRF-3 

Lys22 I B PKA 

Ser385 

IRF-3 

P Ser727 

TyrJAK1 

P 

Tyr697 

Tyr706 

P Tyr Gab2 

Tyr542 SHP-2 

Thr JNK 

Tyr542 SHP-2 

Syk 

Tyr519 

P 

Tyr519 Syk 

Ser386 R 

RSK Ser227 

14.1 

14.2 

14.3 

14.4 

14.5 

14.6 

14.7 

14.8 

14.9 

14.10 

14.11 

14.12 

14.13 

14.14 

14.15 

14.16 

14.17 

14.18 

14.19 

14.20 

CHAPTER OUTLINE 

Introduction 

Cellular signaling is primarily chemical 

Receptors sense diverse stimuli but initiate a limited 

repertoire of cellular signals 

Receptors are catalysts and amplifiers 

Ligand binding changes receptor conformation 

Signals are sorted and integrated in signaling pathways 

and networks 

Cellular signaling pathways can be thought of as 

biochemical logic circuits 

Scaffolds increase signaling efficiency and enhance 

spatial organization of signaling 

Independent, modular domains specify protein-protein 

interactions 

Cellular signaling is remarkably adaptive 

Signaling proteins are frequently expressed as multiple 

species 

Activating and deactivating reactions are separate and 

independently controlled 

Cellular signaling uses both allostery and covalent 

modification 

Second messengers provide readily diffusible pathways 

for information transfer 

Ca2+ signaling serves diverse purposes in all eukaryotic 

cells 

Lipids and lipid-derived compounds are signaling 

molecules 

PI 3-kinase regulates both cell shape and the activation 

of essential growth and metabolic functions 

Signaling through ion channel receptors is very fast 

Nuclear receptors regulate transcription 

G protein signaling modules are widely used and highly 

adaptable 

14.21 

14.22 

14.23 

14.24 

14.25 

14.26 

14.27 

14.28 

14.29 

14.30 

14.31 

14.32 

14.33 

14.34 

14.35 

14.36 

Tyr JAK1 GM-CSFR 

Tyr 

JAK2 

Tyr1007 

Heterotrimeric G proteins regulate a wide variety of 

effectors 

NADP + Cl - 

P Ser727 STAT1 e 

O 2 H 2 O 

Heterotrimeric G proteins are controlled P 

pyruvate 

by a regulatory 

- 

2 

F 

hypoxanthine 

P Tyr STAT2 

NAD + 

Fe 3+ 

H + H + 

e 

GTPase - Tyr542 SHP-2 

cycle 

O 2 

e - 

Small, monomeric GTP-binding proteins are multiuse 

switches 

Protein phosphorylation/dephosphorylation is a major 

regulatory mechanism in the cell 

Two-component protein phosphorylation systems are 

signaling relays 

Pharmacological inhibitors of protein kinases may be 

used to understand and treat disease 

Phosphoprotein phosphatases reverse the actions of 

kinases and are independently regulated 

Covalent modification by ubiquitin and ubiquitin-like 

proteins is another way of regulating protein function 

The Wnt pathway regulates cell fate during development 

and other processes in the adult 

Diverse signaling mechanisms are regulated by protein 

tyrosine kinases 

Src family protein kinases cooperate with receptor 

protein tyrosine kinases 

MAPKs are central to many signaling pathways 

Cyclin-dependent protein kinases control the cell cycle 

Diverse receptors recruit protein tyrosine kinases to the 

plasma membrane 

What’s next? 

Summary 

References 

589


(GPCR) 

G protein 

coupled 

receptor 

Heterotrimeric 

G protein 

14.1 

Introduction 

All cells, from prokaryotes through plants and 

animals, sense and react to stimuli in their environments 

with stereotyped responses that allow 

them to survive, adapt, and function in 

ways appropriate to the needs of the organism. 

These responses are not simply direct physical 

or metabolic consequences of changes in the 

local environment. Rather, cells express arrays 

of sensing proteins, or receptors, that recognize 

specific extracellular stimuli. In response to 

these stimuli, receptors regulate the activities 

of diverse intracellular regulatory proteins that 

in turn initiate appropriate responses by the 

cell. The process of sensing external stimuli and 

conveying the inherent information to intracellular 

targets is referred to as cellular signal 

transduction. 

Cells respond to all sorts of stimuli. Microbes 

respond to nutrients, toxins, heat, light, and 

chemical signals secreted by other microbes. 

Cells in multicellular organisms express receptors 

specific for hormones, neurotransmitters, 

autocrine and paracrine agents (hormonelike 

compounds from the secreting cell or cells 

Overview of major receptor types in a cell 

Receptor 

protein 

kinase 

Ion 

channel 

Transcription 

factor 

Twocomponent 

complex 

( 

Sensor 

Histidine 

kinase 

Response 

regulator 

NUCLEUS 

Transmembrane 

scaffold 

( 

E1 

E2 

E1 

E2 

Guanylyl 

cyclase 

FIGURE 14.1 Receptors form a rather small number of families that share common 

mechanisms of action and overall similar structures. 

nearby), odors, molecules that regulate growth 

or differentiation, and proteins on the outside 

of adjacent cells. A mammalian cell typically 

expresses about fifty distinct receptors that sense 

different inputs, and, overall, mammals express 

several thousand receptors. 

Despite the diversity of cellular lifestyles 

and the enormous number of substances sensed 

by different cells, the general classes of proteins 

and mechanisms involved in signal transduction 

are conserved throughout living cells, as 

shown in FIGURE 14.1. 

• G protein-coupled receptors, 

composed of seven membrane-spanning 

helices, promote activation of heterotrimeric 

GTP-binding proteins called 

G proteins, which associate with the inner 

face of the plasma membrane and 

convey signals to multiple intracellular 

proteins. 

• Receptor protein kinases are often 

dimers of single membrane-spanning 

proteins that phosphorylate their intracellular 

substrates and, thus, change 

the shape and function of the target proteins. 

These protein kinases frequently 

contain protein interaction domains that 

organize complexes of signaling proteins 

on the inner surface of the plasma 

membrane. 

• Phosphoprotein phosphatases reverse 

the effect of protein kinases by removing 

the phosphoryl groups added 

by protein kinases. 

• Other single membrane-spanning enzymes, 

such as guanylyl cyclase, have 

an overall architecture similar to the receptor 

protein kinases but different enzymatic 

activities. Guanylyl cyclase 

catalyzes the conversion of GTP to 3′:5′- 

cyclic GMP, which is used to propagate 

the signal. 

• Ion channel receptors, although diverse 

in detailed structure, are usually 

oligomers of subunits that each contain 

several membrane-spanning segments. 

The subunits change their conformations 

and relative orientations to permit 

ion flux through a central pore. 

• Two-component systems may either 

be membrane spanning or cytosolic. The 

number of their subunits is also variable, 

but each two-component system 

contains a histidine kinase domain or 

subunit that is regulated by a signaling 

molecule and a response regulator that 

590 CHAPTER 14 Principles of cell signaling


contains a phosphorylatable aspartate 

(Asp) residue. 

• Some receptors are transmembrane 

scaffolds that change either the conformation 

or oligomerization of their 

intracellular scaffold domains in response 

to extracellular signaling molecules, 

or ligands, and, thus, recruit 

interacting regulatory proteins to a common 

site on the membrane. 

• Nuclear receptors are transcription 

factors, often heterodimers, that may 

reside in the cytoplasm until activated 

by agonists or may be permanently located 

in the nucleus. 

The biochemical processes of signal transduction 

are strikingly similar among cells. 

Bacteria, fungi, plants, and animals use similar 

proteins and multiprotein modules to detect 

and process signals. For example, evolutionarily 

conserved heterotrimeric G proteins and G 

protein-coupled receptors are found in plants, 

fungi, and animals. Similarly, 3′:5′ cyclic AMP 

(cAMP) is an intracellular signaling molecule 

in bacteria, fungi, and animals; and Ca2+ serves 

a similar role in all eukaryotes. Protein kinases 

and phosphoprotein phosphatases are used to 

regulate enzymes in all cells. 

Although the basic biochemical components 

and processes of signal transduction are conserved 

and reused, they are often used in wildly 

divergent patterns and for many different physiological 

purposes. For example, cAMP is synthesized 

by distantly related enzymes in bacteria, 

fungi, and animals, and acts on different proteins 

in each organism; it is a pheromone in 

some slime molds. 

Cells often use the same series of signaling 

proteins to regulate a given process, such as 

transcription, ion transport, locomotion, and 

metabolism. Such signaling pathways are assembled 

into signaling networks to allow the 

cell to coordinate its responses to multiple inputs 

with its ongoing functions. It is now possible 

to discern conserved reaction sequences 

in and between pathways in signaling networks 

that are analogous to devices within the circuits 

of analog computers: amplifiers, logic gates, 

feedback and feed-forward controls, and memory. 

This chapter discusses the principles and 

strategies of cellular signaling first and then discusses 

the conserved biochemical components 

and reactions of signaling pathways and how 

these principles are applied. 

14.2 

Cellular signaling is 

primarily chemical 

Key concepts 

• Cells can detect both chemical and physical 

signals. 

• Physical signals are generally converted to 

chemical signals at the level of the receptor. 

Most signals sensed by cells are chemical, and, 

when physical signals are sensed, they are generally 

detected as chemical changes at the level 

of the receptor. For example, the visual photoreceptor 

rhodopsin is composed of the protein 

opsin, which binds to a second component, 

the colored vitamin A derivative cis-retinal (the 

chromophore). When cis-retinal absorbs a 

photon, it photoisomerizes to trans-retinal, 

which is an activating ligand of the opsin protein. 

(For more on rhodopsin signaling see 14.20 

G protein signaling modules are widely used and 

highly adaptable). Similarly, plants sense red and 

blue light using the photosensory proteins phytochrome 

and cryptochrome, which detect photons 

that are absorbed by their tetrapyrrole or 

flavin chromophores. Cryptochrome homologs 

are also expressed in animals, where they probably 

mediate adjustment of the diurnal cycle. 

A few receptors do respond directly to physical 

inputs. Pressure-sensing channels, which exist 

in one form or another in all organisms, 

mediate responses to pressure or shear by changing 

their ionic conductance. In mammals, hearing 

is mediated indirectly by a mechanically 

operated channel in the hair cell of the inner ear. 

The extracellular domain of a protein called cadherin 

is pulled in response to acoustic vibration, 

generating the force that opens the channel. 

Cells sense mechanical strain through a 

number of cell surface proteins, including integrins. 

Integrins provide signals to cells based on 

their attachment to other cells and to molecular 

complexes in the external milieu. 

One major group of physically responsive 

receptors is made up of channels that sense electric 

fields. Another interesting group are 

heat/pain-sensing ion channels; several of these 

heat-sensitive ion channels also respond to 

chemical compounds, such as capsaicin, the 

“hot” lipid irritant in hot peppers. 

Whether a signal is physical or chemical, the 

receptor initiates the reactions that change the 

behavior of the cell. We will discuss how these 

effects are generated in the rest of the chapter. 

14.2 Cellular signaling is primarily chemical 591


Ligand A 

14.3 

Receptors sense diverse 

stimuli but initiate a 

limited repertoire of 

cellular signals 

Key concepts 

• Receptors contain a ligand-binding domain and an 

effector domain. 

• Receptor modularity allows a wide variety of 

signals to use a limited number of regulatory 

mechanisms. 

• Cells may express different receptors for the same 

ligand. 

• The same ligand may have different effects on the 

cell depending on the effector domain of its 

receptor. 

Receptors mediate responses to amazingly diverse 

extracellular messenger molecules; hence, 

the cell must express a large number of receptor 

varieties, each able to bind its extracellular 

ligand. In addition, each receptor must be able 

to initiate a cellular response. Receptors, thus, 

contain two functional domains: a ligandbinding 

domain and an effector domain, 

which may or may not correspond to definable 

structural domains within the protein. 

The separation of ligand-binding and effector 

functions allows receptors for diverse ligands 

to produce a limited number of evolutionarily 

conserved intracellular signals through the action 

of a few effector domains. In fact, there are 

Receptors have a ligand-binding domain and an effector domain 

Output 

1 

Output 

2 

Ligand A 

Output 

1 

Ligand B 

Output 

1 

CHIMERIC 

RECEPTOR 

Ligand C 

LBD1 LBD1 LBD1 LBD2 LBD3 

ED1 ED2 

ED1 ED1 

Output 

2 

ED2 

FIGURE 14.2 Receptors can be thought of as composed of two functional domains, 

a ligand-binding domain (LBD) and an effector domain (ED). The twodomain 

property implies that two receptors that respond to different ligands 

(middle) could initiate the same function by activating similar effector domains, 

or that a cell could express two receptor isoforms (left) that respond to 

the same ligand with distinct cellular effects mediated by different effector domains. 

It also implies that one can create an artificial chimeric receptor with 

novel properties. 

only a limited number of receptor families, which 

are related by their conserved structures and signaling 

functions (see Figure 14.1). 

There are several useful correlates to the 

two-domain nature of receptors. For example, 

a cell can control its responsiveness to an extracellular 

signal by regulating the synthesis or 

degradation of a receptor or by regulating the 

receptor’s activity (see 14.10 Cellular signaling is 

remarkably adaptive). 

In addition, the nature of a response is generally 

determined by the receptor and its effector 

domain rather than any physicochemical 

property of the ligand. FIGURE 14.2 illustrates the 

concept that a ligand may bind to more than 

one kind of receptor and elicit more than one 

type of response, or several different ligands 

may all act identically by binding to functionally 

similar receptors. For example, the neurotransmitter 

acetylcholine binds to two classes 

of receptors. Members of one class are ion channels; 

members of the other regulate G proteins. 

Similarly, steroid hormones bind both to nuclear 

receptors, which bind chromatin and regulate 

transcription, and to other receptors in 

the plasma membrane. 

Conversely, when multiple ligands bind to 

receptors of the same biochemical class, they 

generate similar intracellular responses. For example, 

it is not uncommon for a cell to express 

several distinct receptors that stimulate production 

of the intracellular signaling molecule cAMP. 

The effect of the receptor on the cell will also be 

determined significantly by the biology of the 

cell and its state at any given time. 

Ligand binding and effector domains may 

evolve independently in response to varied selective 

pressures. For example, mammalian and 

invertebrate rhodopsins transduce their signal 

through different effector G proteins (G t 

and 

G q 

, respectively). Another example is calmodulin, 

a small calcium-binding regulatory protein 

in animals, which in plants appears as a 

distinct domain in larger proteins. 

The receptor’s two-domain nature allows 

the cell to regulate the binding of ligand and 

the effect of ligand independently. Covalent 

modification or allosteric regulation can alter 

ligand-binding affinity, the ability of the ligand-bound 

receptor to generate its signal or 

both. We will discuss these concepts further in 

14.13 Cellular signaling uses both allostery and covalent 

modification. 

Receptors can be classified either according 

to the ligands they bind or the way in which 

they signal. Signal output, which is character- 



istic of the effector domain, usually correlates 

best with overall structure and sequence conservation. 

(Receptor families grouped by their 

functions are the organizational basis of the second 

half of this chapter.) However, classifying 

receptors pharmacologically, according to their 

specificity for ligands, is particularly useful for 

understanding the organization of endocrine 

and neuronal systems and for categorizing the 

multiple physiological responses to drugs. 

Expression of a receptor that is not normally 

expressed in a cell is often sufficient to 

confer responsiveness to that receptor’s ligand. 

This responsiveness often occurs because the 

cell expresses the other components necessary 

for propagating the intracellular signal from the 

receptor. The precise nature of the response will 

reflect the biology of the cell. Experimentally, 

responsiveness to a compound can be induced 

by introducing the cDNA that encodes the receptor. 

For example, mammalian receptors may 

be expressed in yeast, such that the yeast respond 

visibly to receptor ligands, thus providing 

a way to screen for new chemicals (drugs) 

that activate the receptor. 

Finally, it is possible to create chimeric receptors 

by fusing the ligand-binding domain 

from one receptor with the effector domain 

from a different receptor (Figure 14.2). Such 

chimeras can mediate novel responses to the 

ligand. With genetic modification of the ligandbinding 

domain, receptors can be reengineered 

to respond to novel ligands. Thus, scientists can 

manipulate cell functions with nonbiological 

compounds. 

14.4 

Receptors are catalysts 

and amplifiers 

Key concepts 

• Receptors act by increasing the rates of key 

regulatory reactions. 

• Receptors act as molecular amplifiers. 

Receptors act to accelerate intracellular functions 

and are, thus, functionally analogous to enzymes 

or other catalysts. Some receptors, 

including the protein kinases, protein phosphatases, 

and guanylate cyclases, are themselves 

enzymes and thus classical biochemical catalysts. 

More generally, however, receptors use 

the relatively small energy of ligand binding to 

accelerate reactions that are driven by alternative 

energy sources. For example, receptors that 

are ion channels catalyze the movement of ions 

across membranes, a process driven by the electrochemical 

potential developed by distinct ion 

pumps. G protein-coupled receptors and other 

guanine nucleotide exchange factors catalyze 

the exchange of GDP for GTP on the G protein, 

an energetically favored process dictated by the 

cell’s nucleotide energy balance. Transcription 

factors accelerate the formation of the transcriptional 

initiation complex, but transcription itself 

is energetically driven by multiple steps of 

ATP and dNTP hydrolysis. 

As catalysts, receptors enhance the rates of 

reactions. Most signaling involves kinetic rather 

than thermodynamic regulation; that is, signaling 

events change reaction rates rather than 

their equilibria (see the next section). Thus, signaling 

is similar to metabolic regulation, in 

which specific reactions are chosen according to 

their rates, with thermodynamic driving forces 

playing only a supportive role. 

In all signaling reactions, receptors use their 

catalytic activities to function as molecular amplifiers. 

Directly or indirectly, a receptor generates 

a chemical signal that is huge, both 

energetically and with respect to the number 

of molecules recruited by a single receptor. 

Molecular amplification is a hallmark of receptors 

and many other steps in cellular signaling 

pathways. 

14.5 

Ligand binding changes 

receptor conformation 

Key concepts 

• Receptors can exist in active or inactive 

conformations. 

• Ligand binding drives the receptor toward the 

active conformation. 

A central mechanistic question in receptor function 

is how the binding of a signaling molecule 

to the ligand-binding domain increases the activity 

of the effector domain. The key to this 

question is that receptors can exist in multiple 

molecular conformations, some active for signaling 

and others inactive. Ligands shift the 

conformational equilibrium among these conformations. 

The structural changes that occur 

during the receptor’s inactive-active isomerization 

and how ligand binding drives these 

changes are exciting areas of biophysical research. 

However, the basic concept can be described 

simply in terms of coupling the 

conformational isomerizations of the ligandbinding 

and effector domains. 

14.5 Ligand binding changes receptor conformation 593


How do ligands activate (or not activate) a 

receptor? Most of the basic regulatory activities 

of receptors can be described by a simple scheme 

that considers the receptors as having two interconvertible 

conformations, inactive (R) and 

active (R*). R and R* are in equilibrium, which 

is described by the equilibrium constant J. 

Because unliganded receptors are usually 

minimally active, J1), then ligand binding 

will shift the conformation to the R* state to an 

equivalent extent (i.e., J*/J>>1). The relative 

activation by a saturating concentration of ligand, 

J*/J, will exactly equal the ligand’s relative 

selectivity for the active receptor conformation, 

K*/K. This argument is generally valid for the regulation 

of a protein’s activity by any regulatory 

ligand. 

This model explains many properties of receptors 

and their ligands both simply and quantitatively. 

• First, J must be greater than zero for the 

equilibrium to exist. Thus, even unliganded 

receptor has some activity. 

Overexpressed receptors frequently display 

their intrinsic low activity. 

• Because physiological receptors are 

nearly inactive in the absence of ligand, 

J must be much less than 1 and is probably 

less than 0.01; most receptors are 

less than 1% active without agonist. 

• Ligands can vary in their selectivities 

between R and R*. Their abilities to activate 

will also vary. Some ligands, referred 

to as agonists, can drive formation 

of appreciable R*. Others, known as partial 

agonists, will promote submaximal 

activation. Chemical manipulation 

of a ligand’s structure will often alter its 

activity as an agonist. These relationships 

are depicted graphically in FIGURE 

14.3. 

• A ligand that binds equally well to both 

the R and R* states will not cause activation. 

However, such a ligand may still 

occupy the binding site and thereby 

competitively inhibit binding of an activating 

ligand. Such competitive inhibitors, 

referred to as antagonists, are 

frequently used as drugs to block unwanted 

activation of a receptor in various 

disease states. 

• A ligand that binds preferentially to R 



relative to R* will further shift the conformational 

equilibrium to the inactive 

state and cause net inhibition. Such ligands 

are called inverse agonists. 

Because J is already low, effects of inverse 

agonists may only be noticeable 

if a receptor is overexpressed or if the 

receptor is mutated to increase its intrinsic 

activity (i.e., the mutation increases 

J). 

• The extent to which an agonist stimulates 

a receptor is unrelated to its affinity. 

Both agonists and antagonists may 

bind with either high or low affinity. 

Affinity does determine the receptor’s 

sensitivity—that is, how low a concentration 

of ligand can the receptor detect. 

Affinities of receptors for natural regulatory 

ligands vary enormously, with 

physiologic K d 

values ranging from 


RECEPTORS 

TRANSDUCERS 

EFFECTORS 

Convergent and divergent signaling pathways 

Linear, 

parallel 

Convergent Divergent Multiply 

branched 

FIGURE 14.4 Signaling pathways use convergent and divergent branching to coordinate 

information flow. The diagrams at top show how even a simple, threelevel 

signaling network can sort information. Convergence or divergence can 

take place at multiple points along a signaling pathway. As an example of complexity, 

the lower portion of the figure shows a small segment (~10%) of the G 

protein-mediated signaling network in a mouse macrophage cell line. It omits 

several interpathway regulatory mechanisms and completely ignores inputs from 

non-G protein-coupled receptors. Pathway map courtesy of Lily Jiang, University 

of Texas Southwestern Medical Center. 

maps. Signaling networks are also spatially complex. 

They may include components in various 

subcellular locations, with initial receptors and 

associated proteins in the plasma membrane, but 

with downstream proteins in the cytoplasm or intracellular 

organelles. Such complexity is necessary 

to allow the cells to integrate and sort 

incoming signals and to regulate multiple intracellular 

functions simultaneously. 

The complexity and adaptability of signaling 

networks, like the one shown in the lower 

half of Figure 14.4, make their dynamics at the 

whole-cell level difficult or impossible to grasp 

intuitively. Signaling networks resemble large 

analog computers, and investigators are increasingly 

depending on computational tools to understand 

cellular information flow and its 

regulation. First, many signaling interactions 

that include only two or three proteins exert 

functions analogous to traditional computational 

logic circuits (see the next section). The 

theory and experience with such circuits in electronics 

facilitate understanding biological signaling 

functions as well. 

The enormous complexity of cellular signaling 

networks can be simplified by considering 

them to be composed of interacting signaling 

modules, i.e., groups of proteins that process signals 

in well-understood ways. A cellular signaling 

module is analogous to an integrated circuit 

in an electronic instrument that performs a 

known function, but whose exact components 

could be changed for similar use in another device. 

The concept of modular construction facilitates 

both qualitative and quantitative 

understanding of signaling networks. We will refer 

to many standard signaling modules later in 

the chapter. Examples include monomeric and 

heterotrimeric G protein modules, MAPK cascades, 

tyrosine (Tyr) kinase receptors and their 

binding proteins, and Ca2+ release/uptake modules. 

In each case, despite the numerous phylogenetic, 

developmental, and physiologic 

variations, understanding the basic function of 

that class of module conveys understanding of all 

its incarnations. Last, the evolutionary importance 

of modules is significant; once the architecture 

of a module is established it can be reused. 

For larger-scale networks, multiplexed, 

high-throughput measurements on living cells 

have been combined with powerful kinetic modeling 

strategies to allow an increasingly accurate 

quantitative depiction of information flow 

within signaling modules or entire networks. 

Such models, with sound and experimentally 

based parameter sets, can describe signaling 

processes in systems too complex for intuitive 

or ad hoc analysis. They are also vital as tests of 

understanding because they can predict experimental 

results in ways that can be used to test 

the validity of the model. Well-grounded models 

can then be used (cautiously) to suggest the 

mechanisms of systems for which data sets remain 

unattainable. At even greater levels of 

complexity, the theories and tools of computer 

science are increasingly giving useful systemslevel 

analyses of signal flow in cells. Using computational 

tools to analyze large arrays of 

quantitative data allows us to understand cellular 

information flow and its regulation. 



Developing quantitative models of signaling 

networks is a frontier in signaling biology. These 

models both help describe network function 

and pinpoint experiments to clarify mechanism. 

14.7 

Cellular signaling 

pathways can be thought 

of as biochemical logic 

circuits 

Key concepts 

• Signaling networks are composed of groups of 

biochemical reactions that function as 

mathematical logic functions to integrate 

information. 

• Combinations of such logic functions combine as 

signaling networks to process information at more 

complex levels. 

As introduced in the preceding section, processes 

that signaling pathways use to integrate and direct 

information to cellular targets are strikingly analogous 

to the mathematical logic functions that are 

used to design the individual circuits of electronic 

computers. Indeed, there are biological equivalents 

of essentially all of the functional components 

that computer scientists and engineers 

consider in the design of computers and electronic 

control devices. To understand signaling pathways, 

it is, therefore, useful to consider groups of 

reactions within a pathway as constituting logic circuits 

of the sort used in electronic computing, as 

illustrated in FIGURE 14.5. The simplest example is 

when two stimulatory pathways converge. If sufficient 

input from either is adequate to elicit the 

response, the convergence would constitute an 

“OR” function. If neither input is sufficient by itself 

but the combination of the two elicits the response, 

then the converging pathways would 

create “AND” functions. AND circuits are also referred 

to as coincidence detectors—a response 

is elicited only when two stimulating pathways 

are activated simultaneously. 

AND functions can result from the combination 

of two similar but quantitatively inadequate 

inputs. Alternatively, two mechanistically 

different inputs might both be required to elicit 

a response. An example of the latter would be 

a target protein that is allosterically activated 

only when phosphorylated, or that is activated 

by phosphorylation but is only functional when 

recruited to a specific subcellular location. 

The opposite of an AND circuit is a NOT 

function, where one pathway blocks the stim- 

Logical (Boolean) 

A 

B 

A + B 

A 

B 

A + B 

A 

B 

A + B 

A OR B 

A AND B 

A NOT B 

Response 

Response 

Response 

Response 

Response 

Simple logic circuits 

Response 

Quantitative (Analog) 

A + fixed [B] 

Response 

Response 

Additive 

ulatory effect of another. Simple logic gates are 

observed at many locations in cellular signaling 

pathways. 

We can also think about convergent signaling 

in quantitative rather than Boolean terms 

by considering the additivity of inputs to a distinct 

process (see Figure 14.5, right). The OR 

function referred to above can be considered to 

be the additive positive inputs of two pathways. 

Such additivity could represent the ability of 

several receptors to stimulate a pool of a particular 

G protein or the ability of two protein kinases 

to phosphorylate a single substrate. 

Additivity may be positive, as in the examples 

above, or negative, such as when two inhibitory 

inputs combine. Inhibition and stimulation may 

also combine additively to yield an algebraically 

balanced output. Alternatively, multiple inputs 

can combine with either more or less than an 

additive effect. The NOT function, discussed 

above, is analogous to describing a blockade of 

stimulation. The AND function describes synergism, 

where one input potentiates another 

but alone has little effect. 

Even simple signaling networks can display 

complex patterns of information processing. One 

A 

log (agonist concentration) 

More than additive 


Less than additive 


B 

A + B 

A 

B 

A 

A + B 

B 

FIGURE 14.5 Signaling networks use simple logic functions to process 

information. Boolean OR, AND, and NOT functions (left) correspond to 

the quantitative interactions between converging signals that are shown 

on the right. 

14.7 Cellular signaling pathways can be thought of as biochemical logic circuits 597


Input 

Positive feedback loop : irreversible ON switch 

Positive feed-forward loop : responds to prolonged input 

Input 

OH 

E 

G 

T 

T 

+ 

Output 

Output 

Output 

Input strength 

Conformational lock - Dual control switch 

OH 

E 

OH 

+ 

Kinase 

E 

E 

G 

G 

Phosphatase 

Signal processing circuits 

E 

E 

G 

P 

E 

P 

P 

E 

Output 

Output 

Time 

Time 

input 

G K G P K 

FIGURE 14.6 Relatively complex signal processing can be executed by simple 

multi-protein modules. The figure depicts three types of signaling modules 

(left) and their behavior in response to agonist (right). (top) In a positive 

feed-back module, a transducer protein (T) stimulates an effector (E) to produce 

a cellular output, but the effector also stimulates the activity of the transducer. 

The result can be an all-or-none switch, where input up to a threshold 

has little effect, but then becomes committed when feedback from the effector 

is sufficient to maintain transducer activity even in the absence of continued 

input from the receptor. (center) In a positive feed-forward module, the 

effector requires input both from the transducer and from upstream in the pathway. 

When stimulation is brief (short horizontal bar under trace at right), significant 

amounts of active transducer do not accumulate and output is minimal. 

When stimulation is prolonged (longer bar), signal output is substantial. (bottom) 

In some dual-control switching modules, the binding of one regulator (G) 

can both activate the effector and expose another regulatory site, shown here 

as a Ser substrate site (-OH) for a protein kinase. The effector can only be phosphorylated 

or dephosphorylated when G is bound. Therefore, as shown at the 

right, addition of G alone will activate but activation of the kinase (K) alone 

will not. If kinase is active while G is bound, phosphorylation is resistant to 

phosphatase activity unless G is again present to reexpose the phosphoserine 

residue (shown on the graph at the right as a bold P). 

G P 

good example is the creation of “memory”: making 

the effect of a transient signal more or less 

permanent. Signaling pathways have multiple 

ways of setting memories, and of forgetting. One 

mechanism, common in protein kinase pathways, 

is the positive feedback loop, illustrated 

in the top panel of FIGURE 14.6. In a positive feedback 

loop, the input stimulates a transducer (T), 

which in turn stimulates the effector protein (E) 

to create the output. If the effector can also activate 

the transducer, sufficient initial signal can 

be fed back to the transducer that it can maintain 

the effector's full signal output even when 

input is removed. Such systems typically display 

a threshold behavior, as shown on the right. 

A positive feed-forward loop can generate 

memory of another type (Figure 14.6, middle 

panel), indicating the duration of input. In such 

circuits, the effector requires simultaneous input 

from both the receptor and from the intermediary 

transducer. If the pathway from 

receptor through transducer is relatively slow, 

or if it requires the accumulation of a substantial 

amount of transducer, only a prolonged input 

will trigger a response, as shown in the 

time-base output diagram at the right. 

A third way to establish memory is to allow 

one input to control the reversibility of a second 

regulatory event (Figure 14.6, bottom panel). 

WASP, a protein that initiates the polymerization 

of actin to drive cellular motion and shape 

change, is activated both by phosphorylation 

and by the binding of Cdc42, a small GTP-binding 

protein (G). However, the phosphorylation 

site on WASP is only exposed when WASP is 

bound to Cdc42. Phosphorylation thus requires 

both activated Cdc42 and activated protein kinase. 

If Cdc42 dissociates, the phosphorylated 

state of WASP persists until another signaling 

molecule, whose identity remains uncertain, 

binds again to expose the site to a protein phosphatase. 

As shown in the time-base graph, exposure 

to Cdc42 will activate, but exposure to 

kinase alone will not. If Cdc42 is present, then 

the kinase can activate WASP. Phospho-WASP 

is relatively insensitive to protein phosphatase 

(P) alone, but can be dephosphorylated if Cdc42 

or another G protein binds to expose the site to 

phosphatase. 

14.8 

Scaffolds increase 

signaling efficiency and 

enhance spatial 

organization of signaling 

Key concepts 

• Scaffolds organize groups of signaling proteins and 

may create pathway specificity by sequestering 

components that have multiple partners. 

• Scaffolds increase the local concentration of 

signaling proteins. 

• Scaffolds localize signaling pathways to sites of 

action. 



The INAD signaling complex 

TRP 

Rhodopsin 

Scaffolds concentrate and insulate signaling proteins 

Pheromone 

GPCR 

PKC 

PDZ PDZ 

PDZ 

INAD 

- 

PDZ PDZ 

CaM 

CaM 

PKC 

PDZ 

PDZ 

PDZ 

INAD 

PDZ PDZ 

CYTOSOL 

FIGURE 14.7 The scaffold InaD organizes proteins that transmit visual 

signals in the fly photoreceptor cell. InaD is localized to the photoreceptor 

membrane and coordinates light sensing and visual transduction. In 

invertebrate eyes, the visual signaling pathway goes from rhodopsin 

through G q 

to a phospholipase C-, and Ca2+ release triggered by PLC action 

initiates depolarization. This system is specialized for speed, and requires 

that the relevant proteins are nearby. InaD contains five PDZ 

domains, each of which binds to the C terminus of a signal transducing 

protein. The TRP channel, which mediates Ca2+ entry, PLC-, and a protein 

kinase C isoform that is involved in rapid desensitization all bind constitutively 

to InaD. Rhodopsin and a myosin (NinaC) also bind, and G q 

binds indirectly. 

The proteins in a signaling pathway are frequently 

colocalized within cells such that their 

mutual interactions are favored and their interactions 

with other proteins are minimized. 

Many signaling pathways are organized on scaffolds. 

Scaffolds bind several components of a 

signaling pathway in multiprotein complexes 

to enhance signaling efficiency. Scaffolds promote 

interactions of proteins that have a low 

affinity for each other, accelerate activation (and 

often inactivation) of the associated components, 

and localize the signaling proteins to appropriate 

sites of action. Colocalization may be 

tonic or regulated, and stimulus-dependent scaffolding 

often determines signaling outputs. 

The binding sites on a scaffolding protein 

are often localized in distinct modular proteinbinding 

domains, giving the impression that the 

protein is designed simply to hold the components 

of the pathway together. Many scaffolding 

proteins do lack intrinsic enzymatic activity, 

but some signaling enzymes also act as scaffolds. 

Binding to a scaffold facilitates signaling by 

increasing the local concentrations of the components, 

so that diffusion or transport of molecules 

to their sites of action is not necessary. In 

the photoreceptor cells of Drosophila, scaffolding 

of signaling components is critical for rapid 

signal transmission. These cells contain the InaD 

- 

Ste5p 

Ste11p 

Ste7p 

Fus3p 

G protein 

Pheromone 

Ste5p 

Mating 

response 

Ste11p 

Ste7p 

Fus3p 

Cdc42p 

Ste20p 

Scaffold organizes 

MAPK cascade 

Cdc42p 

Ste20p 

scaffolding protein, which has five modular 

binding domains, known as PDZ domains. Each 

of its PDZ domains binds to a C-terminal motif 

of a target protein, thereby facilitating interactions 

among the associated proteins. FIGURE 14.7 

shows a model for how InaD organizes the signaling 

proteins. The mutational loss of InaD 

produces a nearly blind fly, and deletion of a 

single PDZ domain can yield a fly with a distinct 

visual defect characteristic of the protein 

that binds to the missing domain. 

A second example is Ste5p, a scaffold for the 

pheromone-induced mating response pathway 

in S. cerevisiae. FIGURE 14.8 illustrates how Ste5p 

binds and organizes components of a mitogen- 

Scaffold determines 

specificity of Ste11p 


Ste11p 

Ste7p 

Fus3p 

Mating 

response 

High osmolarity 

Ste11p 

Pbs2p 

Hog1p 

Osmoadaptation 

Cdc42p 

Ste20p 

Cdc42p 

Ste20p 

FIGURE 14.8 The scaffold Ste5p organizes the components of the MAPK 

cascade that mediates the pheromone-induced mating response in 

Saccharomyces cerevisiae. In the top left panel, Ste5p brings the components 

of the MAPK cascade to the membrane in response to pheromone. In 

the top right panel, binding to the heterotrimeric G protein brings loaded 

Ste5p in proximity to the protein kinase Ste20p bound to the activated small 

GTP binding protein Cdc42p. Their colocalization facilitates the sequential 

activation of the cascade components, resulting in activation of the MAPK 

Fus3p and the mating response. The MAP3K Ste11p can regulate not only 

the MAPK Fus3p in the mating pathway, but also the MAPK Hog1p in the 

high osmolarity pathway, as shown in the bottom two panels. The scaffold 

to which Ste11p binds, either Ste5p or Pbs2 (both a scaffold and a MAP2K), 

determines which MAPK and downstream events are activated as the output. 

14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling 599


activated protein kinase (MAPK) cascade, including 

a MAP3K (Ste11p), a MAP2K (Ste7p) 

and a MAPK (Fus3p). (The MAPK cascade will 

be discussed in 14.32 MAPKs are central to many 

signaling pathways). The function of Ste5p is partially 

retained even if the positions of its binding 

sites for the kinases are shuffled in the linear 

sequence of the protein, indicating that a major 

role is to bring the enzymes into proximity, rather 

than to precisely orient them. Ste5p also binds 

to the subunits of the heterotrimeric G protein 

that mediates the actions of mating 

pheromones, linking the membrane signal to 

the intracellular transducers. Yeast that lack 

Ste5p cannot mate, demonstrating that Ste5p is 

required for this biological function (but not all 

functions) carried out by the pathway. 

In addition to facilitating signaling in their 

own pathways, scaffolds can enhance signaling 

specificity by limiting interactions with other 

signaling proteins. Scaffolds thus insulate components 

of a signaling pathway both from activation 

by inappropriate signals and from 

producing incorrect outputs. For example, the 

mating and osmosensing pathways in yeast 

share several components, including the MAP3K 

Ste11p, but each pathway maintains specificity 

because it employs different scaffolds that restrict 

signal transmission. 

In contrast, the presence of excess scaffold 

can inhibit signaling because the individual signaling 

components will more frequently bind 

to distinct scaffold proteins rather than forming 

a functional complex. Such dilution among scaffolds 

causes separation rather than concentration 

of the components, preventing their 

productive interaction. 

14.9 

Independent, modular 

domains specify proteinprotein 

interactions 

Key concepts 

• Protein interactions may be mediated by small, 

conserved domains. 

• Modular interaction domains are essential for 

signal transmission. 

• Adaptors consist exclusively of binding domains or 

motifs. 

Modular protein interaction domains or motifs 

occur in many signaling proteins and confer the 

ability to bind structural motifs in other molecules, 

including proteins, lipids, and nucleic 

acids. Some of these domains are listed in FIGURE 

14.9. In contrast to scaffolds, which bind specific 

proteins with considerable selectivity, modular 

interaction domains generally recognize 

not a single molecule but a group of targets that 

share related structural features. 

Modular interaction domains important for 

signal transduction were first discovered in the 

protein tyrosine kinase proto-oncogene Src, 

which contains a protein tyrosine kinase domain 

and two domains named Src homology 

(SH) 2 and 3 domains. The modular SH2 and 

SH3 domains were originally identified by comparison 

of Src to two other tyrosine kinases, Fps 

and Abl. One or both of these domains appear 

in numerous proteins and both are critically involved 

in protein-protein interactions. 

SH3 domains, which consist of approximately 

50 residues, bind to specific short proline-rich 

sequences. Many cytoskeletal proteins 

and proteins found in focal adhesion complexes 

contain SH3 domains and proline rich sequences, 

suggesting that this targeting motif 

may send proteins with these domains to these 

sites of action within cells. In contrast to phosphotyrosine-SH2 

binding, the proline-rich binding 

sites for SH3 domains are present in resting 

and activated cells. However, SH3-proline interactions 

may be negatively regulated by phosphorylation 

within the proline-rich motif. 

SH2 domains, which consist of approximately 

100 residues, bind to Tyr phosphorylated 

proteins, such as cytoplasmic tyrosine 

kinases and receptor tyrosine kinases. Thus, Tyr 

phosphorylation regulates the appearance of 

SH2 binding sites and, thereby, regulates a set 

of protein-protein interactions in a stimulusdependent 

manner. 

A clever strategy was used to identify the 

binding specificity of SH2 domains. An isolated 

recombinant SH2 domain was incubated with 

cell lysates and then recovered from the lysates 

using a purification tag. The proteins associated 

with the SH2 domain were some of the same 

proteins that were recognized by antiphosphotyrosine 

antibodies. By this and other methods, 

it was discovered that SH2 domains recognize 

sequences surrounding Tyr phosphorylation 

sites and require phosphorylation of the included 

Tyr for high affinity binding. 

Information on specific amino acid sequences 

that recognize and bind to modular 

binding domains is being accumulated as these 

individual interactions are identified. In addition, 

screening programs using cDNA and/or 

peptide libraries to assess binding capabilities 



Characteristics of some common modular protein domains 

Domain Characteristics Cellular involvement 

14-3-3 Binds protein phosphoserine or 

phosphothreonine 

Bromo 

CARD 

C1 

C2 

EF hand 

F-Box 

FHA 

HECT 

LIM 

PDZ 

PH 

RING 

SAM 

SH2 

Binds acetylated lysine residues 

Dimerization 

Binds phorbol esters or diacylglycerol 

Binds phospholipids 

Binds calcium 

Binds Skp1 in a ubiquitin-ligase 

complex 

Binds protein phosphothreonine 

or phosphoserine 

Binds E2 ubiquitin-conjugating 

enzymes to transfer ubiquitin to 

the substrate or to ubiquitin 

chains 

Zinc-binding cysteine-rich motif 

that forms two tandemly 

repeated zinc fingers 

Binds to the C-terminal 4-5 

residues of proteins that have a 

hydrophobic residue at the 

terminus; may bind to PIP2 

Binds to specific phosphoinositides, 

esp. PI-4,5-P 2 , PI-3,4-P 2 or 

PI-3,4,5-P 3 . 

Binds zinc and may be found in 

E3 ubiquitin ligases 

Homo- and heterooligomerization 

Binds to protein phosphotyrosine 

(pY) 

Protein sequestration 

Chromatin-associated 

proteins 

Caspase activation 

Recruitment to membranes 

Signal transduction, 

vesicular trafficking 

Calcium-dependent 

processes 

Ubiquitination 

Various; DNA damage 

FYVE Binds to PI(3)P Membrane trafficking, 

TGF- signaling 

Ubiquitination 

Wide variety of 

processes 

Scaffolding diverse 

protein complexes 

often at the membrane 

Recruitment to membranes 

and motility 

Ubiquitination, 

transcription 


processes 

Tyrosine protein kinase 


SH3 Binds to PXXP motifs Various processes 

TPR 

WW 

Degenerate sequence of ~34 

amino acids with residues 

WL/GYAFAP; forms a scaffold 

Binds proline-rich sequences 


processes 

Alternative to SH3; 

vesicular trafficking 

FIGURE 14.9 The table describes a subset of known modular protein interaction 

domains found in many proteins. Interactions mediated by these 

domains are essential to controlling cell function. Few if any of these domains 

exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction 

Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/). 

yield such motifs. Consensus target sequences 

for individual domains have been identified 

based on the sequence specificity of their binding 

to arrayed sequences. These consensus sequences 

can then be used to predict whether 

the domain will bind a site in a candidate protein. 

Adaptor proteins, which lack enzymatic 

activity, link signaling molecules and target 

them in a manner that is responsive to extracellular 

signals. Adaptor proteins are generally 

made up of two or more modular interaction 

domains or the complementary recognition 

motifs. Unlike scaffolds, adaptors are usually 

14.9 Independent, modular domains specify protein-protein interactions 601


multifunctional because their modular interaction 

domains and motifs are not as highly specific. 

Adaptors bind to two or more other 

signaling proteins via their protein-protein interaction 

domains to colocalize them or to facilitate 

additional interactions. 

Grb2 is a prototypical adaptor protein that 

was identified as a protein that bound to the C- 

terminal region of the EGF receptor. Grb2 has 

one SH2 and two SH3 domains. It binds constitutively 

to specific proline-rich segments of proteins 

through its SH3 domain, although this 

binding can be negatively regulated. One target 

of Grb2 is SOS, a guanine nucleotide exchange 

factor that activates the small GTP-binding protein 

Ras in response to EGF signaling. Through 

its SH2 domain, Grb2 binds Tyr-phosphorylated 

proteins, including the receptors themselves in 

a stimulus-dependent manner. Thus, Tyr phosphorylation 

of these receptors in response to 

ligand will enable the binding of Grb2 to the receptors, 

which, in turn, will recruit SOS to the 

membrane-localized receptor. Once at the membrane, 

SOS can activate its target, Ras. 

14.10 

Cellular signaling is 

remarkably adaptive 

Key concepts 

• Sensitivity of signaling pathways is regulated to 

allow responses to change over a wide range of 

signal strengths. 

• Feedback mechanisms execute this function in all 

signaling pathways. 

• Most pathways contain multiple adaptive feedback 

loops to cope with signals of various strengths and 

durations. 

A universal property of cellular signaling pathways 

is adaptation to the incoming signal. Cells continuously 

adjust their sensitivity to signals to maintain 

their ability to detect changes in input. Typically, 

when a cell is exposed to a new input, it initiates 

a process of desensitization that dampens the cellular 

response to a new plateau lower than the initial 

peak response, as illustrated in FIGURE 14.10. 

When the stimulus is removed, the desensitized 

state can persist, with sensitivity slowly returning 

to normal. Similarly, the removal of a tonic stimulus 

can hypersensitize signaling systems. 

FIGURE 14.10 Top: Upon exposure to 

a stimulus, signaling pathways adjust 

their sensitivities to adapt to the new 

level of input. Thus, the response decays 

after initial stimulation. A second 

similar stimulus will elicit a smaller 

response unless adequate time is allowed 

for recovery. Bottom: Some adaptation 

mechanisms feed back only on 

the receptor that is stimulated and do 

not alter parallel pathways. Such mechanisms 

are referred to as homologous. 

At left, agonist a for receptor R1 can 

initiate either of two feedback events 

that desensitize R1 alone. In other 

cases, a stimulus will also cause parallel 

or related systems to desensitize. 

At the right, agonist a initiates desensitization 

of both R1 and R2. The response 

to agonist b, which binds to 

R2, is also desensitized. Such heterologous 

desensitization is common. 

K 

a 

Homologous 

desensitization 

Initial 

response 

R1 R 2 

R1 R 2 

X 1 X 2 

R esponse 

Response 

Patterns of adaptation in signaling networks 

Agonist a 

for R1 

Agonist Agonist Agonist 

Reapply 

a or b 

Desensitization 

b 

a 

a 

R1 R 2 

X 1 X 2 

Time 

Heterologous 

desensitization 

Response 

R1 

Agonist a 

for R1 

or R1 R 2 

Reapply 

a or b 

Y 

Time 

Y 

Time 

Z 

Z 



Adaptation in signaling is one of the best examples 

of biological homeostasis. The adaptability 

of cellular signaling can be quite impressive. 

Cells commonly regulate their sensitivity to physiological 

stimuli over more than a 100-fold range, 

and the mammalian visual response can adapt to 

incoming light over a 107-fold range. This remarkable 

ability allows a photoreceptor cell to 

detect a single photon, and allows a person to 

read in both very dim light and intense sunlight. 

Adaptability is observed in bacteria, plants, fungi, 

and animals. Many of its properties are conserved 

throughout biology, although the most complex 

adaptive mechanisms are found in animals. The 

general mechanism for adaptation is the negative 

feedback loop, which biochemically samples 

the signal and controls the adaptive process. 

Adaptation varies with both the intensity and 

the duration of the incoming signal. Stronger or 

more persistent inputs tend to drive greater adaptive 

change and, often, adaptation that persists 

for a longer time. Cells can modulate adaptation 

in this way because adaptation is exerted by a 

succession of independent mechanisms, each with 

its own sensitivity and kinetic parameters. 

G protein pathways offer excellent examples 

of adaptation. FIGURE 14.11 shows that the earliest 

step in adaptation is receptor phosphorylation, 

which is catalyzed by G protein-coupled 

receptor kinases (GRKs) that selectively recognize 

the receptor’s ligand-activated conformation. 

Phosphorylation inhibits the receptor’s ability 

to stimulate G protein activation and also promotes 

binding of arrestin, a protein that further 

inhibits G protein activation. Moreover, arrestin 

binding primes receptors for endocytosis, which 

removes them from the cell surface. Endocytosis 

can also be the first step in receptor proteolysis. 

Along with these direct effects, many receptor 

genes display feedback inhibition of transcription, 

such that signaling by a receptor decreases 

its own expression. 

Stimulation thus causes multiple adaptive 

processes that range from immediate (phosphorylation, 

arrestin binding) through delayed (transcriptional 

regulation), and include both reversible 

and irreversible events. This array of adaptive 

events has been demonstrated for many G protein-coupled 

receptors, and many cells may use 

all of them to control output from one receptor. 

The speed, extent, and reversibility of adaptation 

are selected by a cell’s developmental program. 

Cells can change their patterns of adaptation 

both qualitatively and quantitatively by altering 

the points in a pathway where feedback is initiated 

and exerted. In a linear pathway, changing 

Multiple adaptation processes occur after a stimulus 

Relative 

response 

Receptor phosphorylation 

Arrestin binding 

Receptor 

endocytosis 

Endosomal receptor 

degradation 

5 Receptor transcription 

inhibited 

0 1 10 100 1000 

Time (seconds) 

Agonist 

added 

Agonist 

binds 

1 

2 

Agonist 

GPCR 

G protein 

Receptor 

recycling 

Lysosome 

3 

G protein 

active 

4 

EFFECTORS 

CYTOPLASM 

4 Receptor 

degradation 

DNA 

GRK 

1 Receptor 

Arrestin 

phosphorylation 

2 Arrestin 

binding 

NUCLEUS 

Early 

endosome 

GPCR gene 

3 Receptor 

endocytosis 

5 Receptor 

transcription 

inhibited 

FIGURE 14.11 Multiple adaptation processes are invoked during a stimulus, 

and multiple nested mechanisms for adaptation are the rule. They are usually 

invoked sequentially according to the duration and intensity of the stimulus. 

For GPCRs, at least five desensitizing mechanisms are known, with others acting 

on the G protein and effectors. 

these points will alter the kinetics or extent of 

adaptation (Figure 14.10). In branched pathways, 

changing these points can determine whether 

adaptation is unique to one input or is exerted 

for many similar inputs. If receptor activation triggers 

its desensitization directly, or if an event 

downstream on an unbranched pathway triggers 

desensitization, then only signals that initiate with 

that receptor will be altered. Receptor-selective 

adaptation is referred to as homologous adaptation 

(Figure 14.10). 

14.10 Cellular signaling is remarkably adaptive 603


Alternatively, feedback control can initiate 

downstream from multiple receptors in a convergent 

pathway and thus regulate both the 

initiating receptor and the others. Such heterologous 

adaptation regulates all the possible 

inputs to a given control point. A common 

example is the phosphorylation of G proteincoupled 

receptors by either protein kinase A or 

protein kinase C, which are activated by downstream 

signals cAMP or Ca2+ plus the lipid diacylglycerol, 

respectively. Like GRK, these kinases 

both attenuate receptor activity and promote 

arrestin binding. 

Cells also alter their responses to incoming 

signals for homeostatic reasons. These considerations 

include phase of the cell cycle, metabolic 

status, or other aspects of cellular activity. 

Again, all these adaptive processes may be displayed 

to a greater or lesser extent in different 

cells, different pathways within a cell or different 

situations during the cell’s lifetime. 

14.11 

Signaling proteins are 

frequently expressed as 

multiple species 

Key concepts 

• Distinct species (isoforms) of similar signaling 

proteins expand the regulatory mechanisms 

possible in signaling pathways. 

• Isoforms may differ in function, susceptibility to 

regulation or expression. 

• Cells may express one or several isoforms to fulfill 

their signaling needs. 

Cells increase the richness, adaptability, and 

regulation of their signaling pathways by expressing 

multiple species of individual signaling 

proteins that display distinct biochemical 

properties. These species may be encoded by 

multiple genes or by multiple mRNAs derived 

from a single gene by alternative splicing or 

mRNA editing. The numerical complexity implicit 

in these choices is impressive. Consider 

the neurotransmitter serotonin: In mammals, 

there are thirteen serotonin receptors, each of 

which stimulates a distinct spectrum of G proteins 

of the G i 

, G s 

, and G q 

families. (A fourteenth 

serotonin receptor is an ion channel.) 

FIGURE 14.12 shows the relationship of serotonin 

receptors to these G protein families. 

There is also tremendous diversity among 

the G proteins and adenylyl cyclases. There are 

three genes for Gα i 

and one each for the closely 

related Gα z 

and Gα o 

. Furthermore, the Gα o 

mRNA is multiply spliced. There are four G q 

members. In addition, there are five genes for 

Gβ and twelve for Gγ, and most of the possible 

Gβγ dimers are expressed naturally. There are 

ten genes for adenylyl cyclases, which are direct 

targets of G s 

and either direct or indirect targets 

of the other G proteins. While all nine membrane-bound 

adenylyl cyclase isoforms are stimulated 

by Gα s 

, they display diverse stimulatory 

and inhibitory responses to Gβγ, Gα i 

, Ca2+, 

calmodulin, and several protein kinases, as illustrated 

in FIGURE 14.13. Thus, stimulation by 

serotonin can lead to diverse responses depending 

upon the various forms of the proteins that 

are engaged at a particular time and location. 

FIGURE 14.12 Receptors for serotonin have 

evolved in mammals as a family of 13 genes that 

regulate three of the four major classes of G proteins. 

While all respond to the natural ligand 

serotonin, the binding sites have evolved sufficient 

differences that drugs have been developed 

that specifically target one or more 

isoforms. The type 3 serotonin receptors, not 

shown here, are ligand-gated ion channels and 

are not obviously related to the others. 

Evolutionary relationship of serotonin receptor isoforms 

Isoforms G protein 

1B 

1D 

1E G i 

1F 

1A 

5A 

7 

5B 

G s 

4 

2A 

2C 

G q 

6 

2B 

G s 

120 100 80 60 40 20 0 

Nucleotide substitution distance 



Different isoforms of adenylyl cyclase are regulated differently 

Gα s 

Gβγ 

PKC 

Ca 2+ NO 

PKA 

Regulators 

CaMK 

inhibit 

Gα i 

activate 

CaM 

FIGURE 14.13 All of the mammalian membrane-bound adenylyl cyclases are 

structurally homologous and catalyze the same reaction, and all are stimulated 

by G s 

. Their responses to other inputs (protein kinases CaMK, PKA and PKC; 

Ca2+; calmodulin (CaM); NO • ) are specific to each isoform, allowing a rich combinatoric 

input to cellular cAMP signaling. 

Sometimes isoforms of a signaling protein 

are subject to quite different kinds of inputs. 

For example, all of the members of the phospholipase 

C family (PLC) hydrolyze phosphatidylinositol-4,5-bisphosphate 

to form two second 

messengers, diacylglycerol and inositol-1,4,5 

trisphosphate (see 14.16 Lipids and lipid-derived 

compounds are signaling molecules). The distinct 

isoforms may be regulated by diverse combinations 

of Gα q 

, Gβγ, phosphorylation, monomeric 

G proteins, or Ca2+. 

Because a cell has multiple options when 

expressing a form of a signaling protein, it can 

use expression of particular isoforms to alter 

how it performs otherwise identical signaling 

functions. Different cells express one or more 

isoforms to allow appropriate responses, and expression 

can vary according to other inputs or 

the cell’s metabolic status. In addition, signaling 

pathways are remarkably resistant to mutational 

or other injuries because loss of a single species 

or isoform of a signaling protein can often be 

compensated for by increased expression or activity 

of another species. Similarly, engineered 

overexpression can result in the reduced expression 

of endogenous proteins. The existence of 

multiple receptor species can, thus, substantially 

add to adaptability and the consequent resistance 

of signaling networks to damage. 

14.12 

Activating and 

deactivating reactions 

are separate and 

independently controlled 

Key concepts 

• Activating and deactivating reactions are usually 

executed by different regulatory proteins. 

• Separating activation and inactivation allows for 

fine-tuned regulation of amplitude and timing. 

In signaling networks, individual proteins are 

frequently activated and deactivated by distinct 

reactions, a feature that facilitates separate regulation. 

Common examples include using protein 

kinases and phosphoprotein phosphatases 

14.12 Activating and deactivating reactions are separate and independently controlled 605


to catalyze protein phosphorylation and dephosphorylation; 

using adenylyl cyclase to create 

cAMP while using phosphodiesterases to 

hydrolyze it or anion transporters to pump it 

out of the cell; or using GTP/GDP exchange factors 

(GEFs) to activate G proteins and GTPaseactivating 

proteins (GAPs) to deactivate them. 

Depending on stoichiometry and detailed mechanism, 

these strategies can convey either additive 

or nonadditive inputs while maintaining 

fine control over the kinetics of activation and 

deactivation of a signaling pathway. The use of 

distinct reactions for activation and deactivation 

is analogous to the use of distinct anabolic 

and catabolic enzymes in reversible metabolic 

pathways. 

14.13 

Cellular signaling uses 

both allostery and 

covalent modification 

Key concepts 

• Allostery refers to the ability of a molecule to alter 

the conformation of a target protein when it binds 

noncovalently to that protein. 

• Modification of a protein’s chemical structure is 

also frequently used to regulate its activity. 

Cellular signaling uses almost every imaginable 

mechanism for regulating the activities of 

intracellular proteins, but most can be described 

as either allosteric or covalent. Individual signaling 

proteins typically respond to multiple allosteric 

and covalent inputs. 

Allostery refers to the ability of a molecule 

to alter the conformation of a target protein 

when it binds noncovalently to that protein. 

Because a protein’s activity reflects its conformation, 

the binding of any molecule that alters 

conformation can change the target protein’s 

activity. Any molecule can have allosteric effects: 

protons or Ca2+, small organic molecules, 

or other proteins. Allosteric regulation can be 

both inhibitory or stimulatory. 

Covalent modification of a protein’s chemical 

structure is also frequently used to regulate 

its activity. The change in the protein’s chemical 

structure alters its conformation and, thus, 

its activity. Most regulatory covalent modification 

is reversible. The classic and most common 

regulatory covalent event is phosphorylation, 

in which a phosphoryl group is transferred from 

ATP to the protein, most often to the hydroxyl 

group of serine (Ser), threonine(Thr), or tyrosine 

(Tyr). Enzymes that phosphorylate proteins 

are known as protein kinases. Their actions are 

opposed by phosphoprotein phosphatases, which 

catalyze the hydrolysis of the phosphoryl group 

to yield free phosphate and restore the unmodified 

hydroxyl residue. Other forms of covalent 

modification are also common and will be addressed 

throughout the chapter. 

14.14 

Second messengers 

provide readily diffusible 

pathways for information 

transfer 

Key concepts 

• Second messengers can propagate signals between 

proteins that are at a distance. 

• cAMP and Ca2+ are widely used second messengers. 

Signaling pathways make use of both proteins 

and small molecules according to their distinctive 

attributes. A small molecule used as an intracellular 

signal, or second messenger, has a 

number of advantages over a protein as a signaling 

intermediary. Small molecules can be 

synthesized and destroyed quickly. Because they 

can be made readily, they can act at high concentrations 

so that their affinities for target proteins 

can be low. Low affinity permits rapid 

dissociation, such that their signals can be terminated 

promptly when free second messenger 

molecules are destroyed or sequestered. Because 

second messengers are small, they also can diffuse 

quickly within the cell, although many cells 

have developed mechanisms to spatially restrict 

such diffusion. Second messengers are, thus, 

superior to proteins in mediating fast responses, 

particularly at a distance. Second messengers 

are also useful when signals have to be addressed 

to large numbers of target proteins simultaneously. 

These advantages often overcome their 

lack of catalytic activity and their inability to 

bind multiple molecules simultaneously. 

FIGURE 14.14 lists intracellular second messengers 

developed through evolution. This number 

is surprisingly low. Several are nucleotides 

synthesized from major metabolic nucleotide 

precursors. They include cAMP, cyclic GMP, 

ppGppp, and cyclic ADP-ribose. Other soluble 

second messengers include a sugar phosphate, 

inositol-1,4,5-trisphosphate (IP 3 

), a divalent metal 

ion Ca2+, and a free radical gas nitric oxide (NO • ). 

Lipid second messengers include diacylglycerol 

and phosphatidylinositol-3,4,5-trisphosphate, 



Second 

messenger 

3':5'-cyclic AMP 

(cAMP) 

RNA polymerase 

Magic spot 

(ppGpp, ppGppp) ObgE transcription 

arrest 

detector 

Cyclic di-GMP 

phosphodiesterase 

Inositol-1,3,5- 

trisphosphate 

(IP 3 ) 

Diacylglycerol 

(DAG) 

Phosphatidyl- 

inositol-4,5- 

bisphosphate 

(PIP 2 ) 

3':5'-Cyclic GMP 

(cGMP) 

Cyclic ADP-ribose 

Nitric oxide (NO. ) 

Ca2+ 

Cyclic 

diguanosinemonophosphate 

Phosphatidyl- 

inositol-3,4,5- 

trisphosphate 

Adenylyl 

Protein kinase A 

cyclase 

Bacterial transcription 

factors 

Cation channel 

Cyclic nucleotide 


Rap GDP/GTP 

exchange factor 

(Epac) 

IP 3 -gated Ca 2+ 

channel 

Protein 

kinase C 

Trp cation 

channel 

Ion channel 

Transporters 

Protein kinase G 

Ca2+ channel 

Various two 

component 

system proteins 

Guanylyl cyclase 

Numerous 

calmodulin 

Akt (protein 

kinase B) 

Second messengers 

Targets 

Other PH 

domains/proteins 

Synthesis/ 

Release 

Rel1A 

SpoT 

Cation channel 

Cyclic nucleotide 


Phospholipase 

C 

Phospholipase 

C 

PIP 5-kinase 

Guanylyl 

cyclase 

ADP-ribose 

cyclase 

Diguanylate 

cyclase 

PI 3-kinase 

GTP 

PIP 2 

PIP 2 

PI-4-P 

GTP 

NAD 

GTP 

Stored 

Ca2+ 

PIP 2 

phosphatidylinositol-4,5-diphosphate, sphingosine-1-phosphate 

and phosphatidic acid. 

The first signaling compound to be described 

as a second messenger was cAMP. The name 

arose because cAMP is synthesized in animal 

cells as a second, intracellular signal in response 

to numerous extracellular hormones, the first 

messengers in the pathway. cAMP is used by 

prokaryotes, fungi, and animals to convey information 

to a variety of regulatory proteins. 

(Its occurrence in higher plants has still not been 

proved.) 

Adenylyl cyclases, the enzymes that synthesize 

cAMP from ATP, are regulated in various 

ways depending on the organism in which 

they occur. In animals, adenylyl cyclase is an 

integral protein of the plasma membrane whose 

multiple isoforms are stimulated by diverse 

agents (see Figure 14.13). In animal cells, adenylyl 

cyclase is generally stimulated by G s 

, which 

was originally discovered as an adenylyl cyclase 

regulator. Some fungal adenylyl cyclases are 

also stimulated by G proteins. Bacterial cyclases 

are far more diverse in their regulation. 

cAMP is removed from cells in two ways. 

It may be extruded from cells by an ATP-driven 

anion pump but is more often hydrolyzed to 5′- 

AMP by members of the cyclic nucleotide phosphodiesterase 

family, a large group of proteins 

that are themselves under multiple regulatory 

controls. 

The prototypical downstream regulator for 

cAMP in animals is the cAMP-dependent protein 

kinase, but a bacterial cAMP-regulated transcription 

factor was discovered shortly thereafter, 

and other effectors are now known (Figure 

14.14). The cAMP system remains the prototypical 

eukaryotic signaling pathway in that its 

components exemplify almost all of the recognized 

varieties of signaling molecules and their 

interactions: hormone, receptor, G protein, 

adenylyl cyclase, protein kinase, phosphodiesterase, 

and extrusion pump. 

The second messenger-stimulated protein 

kinase PKA is a tetramer composed of two catalytic 

(C) subunits and two regulatory (R) subunits, 

as illustrated in FIGURE 14.15. The R subunit 

binds to the catalytic subunit in the substratebinding 

region, maintaining C in an inhibited 

state. Each R subunit binds two molecules of 

cAMP, four cAMP molecules per PKA holoenzyme. 

When these sites are filled, the R subunit 

dimer dissociates rapidly, leaving two free catalytic 

subunits with high activity. The difference 

in affinity of R for C in the presence and absence 

of cAMP is ~10,000-fold. The strongly cooperative 

binding of cAMP generates a very steep 

activation curve with an apparent threshold below 

which no significant activation of PKA occurs, 

as illustrated in Figure 14.15. PKA activity, 

thus, increases dramatically over a narrow range 

of cAMP concentrations. PKA is also regulated 

Precursor 

Removal 

Phosphodiesterase 

ATP 

Organic 

anion 

transporter 

SpoTcatalyzed 

hydrolysis 

Phosphatase 


kinase 


lipase 

Phospholipase 

C 

Phosphatase 

Phosphodiesterase 

Hydrolysis 

NO . synthase arginine Reduction 

Release from 

storage 

organelles 

or plasma 

membrane 

channels 

Reuptake 

and 

extrusion 

pumps 

Phosphatase 

FIGURE 14.14 Major second messengers, some of the proteins that they regulate, 

their sources and their disposition. 

14.14 Second messengers provide readily diffusible pathways for information transfer 607


(C) 

Catalytic 

subunits 

PKA 

C 

R 

R 

C 

Activation of PKA by cAMP 

(R) 

R egulatory 

subunits 

4 cAMP 

R 

R 

- cAMP 

- cAMP 

- cAMP 

- cAMP 

Activated 

PKA 

C 

C 

teins throughout the cell ranging from ion channels 

to transcription factors, and its conserved 

substrate preference frequently permits prediction 

of substrates by sequence analysis. The 

cAMP response element binding protein CREB 

is phosphorylated by PKA on Ser 133 and is 

largely responsible for the impact of cAMP on 

transcription of numerous genes. 

R 2 C 2 + 4 cAMP 

Kinase activity as a 

function of [cAMP] (%) 

100 

80 

60 

40 

20 

10% 

R 2 

. cAMP 4 + 2C 

90% 

14.15 

Ca2+ signaling serves 

diverse purposes in all 

eukaryotic cells 

Key concepts 

• Ca2+ serves as a second messenger and regulatory 

molecule in essentially all cells. 

• Ca2+ acts directly on many target proteins and also 

regulates the activity of a regulatory protein 

calmodulin. 

• The cytosolic concentration of Ca2+ is controlled by 

organellar sequestration and release. 

2 x 10 -9 2 x 10 -8 2 x 10 -7 

[cAMP] 

FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and 

two regulatory (R) subunits. Binding of four molecules of cAMP to the regulatory 

subunits induces dissociation of two molecules of C, the active form 

of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel, 

the cooperative binding of four molecules of cAMP generates a steep activation 

profile. Activity increases from approximately 10% to 90% as the 

cAMP concentration increases only 10-fold. An apparent threshold is introduced 

because there is little change in activity at low concentrations of 

cAMP. 

by phosphorylation of its activation loop. 

Phosphorylation occurs cotranslationally, and 

the activation loop phosphorylation is required 

for assembly of the R 2 

C 2 

tetramer. 

The PKAs are mostly cytosolic and are also 

targeted to specific locations by binding organelle-associated 

scaffolds (A-kinase anchoring 

proteins, or AKAPs). These AKAPs facilitate 

phosphorylation of membrane proteins including 

GPCRs, transporters, and ion channels. 

AKAPs can also target PKA to other cellular locations 

including mitochondria, the cytoskeleton, 

and the centrosome. AKAPs often harbor 

binding sites for other regulatory molecules 

such as phosphoprotein phosphatases and additional 

protein kinases, which allows for coordination 

of multiple signaling pathways and 

integration of their outputs. 

PKA generally phosphorylates substrates 

with a primary consensus motif of Arg-Arg- 

Xaa-Ser-Hydrophobic, placing it in a large group 

of kinases that recognize basic residues preceding 

the phosphorylation site. PKA regulates pro- 

Ca2+ is used as a second messenger in all cells, 

and is, thus, an even more widespread second 

messenger than cAMP. Many proteins bind Ca2+ 

with consequent allosteric changes in their enzymatic 

activities, subcellular localization, or 

interaction with other proteins or with lipids. 

Direct targets of Ca2+ regulation include almost 

all classes of signaling proteins described in this 

chapter, numerous metabolic enzymes, ion 

channels and pumps, and contractile proteins. 

Most noteworthy may be muscle actomyosin 

fibers, which are triggered to contract in response 

to cytosolic Ca2+ (see 8.21 Myosin-II functions 

in muscle contraction). 

Although free Ca2+ is found at concentrations 

near 1 mM in most extracellular fluids, intracellular 

Ca2+ concentrations are maintained 

near 100 nanomolar levels by the combined action 

of pumps and transporters that either extrude 

free Ca2+ or sequester it in the endoplasmic 

reticulum or mitochondria. Ca2+ signaling is initiated 

when Ca2+-selective channels in the endoplasmic 

reticulum or plasma membrane are 

opened to allow Ca2+ to enter the cytoplasm. 

The most important entrance channels include 

electrically gated channels in animal plasma 

membranes; a Ca2+ channel in the endoplasmic 

reticulum that is opened by another second messenger, 

inositol 1,4,5-trisphosphate (see below); 

and an electrically gated channel in the endoplasmic 

(sarcoplasmic) reticulum of muscle 

that opens in response to depolarization of nearby 

plasma membrane, a process known as excitation-contraction 

coupling (see 2.9 Plasma mem- 



Calcium binding causes a conformational change in calmodulin 

Calcium-free 

calmodulin 

calmodulin free + 4 Ca 2+ 

Calcium-bound 

calmodulin bound to 

target peptide of CaMK 

Ca 2+ 

target 

(Ca 2+ ) 4 

. calmodulin . active target 

FIGURE 14.16 Ribbon diagrams representing 

the crystal structures of calmodulin free 

of Ca2+ and bound to four Ca2+ ions reveal 

the huge conformational change that 

calmodulin undergoes upon Ca2+ binding. 

Ca2+-calmodulin causes activity changes in 

target proteins. The bottom panel shows 

the activation of a target by calmodulin as 

a function of the intracellular free Ca2+ concentration. 

The requirement for binding 

four Ca2+ ions to induce the conformational 

transition results in cooperative activation 

of targets. Activity increases from 10% to 

90% as the Ca2+ concentration increases 

only 10-fold. Structures generated from 

Protein Data Bank files 1CFD and 1MXE. 

Activation of target 

by calmodulin (%) 

100 

90% 

80 

60 

40 

20 

10% 

3 x 10 -8 3 x 10 -7 3 x 10 -6 

[Ca 2+ ] 

brane Ca2+ channels activate intracellular functions). 

In addition to the proteins that are regulated 

by binding Ca2+ directly, many other proteins respond 

to Ca2+ by binding a widespread Ca2+ sensor, 

the small, ~17 kDa protein calmodulin. 

Calmodulin requires the binding of four molecules 

of Ca2+ to become fully active, and binding 

is highly cooperative, generating a sigmoid 

activation profile illustrated in FIGURE 14.16. 

Calmodulin generally binds its targets in a Ca2+dependent 

manner, but Ca2+-free calmodulin 

may remain bound but inactive in some cases. 

For example, calmodulin is a constitutive subunit 

of phosphorylase kinase that is activated 

upon Ca2+ binding. Higher plants again make 

major modifications to this paradigm. Calmodulin 

is not expressed as a distinct protein but, instead, 

is found as a domain in Ca2+-regulated proteins. 

In yet another variation, the adenylyl cyclase secreted 

by the pathogenic bacterium Bordetella pertussis 

is inactive outside cells but is activated by 

Ca2+-free calmodulin in animal cells, where its 

rapid production of cAMP is highly toxic. 

14.16 

Lipids and lipid-derived 

compounds are signaling 

molecules 

Key concepts 

• Multiple lipid-derived second messengers are 

produced in membranes. 

• Phospholipase Cs release soluble and lipid second 

messengers in response to diverse inputs. 

• Channels and transporters are modulated by 

different lipids in addition to inputs from other 

sources. 

• PI 3-kinase synthesizes PIP 3 

to modulate cell 

shape and motility. 

• PLD and PLA 2 

create other lipid second 

messengers. 

Signals that originate at the plasma membrane 

may have soluble regulatory targets in the cytoplasm 

or intracellular organelles, but integral 

plasma membrane proteins are also subject to 

acute controls. For these targets, lipid second 

messengers may be primary inputs. Lipids derived 

from membrane phospholipids or other 

14.16 Lipids and lipid-derived compounds are signaling molecules 609


lipid species play numerous roles in cell signaling. 

Because their analysis has been more difficult 

than for soluble messengers, many 

probably remain to be discovered and understood. 

FIGURE 14.17 shows the structure of some 

of these lipids. 

Phospholipase Cs (PLCs) are the prototypical 

lipid signaling enzymes. PLC isoforms catalyze 

the hydrolysis of phospholipids between 

the 3-sn-hydroxyl and the phosphate group to 

yield a diacylglycerol and phosphate ester. In 

animals and fungi, PLCs specific for the substrate 

Structures of some lipid second messengers 

O 

Phosphatidylinositol (PI) 

O 

O 

O 

HO 

OH 

O 

2 

O 

3 

P 

1 

O - 

O 

6 OH 

4 5 

OH 

OH 

O 

Phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) 

O 

O 

O 

H - O 3 PO 

HO 

OH 

OH 

O - 

O 

P 

O 

O 

6 OH 

2 1 4 5 

3 

OPO 3 H - 

OPO 3 H - 

O 

Diacylglycerol (DAG) 

O 

O 

O 

OH 

OPO 3 H - 

6 OH 

2 1 4 5 

3 

Inositol trisphosphate (IP 3 ) 

OPO 3 H - 

OPO 3 H - 

O 

Phosphatidic acid (PA) 

O 

O 

O 

O 

O- 

P 

O - 

O 

FIGURE 14.17 Structures of some lipid second messengers and the common precursor phosphatidylinositol. 

The acyl side chain structures shown here are the most common for mammalian PI lipids. Much of the PA in 

cells is derived from PC, and its acyl chains may differ from those shown. 



phosphatidylinositol-4,5-bisphosphate (PIP 2 

) 

hydrolyze PIP 2 

to form two second messengers: 

1,2-sn-diacylglycerol (DAG) and inositol-1,4,5- 

trisphosphate (IP 3 

). The PLC substrate PIP 2 

is itself 

an important regulatory ligand that 

modulates the activity of several ion channels, 

transporters, and enzymes. Thus, PLC alters concentration 

of three second messengers; its net 

effect depends on the net turnover of the substrate 

and products. 

DAG is probably the best known lipid second 

messenger; its hydrophobicity limits it to action 

in membranes. DAG activates some isoforms 

of protein kinase C (PKC), modulates the activity 

of several cation channels and activates at 

least one other protein kinase. DAG can be further 

hydrolyzed to release arachidonic acid, 

which can regulate some ion channels. 

Arachidonic acid is also the precursor of oxidation 

products, such as prostaglandins and thromboxanes, 

which are potent extracellular signaling 

agents. In addition to DAG, PKCs require interaction 

with Ca2+ and an acidic phospholipid, 

such as phosphatidylserine, to become activated. 

Thus, activation of PKC requires the coincidence 

of multiple inputs both to generate DAG and to 

increase intracellular Ca2+. There are more than 

a dozen PKCs, classified together according to 

highly conserved sequences in the catalytic domain. 

Three subgroups of PKCs, also identifiable 

by sequence, share different patterns of regulation. 

Their regulation provides examples of 

many ways in which other mammalian protein 

kinases are regulated. 

The first of these groups, canonical PKCs, 

are generally soluble or very loosely associated 

with membranes prior to the appearance of 

DAG. DAG causes their association with membranes 

and permits activation upon binding of 

other regulators. The second group of PKCs requires 

similar lipids but not Ca2+, and the third 

group requires other lipids but neither DAG nor 

Ca2+ for activation. 

The N-terminal region of PKCs contains a 

pseudosubstrate domain, a sequence that resembles 

that of a typical substrate except that 

the target Ser is replaced with Ala. The pseudosubstrate 

region binds to the active site to inhibit 

the kinase. Activators cause the 

pseudosubstrate domain to flip out of the active 

site. PKCs are also activated by proteolysis, 

as are many protein kinases with discrete 

autoinhibitory domains. Proteases clip a flexible 

hinge region, which results in loss of the 

regulatory domain and consequent activation 

of the kinase. 

PKC is the major receptor for phorbol esters, 

a class of powerful tumor promoters. Phorbol 

esters mimic DAG and cause a more massive 

and prolonged activation than physiological 

stimuli. This massive stimulation can induce 

proteolysis of PKC, resulting in downregulation, 

or loss of the kinase. (For a personal description 

on the discovery of protein kinase C 

see EXP : 14-0001 ) 

IP 3 

, the second product of the PLC reaction, 

is a soluble second messenger. The most significant 

IP 3 

target is a Ca2+ channel in the endoplasmic 

reticulum. IP 3 

causes this channel to 

open and release stored Ca2+ into the cytoplasm, 

thereby rapidly elevating the cytosolic Ca2+ over 

100-fold and, in turn, causing the activation of 

numerous targets of Ca2+ signaling. 

There are at least six families of PIP 2 

-selective 

PLC enzymes, defined by their distinct forms 

of regulation, domain compositions, and overall 

sequence conservation. Their catalytic domains 

are all quite similar. The PLC-βs are 

stimulated primarily by Gα q 

and Gβγ (to individually 

varying extents). Several are also modulated 

by phosphorylation. PLC-γ isoforms are 

stimulated by phosphorylation on Tyr residues, 

frequently by receptor tyrosine kinases. The 

PLC-ε isoforms are regulated by small, 

monomeric G proteins of the Rho family. The 

regulation of the PLC-δs is still incompletely understood. 

Two other classes similar to the PLCδs, 

PLC-η and -ζ, have also been defined recently. 

(There is no PLC-α.) In addition to their distinct 

modes of regulation, all of the PLCs are stimulated 

by Ca2+, and Ca2+ often acts synergistically 

with other stimulatory inputs. This synergy 

underlies the intensification and prolongation 

of Ca2+ signaling observed in many cells. 

Phospholipases A 2 

and D (PLA 2 

and PLD) 

also hydrolyze glycerol phospholipids in cell 

membranes to form important signaling compounds. 

PLA 2 

hydrolyzes the fatty acid at the sn- 

2 position of multiple phospholipids to produce 

the cognate lysophospholipid and the free fatty 

acid, which is generally unsaturated. The free 

fatty acid is often arachidonic acid, a precursor 

of extracellular signals. The biological roles of 

free lysophospholipids are not understood in 

detail but have been linked to effects on the 

structure of the membrane bilayer. 

PLD catalyzes a reaction much like that of 

PLC but instead hydrolyzes the phosphodiester 

on the substituent side of the phosphate group 

to form 3-sn-phosphatidic acid. Cellular PLDs act 

on multiple glycerol phospholipid substrates, 

but phosphatidylcholine is probably the sub- 

14.16 Lipids and lipid-derived compounds are signaling molecules 611


FIGURE 14.18 Activated PI 3- 

kinase phosphorylates PIP 2 

to produce 

PIP 3 

. The PH domain-containing 

protein kinases PDK1 and Akt 

bind to PIP 3 

at the plasma membrane. 

Their colocalization facilitates 

the phosphorylation of Akt 

by PDK1. A second phosphorylation 

within a hydrophobic motif results 

in Akt activation by one of 

several candidate protein kinases. 

The Akt-2 isoform is required to 

elicit hallmark actions of insulin. 

p85 p110 

PI 3-kinase 

PIP 2 PIP 3 

strate most relevant to signaling functions. The 

functions of the phosphatidic acid product, 

which is also formed by phosphorylation of 

DAG, remain poorly understood but appear to 

include a role in secretion and the fusion of intracellular 

membranes. 

14.17 

PI 3-kinase regulates 

both cell shape and the 

activation of essential 

growth and metabolic 

functions 

Key concepts 

• Phosphorylation of some lipid second messengers 

changes their activity. 

• PIP 3 

is recognized by proteins with a pleckstrin 

homology domain. 

Lipid second messengers may also be modified 

by phosphorylation. PI 3-kinase phosphorylates 

PIP 2 

on the 3-position of the inositol ring to 

form PI 3,4,5-P 3 

, another lipid second messenger. 

The total activity of PI 3-kinase is too low 

to significantly deplete total PIP 2 

, but formation 

of small amounts of PIP 3 

in localized membrane 

domains is vital for altering cell shape 

and cellular motility. 

PIP 3 

acts by recruiting proteins that contain 

PIP 3 

binding domains, including pleckstrin 

homology (PH) and FYVE domains, to sites 

where they regulate cytoskeletal remodeling, 

contractile protein function, or other regulatory 

events. These proteins anchor and/or orient 

the structural or motor proteins involved 

in cellular movement and localize signaling proteins 

to sites of action at the membrane. PIP 3 

PIP 3 binding brings Akt and PDK1 to the membrane 

PIP 2 phosphorylated 

Akt 

Akt and PDK1 

bind PIP3 through 

PH domains 

Akt 

PH 

domains 

PDK1 

PDK1 

Akt is activated by 

phosphorylation 

Other 

kinase 

Glucose uptake 

Glycogen synthesis 

Antilipolysis 

Antiapoptosis 

signaling can be fast and dramatic; it largely accounts 

for directing the mobility of motile mammalian 

cells. 

Lipid mediators are essential in the insulin 

signaling pathway. The binding of insulin stimulates 

the Tyr autophosphorylation of its receptor 

and the activation of effectors through insulin receptor 

substrate (IRS) proteins (see 14.30 Diverse 

signaling mechanisms are regulated by protein tyrosine 

kinases). PI 3-kinase is activated when its p85 

subunit binds to IRS1. The PIP 3 

generated by PI3- 

kinase binds the protein kinases Akt and phosphoinositide-dependent 

kinase-1 (PDK-1) via their PH 

domains. This interaction results in the localization 

of Akt to the membrane where it is activated 

by PDK1, as illustrated in FIGURE 14.18. Akt phosphorylates 

downstream targets, including protein 

kinases, GAPs, and transcription factors. 

Activation of Akt, specifically Akt-2, is required 

for the hallmark actions of insulin including regulation 

of glucose transporter translocation, enhanced 

protein synthesis, and expression of 

gluconeogenic and lipogenic enzymes. 

14.18 

Signaling through ion 

channel receptors is very 

fast 

Key concepts 

• Ion channels allow the passage of ions through a 

pore, resulting in rapid (microsecond) changes in 

membrane potential. 

• Channels are selective for particular ions or for 

cations or anions. 

• Channels regulate intracellular concentrations of 

regulatory ions, such as Ca2+. 

Ligand-gated ion channels are multisubunit, 

membrane-spanning proteins that create and 

regulate a water-filled pore through the membrane, 

as illustrated in the X-ray crystal structure 

of the nicotinic acetylcholine receptor in 

FIGURE 14.19. When stimulated by extracellular 

agonists, the subunits rearrange their conformations 

and orientations to open the pore and, thus, 

connect the aqueous spaces on either side of the 

membrane. The pore has a diameter that allows 

ions to diffuse freely from one side of the membrane 

to the other, driven by the electrical and 

chemical gradients that have been established 

by ion pumps and transporters. (For more about 

channel, pump and transporter mechanics see 2 

Transport of ions and small molecules across membranes.) 

Channels maintain selectivity among 

ions by regulating the pore diameter precisely 



and by lining the walls of the pore with appropriate 

hydrophilic residues. Receptor ion channels 

can, thus, provide a diffusion path for only 

cations or anions, or select among different ions. 

Ligand-gated ion channels provide the 

fastest signal transduction mechanism found in 

biology. Upon binding an agonist ligand, channels 

open within microseconds. At synapses, 

where neurotransmitters need to diffuse less 

than 0.1 micron, a signal in the postsynaptic 

cell can be generated in 100 microseconds. In 

contrast, receptor-stimulated G proteins require 

about 100 milliseconds to exchange GDP for 

GTP, and the action of receptor protein kinases 

is even slower. Ligand-gated ion channels are 

important receptors in many cells in addition 

to neurons and muscle, and other ion channels 

play equally vital roles in signaling pathways 

triggered by other classes of ligands. 

Ion channel signaling differs from that of 

the other receptors mentioned in this chapter 

in that there is no immediate protein target nor, 

in most cases, is there a specific second messenger 

involved. In most cases, channel-mediated 

ion flow acts to increase or decrease the cell’s 

membrane potential and, thus, modulates all 

transport processes for metabolites or ions that 

are electrically driven. 

Animal cells maintain an inside-negative 

membrane potential by pumping out Na + ions 

and pumping in K + ions (for more on membrane 

potential see 2.4 Electrochemical gradients 

across the cell membrane generate the membrane potential). 

The opening of a channel selective for 

Na+ will thus depolarize cells, and the opening 

of a channel for K + will hyperpolarize cells. 

Similarly, because Cl - is primarily extracellular, 

opening Cl - channels will also cause hyperpolarization. 

These electrical effects convey information 

to effector proteins that are energetically 

coupled to the membrane potential, or to specific 

ion gradients, or that bind a specific ion 

(such as Ca2+) whose concentration changes 

upon channel opening. 

The nicotinic acetylcholine receptor is the prototypical 

receptor ion channel and was the first 

receptor that was shown to be a channel. It is a relatively 

unselective cation channel that causes depolarization 

of the target cell by allowing Na+ 

influx. It is best known as the excitatory receptor 

at the neuromuscular synapse, where it triggers 

contraction, but alternative isoforms are also active 

in neurons and many other cells. In muscles, 

nicotinic depolarization acts via a voltage-sensitive 

Ca2+ channel to allow Ca2+ release from the sarcoplasmic 

reticulum into the cytosol. Calcium acts 

Nicotinic acetylcholine receptor structure 

CLOSED 

CYTOSOL 

Pore 

OPEN 

Pore 

FIGURE 14.19 The nicotinic cholinergic receptor is a cation-selective channel 

that is composed of five homologous but usually nonidentical subunits that 

oligomerize to form a primarily -helical membrane-spanning core. The channel 

itself is created within this core, and its opening and closing are executed 

by cooperative changes in subunit arrangement. Structure generated from 

Protein Data Bank file 2BG9. 

as a second (or third) messenger to initiate contraction 

(see 2.13 Cardiac and skeletal muscles are activated 

by excitation-contraction coupling). Nicotinic 

receptors promote exocytosis in some secretory 

cells by a similar mechanism, where Ca2+ triggers 

the exocytic event. In neurons, where nicotinic 

stimulation causes an action potential (depolarization 

that is rapidly propagated along the neuron), 

the initial depolarization is sensed by 

voltage-sensitive Na+ channels. Their opening 

(along with the action of other channels) propagates 

the action potential along the neuron. 

The nervous system is rich in receptor cation 

channels that respond to other neurotransmitters, 

the most common of which is the amino 

acid glutamate (Glu). The three different families 

of glutamate receptors share the property 

of cation conductance, but each family has its 

own spectrum of drug responses. All operate as 

neuronal activators, with one interesting twist: 

The NMDA family of receptors, named for their 

response to a selective drug, is permeant to Ca2+ 

in addition to Na+. A significant component of 

its activity is to permit the inward flow of Ca2+, 

which acts as a second messenger on a wide variety 

of targets. Persistant stimulation of NMDA 

channels by glutamate released during injury, 

or by drugs, can cause toxic amounts of Ca2+ to 

enter, resulting in neuronal death. 

A second functional group of receptor channels 

is selective for anions and, by allowing inward 

flux of Cl - , hyperpolarizes the target cell. 

Anion-selective receptors include those for γ- 

aminobutyric acid (GABA) and glycine (Gly). In 

neurons, hyperpolarization can inhibit the initiation 

of an action potential and/or neurotransmitter 

release. 

14.18 Signaling through ion channel receptors is very fast 613


Perhaps the most diverse family of ligandgated 

channels is that of the TRP and TRP-like 

family, of which about 30 have been found in 

mammals. Distinct forms are found in invertebrates. 

The TRP channels are Ca2+-selective 

channels that are formed by tetramers of identical 

subunits that surround the central channel. 

Each subunit is composed of a homologous 

bundle of six membrane-spanning helices, but 

the N and C termini contain a diverse collection 

of regulatory and protein interaction domains, 

including protein kinase domains (whose 

substrates are currently unknown). 

All TRP channels allow transmembrane flux 

of Ca2+ to permit its action as a second messenger, 

but different TRP isoforms serve numerous 

physiological functions. The prototypical TRP, 

found in invertebrate photoreceptors, gates Ca2+ 

flow from intracellular stores into the cytoplasm 

to initiate visual signaling. Others admit Ca2+ 

from outside the cell, and still others allow Ca2+ 

to enter the endoplasmic reticulum virtually directly 

from the extracellular space because they 

form a bridge between the plasma membrane 

and channels in the endoplasmic reticulum at 

points where the membranes abut each other. 

Regulation of TRP channels is perhaps even 

more diverse. Various TRP channels respond to 

heat, cold, painful stimuli, pressure, and high 

or low osmolarity. Many TRPs are regulated either 

positively or negatively by lipids, such as 

eicosanoids, diacylglycerol, and PIP 2 

. For example, 

capsaicin, the hot compound in chilis, is 

an agonist for some vanilloid receptors (TRPVs). 

Still other TRP channels are mechanosensors 

that allow cilia to sense fluid flow. The most famous 

of these is the sensory channel of the hair 

cell of the inner ear. This channel opens when 

the apical cilia on the hair cell are bent in response 

to sound-driven fluid flow. 

14.19 

Nuclear receptors 

regulate transcription 

Key concepts 

• Nuclear receptors modulate transcription by 

binding to distinct short sequences in 

chromosomal DNA known as response elements. 

• Receptor binding to other receptors, inhibitors, or 

coactivators leads to complex transcriptional 

control circuits. 

• Signaling through nuclear receptors is relatively 

slow, consistent with their roles in adaptive 

responses. 

Nuclear receptors are unique among cellular 

receptors in that their ligands pass unaided 

through the plasma membrane. These receptors, 

when complexed with their ligands, enter 

the nucleus and regulate gene transcription. 

Ligands for nuclear receptors include sex steroids 

(estrogen and testosterone) and other steroid 

hormones, vitamins A and D, retinoids and 

other fatty acids, oxysterols, and bile acids. 

Nuclear receptors are structurally conserved. 

They consist of a C-terminal ligand binding domain, 

an N-terminal interaction region that recognizes 

components of the transcriptional 

machinery and acts as a transactivation domain, 

a centrally located zinc finger domain that binds 

DNA, and, often, another transactivation domain 

nearer the C-terminus. In the absence of 

ligand, these receptors are bound to corepressor 

proteins that suppress their activity. Upon 

hormone binding, corepressors dissociate and 

the receptors are assembled in multiprotein 

complexes with coactivators that modulate receptor 

action and facilitate transcriptional regulation. 

As illustrated in FIGURE 14.20, agonists 

and antagonists bind to distinct receptor conformations 

(see 14.5 Ligand binding changes receptor 

conformation). Receptor agonists favor the binding 

of receptors to coactivators and DNA, and 

antagonists favor conformations that block coactivator-receptor 

binding. 

Nuclear receptors bind with high specificity 

to hormone response elements in the 5’ untranscribed 

region of regulated genes. Response 

elements are typically short direct or inverted 

repeat sequences, and a gene may contain response 

elements for several different receptors 

in addition to binding sites for other transcriptional 

regulatory proteins. 

The sex steroid estrogen can bind to two 

different nuclear receptors, the estrogen receptors 

ER and ER. Coactivator and corepressor 

proteins differentially regulate ER and ER in 

transcriptional complexes that are expressed in 

specific cell types. Other ligands that bind to 

these receptors include valuable therapeutic 

agents. For example, 4 hydroxy-tamoxifen is 

an estrogen receptor antagonist used in the therapy 

of estrogen-receptor-positive breast cancer 

to inhibit growth of residual cancer cells. 

However, unlike its antagonistic effects on the 

estrogen receptor in breast, 4 hydroxy-tamoxifen 

displays weak partial agonist activity in 

uterus. In the estrogen receptor system, partial 

agonists are known as selective estrogen receptor 

modulators (SERMs). Properties that contribute 

to partial agonist activity include the 

relative expression of the two estrogen recep- 



Estrogen receptor conformation depends on which ligand is bound 

Agonist-bound 

conformation 

Antagonist-bound 

conformation 

N 

N 

K362 

H11 

H5 

K362 

H5 

545 

C 

H6 

545 

542 

H3 

H11 

H6 

538 

542 

H3 

538 

FIGURE 14.20 The estrogen receptor adopts different conformations when 

bound to agonists and antagonists. The ligand-binding domain of the estrogen 

receptor is bound to the agonist estradiol on the left and to the antagonist 

raloxifene on the right. Note the marked difference in position of helix 12, 

shown in blue in the active structure and green in the inhibited structure. 

Reproduced from Brzozowski, A. M., et al. 1997. Molecular basis of agonism and 

antagonism in the oestrogen receptor. Nature. 389: 753–758. Photo courtesy 

of M. Brzozowski, University of New York. 

tors, ER and ER, as well as the expression of 

repressors and coactivators that interact with 

each receptor type. Thus, the behavior of nuclear 

receptor ligands must be considered in the 

tissue, cellular, and signaling context. 

14.20 

G protein signaling 

modules are widely used 

and highly adaptable 

Key concepts 

• The basic module is a receptor, a G protein and an 

effector protein. 

• Cells express several varieties of each class of 

proteins. 

• Effectors are heterogeneous and initiate diverse 

cellular functions. 

Activation of G protein-coupled receptors 

(GPCRs) and their associated heterotrimeric G 

proteins is one of the most widespread mechanisms 

of communicating extracellular signals 

to the intracellular environment. G protein signaling 

modules are found in all eukaryotes. 

Depending on the species, mammals express 

500-1000 GPCRs that respond to hormones, 

neurotransmitters, pheromones, metabolites, 

local signaling substances, and other regulatory 

molecules. Essentially all chemical classes are 

represented among the GPCR ligands. In addition, 

a roughly equal number of olfactory GPCRs 

are expressed in olfactory neurons and work in 

combination to screen compounds in the animal’s 

environment via the sense of smell. 

Because GPCRs are involved in many kinds of 

physiologic responses, they are also one of the 

most widely used targets for drugs. 

A minimal G protein signaling module consists 

of three proteins: a G protein-coupled receptor, 

the heterotrimeric G protein, and an 

effector protein, as illustrated in FIGURE 14.21. The 

receptor activates the G protein on the inner face 

of the plasma membrane in response to an extracellular 

ligand. The G protein then activates (or 

occasionally inhibits) an effector protein that 

propagates a signal within the cell. Thus, signal 

conduction in the simplest G protein module is 

linear. However, as depicted in FIGURE 14.22, a 

typical animal cell may express a dozen GPCRs, 

more than six G proteins, and a dozen effectors. 

Each GPCR regulates one or more G proteins, 

and each G protein regulates several effectors. 

Moreover, distinct efficiencies and rates govern 

each interaction. Thus, a cell’s G protein network 

is actually a signal-integrating computer whose 

14.20 G protein signaling modules are widely used and highly adaptable 615


Agonist 

GPCR 

Trimeric 

G protein 

Receptor 

activated 

CYTOSOL 

Heterotrimeric G protein signaling 

Activated 

G protein 

dissociates 

IP 3 -gated 

Ca 2+ channel 

ENDOPLASMIC 

RETICULUM 

PIP 2 

IP 3 

Ca 2+ 

DAG 

Hydrolysis of PIP 2 

to IP 3 and DAG 

Release of Ca 2+ 

FIGURE 14.21 G protein-mediated signal transduction follows a path of agonist 

to receptor to heterotrimeric G protein to effector to the effector's output. 

Both G and G subunits regulate distinct effectors. In the example 

shown here, G q 

regulates a phospholipase C- to produce two second messengers, 

diacyglycerol (DAG) and inositol-trisphosphate (IP 3 

). IP 3 

triggers Ca2+ release 

from the endoplasmic reticulum. 

- 

output is a spectrum of cellular signals that is 

complex in both amplitude and kinetics. Because 

of their conserved parts list, G protein modules 

are well suited to initiating a wide variety of intracellular 

signals in response to diverse molecular 

inputs and can do so over a wide range of 

time scales (milliseconds to minutes). 

GPCRs are integral plasma membrane proteins 

composed of a bundle of seven hydrophobic 

membrane-spanning helices with an 

extracellular N terminus and cytosolic C terminus, 

as depicted in FIGURE 14.23. Based on the 

three-dimensional structure of rhodopsin and 

on copious biochemical and genetic data, it is 

likely that all GPCRs share the same basic mechanism 

of conformational activation and deactivation 

in response to activating ligands (see 14.5 

Ligand binding changes receptor conformation). 

Binding of agonist ligand on the extracellular 

face of the receptor drives realignment of the helices 

to alter the structure of a binding site for the 

heterotrimeric G protein on the cytoplasmic face, 

and this altered conformation of the G proteinbinding 

surface promotes G protein activation. 

FIGURE 14.22 A portion of the G protein-mediated 

signaling network in 

macrophages highlights some of the 

complexity of interactions possible in 

such systems. Several receptors and G 

protein subunits are omitted. Where a 

named G protein is shown, its signaling 

output is probably mediated by its G 

subunit. Activation of any G protein also 

activates its G subunit, although Gmediated 

signaling is usually most prominent 

from G i 

trimers. In addition, several 

G proteins modulate the activities of 

others through poorly understood pathways. 

Only a small sampling of effectors 

is shown, and the only adaptive mechanism 

shown is GRK-catalyzed phosphorylation 

of receptors. Data from Paul 

Sternweis, Alliance for Cellular Signaling. 

Agonist C5a ISO PGE S1P UDP UTP PAF LPA 

GPCR 

G Protein 

Effector 

Partial G protein signaling network in mouse macrophages 

C5aR β 2 AR E2R EDG P2YR P2YR PAFR EDG 

Gi Gs G12 

Gq 

Ad 

Cyc 

cAMP 

ATP 

PI 3- 

Kinase 

PIP 2 

PLC-β 

G12 

?? 

PIP 3 

DAG + 

IP 3 IP 2 + P i 

Phosphatase 

Inactivation 

mechanisms 

cAMP 

AMP 

PDE 

Ca2+ 

Ca2+ 

pump 

IP 3 R 

GRK 



Structure of rhodopsin 

Heterotrimeric G protein structure 

CYTOPLASM 

MEMBRANE 

Retinal 

FIGURE 14.23 The figure shows the crystal structure 

of the GPCR rhodopsin. Each membrane-spanning helix 

is a different color; most structures on the cytoplasmic 

face are not shown. The retinal chromophore is 

shown within the helix bundle. GPCR sequence similarity 

separates the mammalian GPCRs into at least four 

structural families that are so diverse that there may 

be little sequence similarity among the classes. Within 

a family, similarity is greatest in the membrane-spanning 

helices, less in the interhelical loops, and least in 

the N- and C-terminal domains and in the cytoplasmic 

loop that connects spans five and six. Regardless, the 

generalizations about functional domains in receptors 

seem to hold true within different families. GPCRs frequently 

form dimers, occasionally heterodimers, and 

dimerization can be crucial for function. Structure generated 

from Protein Data Bank file 1F88. 

The heterotrimeric G proteins to which 

GPCRs are coupled are composed of a nucleotide-binding 

Gα subunit and a Gβγ subunit 

dimer, as illustrated in FIGURE 14.24. The structure 

of the trimer and each subunit is known for 

several states of activation and in complex with 

several interacting proteins. A Gαβγ heterotrimer 

is named according to its α subunit, which largely 

defines the G protein’s selectivity among receptors. 

Each subunit also regulates a distinct group 

of effector proteins. 

Gα subunits are globular, two-domain proteins 

of 38-44 kDa. The GTP-binding domain 

belongs to the GTP-binding protein superfamily 

that includes the small, monomeric G proteins 

(such as Ras, Rho, Arf, Rab; see 14.23 Small, 

monomeric GTP-binding proteins are multiuse 

switches) as well as the GTP-binding translational 

initiation and elongation factors. A second domain 

modulates GTP binding and hydrolysis. 

Gα subunits are only slightly hydrophobic, but 

they are predominantly membrane-associated 

FIGURE 14.24 The structure of the nonactivated G i 

heterotrimer, 

the G protein that is responsible for inhibition of 

adenylyl cyclase and for most G-mediated signaling, is 

shown with each subunit colored as shown. GDP is shown 

bound to the G i 

subunit. Structure generated from Protein 

Data Bank file 1GP2. 

G 

protein 

Gs 

Golf 

Gi (3) 

Go 

Gz 

Ggus 

Gt (2) 

Gq (4) 

G 12 

G 13 

Adenylyl cyclase 

EFFECTOR PROTEIN 

K+channel, PI 3-kinase 

Other cation channel 

Rho GEF 

G protein targets 

Stimulated 

Cyclic GMP phosphodiesterase 

Phospholipase-Cβ 

Inhibited 

Adenylyl cyclase 

FIGURE 14.25 G protein-regulated effectors do not share structural 

similarities. They may be ion channels or membrane spanning 

enzymes in the plasma membrane, peripheral proteins on the 

inner face of the membrane, or fundamentally soluble proteins 

that can bind to G subunits. The chart shows the major groups 

of G proteins, sorted according to sequence similarity, and some 

of the effectors that they are known to regulate. 

because of constitutive N-terminal fatty acylation 

and because they bind to the membraneattached 

Gβγ subunits. Mammals have 16 Gα 

genes that are grouped in subfamilies according 

to similar sequence and function (e.g., s, i, q, 

and 12). These subfamilies are listed in FIGURE 

14.25. 

Gβ and Gγ subunits associate irreversibly 

soon after translation to form stable Gβγ dimers, 

which then associate reversibly with a Gα. Gβ 

subunits are 35 kDa proteins composed of seven 

14.20 G protein signaling modules are widely used and highly adaptable 617


-strand repeats that form a cylindrical structure 

known as a propeller. There are five Gβ genes 

in mammals. Four encode strikingly similar proteins 

that naturally dimerize with the twelve Gγ 

subunits (Figure 14.24). The fifth, Gβ5, is less 

closely related to the others and interacts primarily 

with a Gγ-like domain in other proteins rather 

than with Gγ subunits themselves. 

Gγ subunits are smaller (~7 kDa) and far 

more diverse in sequence than are the Gβ’s. The 

last three amino acid residues of Gγ subunits 

are proteolyzed to leave a conserved C-terminal 

cysteine that is irreversibly S-prenylated 

and carboxymethylated, helping to anchor Gβγ 

to the membrane. Gβ and Gγ subunits can associate 

in most possible combinations. Because 

almost all cells express multiple Gβ and Gγ subunits, 

it has been difficult to assign specific roles 

to individual Gβγ combinations. The best recognized 

interactions of Gβγ subunits occur at 

sites on Gβ, although distinct functions of Gγ 

have also been supported. 

14.21 

Heterotrimeric G proteins 

regulate a wide variety of 

effectors 

Key concepts 

• G proteins convey signals by regulating the 

activities of multiple intracellular signaling 

proteins known as effectors. 

• Effectors are structurally and functionally diverse. 

• A common G-protein binding domain has not been 

identified among effector proteins. 

• Effector proteins integrate signals from multiple G 

protein pathways. 

G protein-regulated effectors include enzymes 

that create or destroy intracellular second 

messengers (adenylyl cyclase, cyclic GMP phosphodiesterase, 

phospholipase C-β, phosphatidylinositol-3-kinase), 

protein kinases, ion channels 

(K+, Ca2+) and possibly membrane transport 

proteins (see Figure 14.25). Effectors may be 

integral membrane proteins or intrinsically soluble 

proteins that bind G proteins at the membrane 

surface. No conserved G protein-binding 

domain or sequence motif has been identified 

among effector proteins, and most effectors are 

related to proteins that have similar functions 

but that are not regulated by G proteins. 

Sensitivity to G protein regulation, thus, evolved 

independently in multiple families of regulatory 

proteins. 

Because they can respond to a variety of 

Gα and Gβγ subunits, effector proteins can integrate 

signals from multiple G protein pathways. 

The different Gα or Gβγ subunits may 

have opposite or synergistic effects on a given 

effector. For example, some of the membranebound 

adenylyl cyclases in mammals are stimulated 

by Gα s 

and inhibited by Gα i 

(see Figure 

14.13). Many effectors are further regulated by 

other allosteric ligands (e.g., lipids, calmodulin) 

and by phosphorylation, contributing even more 

to integration of information. 

Effectors are usually represented as multiple 

isoforms, and each isoform may be regulated 

differently, adding to the complexity of G 

protein networks. For example, some isoforms 

of adenylyl cyclase are stimulated by Gβγ, 

whereas others are inhibited. All phospholipase 

C-βs are stimulated both by Gα q 

family members 

and by Gβγ, but the potency and maximal 

effect of these two inputs vary dramatically 

among the four PLC-β isoforms. 

14.22 

Heterotrimeric G proteins 

are controlled by a 

regulatory GTPase cycle 

Key concepts 

• Heterotrimeric G proteins are activated when the 

Gα subunit binds GTP. 

• GTP hydrolysis to GDP inactivates the G protein. 

• GTP hydrolysis is slow, but is accelerated by 

proteins called GAPs. 

• Receptors promote activation by allowing GDP 

dissociation and GTP association; spontaneous 

exchange is very slow. 

• RGS proteins and phospholipase C-βs are GAPs for 

G proteins. 

The key event in heterotrimeric G protein signaling 

is the binding of GTP to the Gα subunit. 

GTP binding activates the Gα subunit, which 

allows both it and the Gβγ subunit to bind and 

regulate effectors. The Gα subunit remains active 

as long as GTP is bound, but Gα also has 

GTPase activity and hydrolyzes bound GTP to 

GDP. Gα-GDP is inactive. G proteins thus traverse 

a GTPase cycle of GTP binding/activation and hydrolysis/deactivation, 

as depicted in FIGURE 14.26. 

Therefore, the control of G protein signaling is 

intrinsically kinetic. The relative signal strength, 

or amplitude, is proportional to the fraction of 

G protein that is in the active, GTP-bound form. 

This fraction equals the balance of the rates of 

GTP binding and GTP hydrolysis, the activating 



and deactivating arms of the GTPase cycle. Both 

limbs are highly regulated over a range of rates 

greater than 1000-fold. 

Receptors promote G protein activation by 

opening the nucleotide-binding site on the G 

protein, thus accelerating both GDP dissociation 

and GTP association. This process is referred to 

as GDP/GTP exchange catalysis. Exchange proceeds 

in the direction of activation because the 

affinity of G proteins for GTP is much higher than 

that for GDP and because the cytosolic concentration 

of GTP is about 20-fold higher than that 

of GDP. Spontaneous GDP/GTP exchange is very 

slow for most G proteins (many minutes), which 

maintains basal signal output at a low level. In 

contrast, receptor-catalyzed exchange can take 

place in a few tens of milliseconds, which allows 

rapid responses in cells such as visual photoreceptors, 

other neurons, or muscle. 

Because receptors are not directly required 

for a G protein’s signaling activity, a receptor can 

dissociate after GDP/GTP exchange and catalyze 

the activation of additional G protein molecules. 

In this way, a single receptor may maintain the 

activation of multiple G proteins, providing molecular 

amplification of the incoming signal. 

Other receptors may remain bound to their G 

protein targets, which means that they do not 

act as amplifiers. However, more tightly bound 

receptors can initiate signaling more quickly and 

promote G protein reactivation when hydrolysis 

of bound GTP is rapid. 

In the absence of stimulus, Gα subunits 

hydrolyze bound GTP slowly. The average activation 

lifetime of the Gα-GTP complex is 

about 10-150 seconds, depending on the G 

protein. This rate is far slower than rates of deactivation 

often observed in cells when an agonist 

is removed. For example, visual signaling 

terminates in about 10 ms after stimulation by 

a photon, and many other G protein systems 

are almost as fast. GTP hydrolysis is accelerated 

by GTPase-activating proteins (GAPs), 

which directly bind Gα subunits. In some cases 

acceleration exceeds 2000-fold. Such speed is 

necessary in systems like vision or neurotransmission, 

which must respond to quickly changing 

stimuli. Because G protein signaling is a 

balance of activation and deactivation, GAPs 

deplete the pool of GTP-activated G protein 

and can thereby also act to inhibit G protein 

signaling. GAPs can thus inhibit signaling, 

quench output upon signal termination, or 

both. What behavior predominates depends 

on the GAP’s intrinsic activity and its regulation. 

Receptor 

+ agonist 

GDP 

G protein-GDP 

Pi 

The regulatory GTPase cycle 

Receptor 

- agonist 

G protein 

GAP 

GTP 

G protein-GTP 

*ACTIVE* 

Effector protein 

There are two families of GAPs for heterotrimeric 

G proteins. The RGS proteins (regulators 

of G protein signaling) are a family of 

about 30 proteins, most or all of which have 

GAP activity and regulate G protein signaling 

rates and amplitudes. The role of RGS proteins 

in terminating the G protein signal can be seen 

in FIGURE 14.27. Some proteins with RGS domains 

also act as G protein-regulated effectors. 

These include activators of the Rho family of 

monomeric GTP-binding proteins (see below) 

and GPCR kinases, which are feedback regulators 

of GPCR function. The second group of G 

protein GAPs are phospholipase C-βs. These enzymes 

are effectors that are stimulated by both 

Gα q 

and by Gβγ, but they also act as G q 

GAPs, 

probably to control output kinetics. 

G protein-GTP- 

Effector protein 

*ACTIVE* 

FIGURE 14.26 G proteins are activated when GTP binds to the G subunit, such 

that both G-GTP and G can bind and regulate the activities of appropriate 

effector proteins. G subunits also have intrinsic GTPase activities, and the primary 

deactivating reaction is hydrolysis of bound GTP to GDP (rather than GTP 

dissociation). Thus, the steady-state signal output from a receptor-G protein 

module is the fraction of the G protein in the GTP-bound state, which reflects 

the balance of the activation and deactivation rates. Both GTP binding and GTP 

hydrolysis are intrinsically slow and highly regulated. GDP binds tightly to G, 

such that GDP dissociation is rate-limiting for binding of a new molecule of 

GTP and consequent reactivation. Both GDP release and GTP binding are catalyzed 

by GPCRs. Hydrolysis of bound GTP is accelerated by GTPase-activating 

proteins (GAPs). Receptors and GAPs coordinately control both the steady-state 

level of signal output and the rates of activation and deactivation of the module. 

14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle 619


Single photon responses of GAP-deficient mice 

Current (pA) 

0.50 

0.25 

0.00 

knockout 

heterozygous 

wild-type 

0 2 Time (s) 4 

Light flash 

FIGURE 14.27 G protein GAPs can accelerate signal termination 

upon removal of agonist, and often do not act as inhibitors 

during the response to receptor. The figure shows 

the electrical response of a mouse photoreceptor (rod) cell 

to a single photon of light. In mice that lack RGS9, the GAP 

for the photoreceptor G protein G t 

, the signal is prolonged 

for many seconds because hydrolysis of GTP bound to G t 

is 

slow. In wild-type or heterozygous mice, hydrolysis takes 

place in about 15 milliseconds, and the decay of the signal 

is much faster. Note that the maximal output is similar in 

wild-type and mutant mice, indicating that the GAP does 

not act as an inhibitor in rod cells. In humans, genetic loss 

of RGS9 leads to severe loss of vision that is particularly 

marked in bright light. Reproduced from Chen et al. Nature. 

2000. 403:557–560. Permission also granted by Ching-Kang 

Jason Chen, Virginia Commonwealth University. 

While the GTPase cycle described in Figure 

14.26 is general, it is highly simplified. Interactions 

among receptor, Gα, Gβγ, GAP, and effector are 

frequently simultaneous and often demonstrate 

complex cooperative interactions. For example, 

Gβγ inhibits the release of GDP (to minimize 

spontaneous activation), promotes the exchange 

catalyst activity of the receptor, inhibits GAP activity, 

and helps initiate receptor phosphorylation 

that leads to desensitization. The other 

components can be nearly this multifunctional. 

In addition, inputs from other proteins can alter 

the dynamics of the GTPase cycle at several points. 

The core G protein module is, thus, functionally 

versatile as a signal processor in addition to being 

versatile in the scope of its targets. 

14.23 

Small, monomeric GTPbinding 

proteins are 

multiuse switches 

Key concepts 

• Small GTP-binding proteins are active when bound 

to GTP and inactive when bound to GDP. 

• GDP/GTP exchange catalysts known as GEFs 

(guanine nucleotide exchange factors) promote 

activation. 

• GAPs accelerate hydrolysis and deactivation. 

• GDP dissociation inhibitors (GDIs) slow 

spontaneous nucleotide exchange. 

Monomeric GTP-binding proteins, which are 

encoded by about 150 genes in animals, modulate 

a wide variety of cellular processes including 

signal transduction, organellar trafficking, 

intra-organellar transport, cytoskeletal assembly, 

and morphogenesis. The small GTP-binding 

proteins that most clearly function in signal 

transduction are the Ras and Ras-related proteins 

(Ral, Rap) and the Rho/Rac/Cdc42 proteins, 

about 10-15 in all. They are usually about 

20-25 kDa in size and are homologous to the 

GTP-binding domains of Gα subunits. 

The regulatory activities of the small GTPbinding 

proteins are controlled by a GTP binding 

and hydrolysis cycle like that of the 

heterotrimeric G proteins, with similar regulatory 

inputs. They are activated by GTP, and hydrolysis 

of bound GTP to GDP terminates 

activation. GDP/GTP exchange catalysts, known 

as GEFs (guanine nucleotide exchange factors, 

functionally analogous to GPCRs) promote activation, 

and GAPs accelerate hydrolysis and 

consequent deactivation. In addition, GDP dissociation 

inhibitors (GDIs) slow spontaneous 

nucleotide exchange and activation to dampen 

basal activity, an activity shared by Gβγ subunits 

for the heterotrimeric G proteins. 

While the underlying biochemical regulatory 

events are essentially identical for 

monomeric and heterotrimeric G proteins, 

monomeric G proteins use the basic GTPase cycle 

in additional ways. Signal output by heterotrimeric 

G proteins and many monomeric 

G proteins is usually thought to reflect a balance 

of their active (GTP-bound) and inactive 

(GDP-bound) states in a rapidly turning-over 

GTPase cycle. GEFs favor formation of more active 

G protein, and GAPs favor the inactive state. 

In contrast, probably an equal number of the 

monomeric G proteins behave as acute on-off 

switches. Upon binding GTP, they initiate a 

process (regulation, recruitment of other pro- 



teins). They then maintain this activity, sometimes 

for many seconds or minutes, until they 

are acted upon by a GAP. For example, the 

monomeric G protein Ran regulates nucleocytoplasmic 

trafficking of protein and RNA in both 

directions, cooperating with carrier proteins 

known as karyopherins (see 5.15 The Ran GTPase 

controls the direction of nuclear transport). In the 

nucleus, high Ran GEF activity promotes GTP 

binding. Nuclear Ran-GTP then binds import 

karyopherins to drive dissociation of newly arrived 

cargo and promote return of the karyopherin 

to the cytoplasm. It also binds export 

karyopherins to permit binding of outgoing 

cargo. Outside the nucleus, high Ran GAP activity 

promotes GTP hydrolysis. Cytoplasmic 

Ran-GDP dissociates from both the export karyopherins 

to allow dissociation of outgoing cargo 

and from the import karyopherins to allow them 

to bind cargo for import. Thus, for monomeric 

G proteins such as Ran, each phase of the GTPase 

cycle determines a specific, coupled step in a 

parallel regulatory cycle. 

A second major difference between the 

monomeric and the heterotrimeric G proteins 

is the structures of the GEFs, GAPs, and GDIs. 

Both GEFs and GAPs for monomeric GTP-binding 

proteins are structurally heterogeneous (although 

some clearly related families are 

evident). In addition, mechanisms for regulating 

these GEFs and GAPs are equally diverse. 

They include phosphorylation by protein kinases; 

allosteric regulation by heterotrimeric 

and/or monomeric G proteins, by second messengers 

and by other regulatory proteins; subcellular 

sequestration or recruitment to scaffolds; 

and assorted other mechanisms. 

The Ras proteins were the first small GTPbinding 

proteins to be discovered. They were 

identified as oncogene products because they 

cause malignant growth if they are either overexpressed 

or persistently activated by mutation; 

they are among the most commonly mutated 

genes in human tumors. Several viral ras genes 

figure prominently as oncogenes. 

Mammalian cells contain three ras genes (H, 

N, and K). They may share inputs and outputs 

to varying extents, and they can compensate for 

each other in some genetic screens. It has been 

difficult to assign unique functions to the individual 

Ras proteins. Inputs to the Ras proteins 

are diverse and speak to the importance of Ras 

proteins as a crucial node in signaling. 

Ras GEFs and GAPS are regulated by both 

receptor and nonreceptor Tyr kinases through 

direct phosphorylation and by recruitment of 

the regulators to the plasma membrane. Other 

cytoplasmic serine/threonine kinases also converge 

on Ras activation. Rap1, another member 

of the Ras family, may also fit directly into 

this network because it is suspected of competing 

with Ras proteins for protein kinase targets; 

in vivo it can suppress the oncogenic activity of 

Ras. Rap1 is regulated independently, however, 

and acts on independent signaling pathways as 

well. One of its GAPs is stimulated by the G i 

class of G proteins, for example, and its several 

GEFs are stimulated by Ca2+, diacylglycerol, and 

cAMP. 

Ras proteins generally regulate cell growth, 

proliferation, and differentiation by modulating 

the activities of multiple effector proteins. 

The best known and best studied Ras effector is 

the protein kinase Raf, which initiates a MAPK 

cascade. FIGURE 14.28 shows well established Ras 

effectors. 

Rho, Rac, and Cdc42 are related monomeric 

GTP-binding proteins that are involved in generating 

signals that affect cell morphology. Each 

class of proteins regulates its own array of effectors 

and is controlled by separate groups of GEFs, 

GAPs, and GDIs. Effectors regulated by this family 

include phospholipases C and D, multiple 

protein and lipid kinases, proteins that nucleate 

or reorganize actin filaments, and components 

of the neutrophil oxygen activating 

system, among others (see 8.14 Small G proteins 

regulate actin polymerization). 

14.24 

Ras has three main effectors 

Function 

Effector Target 

Protein kinase cascade Raf MAPK 

Lipid kinase PI 3-kinase Akt 

Exchange factor RalGDS Exocyst 

Protein phosphorylation/ 

dephosphorylation is a 

major regulatory 

mechanism in the cell 

Key concepts 

• Protein kinases are a large protein family. 

• Protein kinases phosphorylate Ser and Thr, or Tyr, 

or all three. 

• Protein kinases may recognize the primary 

sequence surrounding the phosphorylation site. 

• Protein kinases may preferentially recognize 

phosphorylation sites within folded domains. 

FIGURE 14.28 Ras-GTP binds 

to many proteins. Three well 

established effectors include 

Raf, PI 3-kinase, and RalGDS. 

Activation of these effectors 

activates a MAPK pathway, increases 

PI 3-kinase activity, 

and promotes assembly of a 

protein complex involved in 

exocytosis of secretory vesicles. 

14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 621


SUBSTRATE 1 

(Protein SER) 

H 

OH 

PRODUCT 1 

(Phosphorylated protein) 

H 

N 

C 

C 

N 

C 

N 

C 

C 

N 

C 

H 

O 

CH 2 

H 

O 

H 

O 

CH 2 

H 

O 

Protein kinases are two substrate enzymes 

O - P O - 

O 

+ 

O - P O 

O 

SUBSTRATE 

(Mg 2+. 2 

ATP) 

Mg 2+ 

O - P O 

O 

O - O - 

O - P O 

O 

O - P O 

O 

CH 2 

(Mg 2+. Triphosphate) 

Ribose 

PROTEIN KINASE 

PRODUCT 

(Mg 2+. 2 

ADP) 

Mg 2+ 

O - P O 

O 

CH 2 

Adenine 

Adenine 

O 

(Mg 2+. Diphosphate) 

Rib 

FIGURE 14.29 Protein kinases transfer the -phosphoryl group from ATP to 

serine, threonine, or tyrosine residues in protein substrates. 

Protein phosphorylation is the most common 

form of regulatory posttranslational modification. 

It occurs in all organisms, and it is estimated 

that about one-third of proteins in animals are 

at some time phosphorylated. Phosphorylation 

can stimulate or inhibit the catalytic activity of 

an enzyme, the affinity with which a protein 

binds other molecules, its subcellular localization, 

its ability to be further covalently modified, 

or its stability. Single phosphorylations may cause 

500-fold or greater changes in activity, and proteins 

are often phosphorylated on multiple 

residues in complex and interacting patterns. 

Most protein phosphorylation in eukaryotes, 

and essentially all in animals, is catalyzed 

by protein kinases; dephosphorylation is catalyzed 

by phosphoprotein phosphatases. Both 

classes of enzymes are controlled by diverse 

mechanisms. In addition, proteins are often 

phosphorylated by multiple protein kinases, resulting 

in the generation of a range of activity 

states. This complexity allows inputs from different 

signaling pathways to be integrated into 

the resulting activity of the target. 

In bacteria, plants, and fungi, an additional 

protein phosphorylating system known as twocomponent 

signaling is vital. The protein kinases 

involved in two-component signaling are 

unrelated to the eukaryotic protein kinase superfamily 

and phosphorylate aspartate residues 

rather than serine, threonine, or tyrosine. 

Protein kinases transfer a phosphoryl group 

from ATP to Ser, Thr, and Tyr residues of protein 

substrates to form chemically stable phosphate 

esters, as shown in FIGURE 14.29. In 

animals, the distribution of phosphate among 

these three amino acid residues is uneven: 

~90%-95% is on Ser, 5%-8% on Thr, and less 

than 1% on Tyr residues. The human genome 

contains approximately 500 genes that encode 

protein kinases, and many protein kinase 

mRNAs undergo alternative splicing. This makes 

the protein kinase gene superfamily one of the 

largest functional gene groups. The number and 

diversity of these enzymes emphasize the great 

and varied uses of protein kinases to regulate cellular 

functions. Although some protein kinases 

have a limited tissue and/or developmental distribution, 

many are ubiquitously expressed. 

Protein kinases are grouped according to 

their residue specificity. Protein kinases that 

phosphorylate Ser will usually also recognize 

Thr, hence the name protein Ser/Thr kinase. 

Multicellular organisms have protein Tyr kinases, 

which only recognize Tyr. Dual specificity 

protein kinases can phosphorylate Ser, 

Thr, and Tyr in the appropriately restricted substrate 

conformational context and are generally 

the most selective of the protein kinases. 

The analysis of the kinomes of several organisms 

has led to a more elaborate grouping 

derived from sequence relationships, shown in 

FIGURE 14.30, that also reflects to some extent 

on regulatory mechanisms and substrate specificity. 

For example, the AGC group is named 

for its founding members, cAMP-dependent 

protein kinase (PKA), cyclic GMP-dependent 

protein kinase (PKG), Ca2+, and phospholipiddependent 

protein kinase (PKC). These protein 

kinases are regulated by second messengers and 

prefer substrates that contain basic residues near 

the phosphorylation site. 

In addition to substrate specificity for amino 

acid residues, most protein kinases are also selective 

for local sequence surrounding the substrate 

site. Screening strategies have resulted in methods 

to predict if proteins contain consensus substrate 

sites for a wide variety of protein kinases. 

Antibodies can be used to identify and roughly 

quantitate protein phosphorylation at specific 

sites in proteins. Beyond local recognition, protein 

kinases may display marked substrate selectivity 

among similar proteins based on overall 



Human kinome tree 

FIGURE 14.30 The protein kinases in 

the human genome can be grouped according 

to sequence relationships that 

reveal seven major branches. The tyrosine 

kinases are contained within one 

major branch. The others are Ser/Thrspecific 

or dual specificity, and are named 

for the best described members: AGC 

from PKA, PKG, and PKC; CAMK from the 

calcium, calmodulin-dependent kinases; 

CMGC from CDKs, MAPKs, GSK3, Clks; CK1 

from casein kinase 1; STE from Ste20, 

Ste11, and Ste7, the MAP4K, MAP3K, 

and MAP2K in the yeast mating pathway; 

and TKL, the Tyr kinase-like enzymes. 

Reproduced with permission from 

G. Manning, et al. 2002. Science. 298: 

1912-1934. © 2002 AAAS. Photo courtesy 

of Gerard Manning, Salk Institute, 

and reprinted with permission of Cell 

Signaling Technology, Inc. (www.cellsignal.com). 

three-dimensional structure, for example, or 

among proteins that have been differentially covalently 

modified by phosphorylation or ubiquitination. 

In animal cells, some protein kinases are 

hormone receptors that span the plasma membrane. 

Some protein kinase receptors are protein 

serine/threonine kinases, such as the 

transforming growth factor- (TGF-)receptor, 

but the majority are protein tyrosine kinases, including 

receptors for insulin, epidermal growth 

factor (EGF), platelet-derived growth factor 

(PDGF), and other regulators of cell growth and 

differentiation. Other protein kinases are intrinsically 

soluble intracellular enzymes, although 

they may bind to one or more organellar 

membranes. 

X-ray crystallographic structures of protein 

kinases have revealed a wealth of information 

about their mechanism of activation. The conserved 

minimum catalytic core of a protein kinase 

contains about 270 amino acids, yielding 

a minimum molecular mass of about 30,000 

Da. Within this core, there are two folded domains 

that form the active site at their interface, 

as shown in FIGURE 14.31. One or both of 

the conserved lysine (Lys) or aspartate (Asp) 

residues that are required for phosphoryl transfer 

are frequently mutated to disrupt kinase activity. 

A sequence near the active site, referred 

to as the activation loop, often undergoes a conformational 

rearrangement to generate active 

forms of the protein kinases and is the most 

common site of regulatory phosphorylation in 

14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell 623


ERK2 inactive and active conformations 

INACTIVE (ERK2) 

N terminal 

domain 

Tyr185 

Thr183 

C terminal 

domain 

ACTIVE (ERK2-P2) 

Tyr185 

Thr183 

FIGURE 14.31 The structures of unphosphorylated, inactive MAPK ERK2 and 

phosphorylated, active ERK2 are compared. ERK2 has a typical protein kinase 

structure. The smaller N-terminal domain is composed primarily of strands 

and the larger C-terminal domain is primarily -helical. The active site is formed 

at the interface of the two domains. The activation loop emerges from the active 

site and is refolded following phosphorylation of the Tyr and Thr residues, 

inducing the repositioning of active site residues. ATP (not shown) binds in 

the interior of the active site; productive binding of protein substrates to the 

surface of the C-terminal domain is also facilitated by the reorganization of 

the activation loop. Structures generated from Protein Data Bank files 1ERK 

and 2ERK. 

Sensor/ 

Histidine 

kinase 

Response 

regulator 

His 

Asp 

Ligand 

P 

ATP 

His phosphorylation 

Two-component signaling systems 

ADP 

His 

Asp 

P 

P 

Transfer of phosphate 

to Asp: response 

regulator active 

His 

Asp 

P 

P 

H 2 O 

Response regulator 

deactivated 

FIGURE 14.32 The basic two-component system is composed of a signal-activated 

histidine kinase, referred to as a sensor, and an effector protein, the response 

regulator, that is activated when it is phosphorylated on an aspartate 

residue by the sensor. The activity of the response regulator is terminated when 

the aspartyl-phosphate is hydrolyzed. 

the protein kinase family. There are unique inserts 

on the surface of protein kinases that generate 

specificity in localization, interaction with 

other regulatory molecules, and recognition of 

substrates. These landmarks allow both classification 

and genetic manipulation of protein kinases. 

Protein kinases have evolved numerous 

and diverse regulatory mechanisms to complement 

their number and multiple functions. 

These mechanisms include allosteric activation 

and inhibition by lipids, soluble small molecules 

and other proteins; activating and inhibitory 

phosphorylation and other covalent modifications, 

including proteolysis; and binding to scaffolds 

and adaptors to enhance activity or limit 

nonspecific activities. Many such inputs may 

regulate a single protein kinase in a complex 

combinatoric code. Further, multiple protein 

kinases that act sequentially, such as in a protein 

kinase cascade (see Figure 14.38), can create 

uniquely complex signaling patterns. 

14.25 

Two-component protein 

phosphorylation systems 

are signaling relays 

Key concepts 

• Two-component signaling systems are composed of 

sensor and response regulator components. 

• Upon receiving a stimulus, sensor components 

undergo autophosphorylation on a histidine (His) 

residue. 

• Transfer of the phosphate to an aspartyl residue on 

the response regulator serves to activate the 

regulator. 

Prokaryotes, plants, and fungi share an alternative 

mechanism for regulatory phosphorylation 

and dephosphorylation known as two-component 

signaling. FIGURE 14.32 shows a typical twocomponent 

system. In this system, the receptor, 

referred to as a sensor, responds to a stimulus by 

catalyzing its own phosphorylation on a His 

residue. Sensors include chemoattractant receptors 

in bacteria, a regulator of osmolarity in fungi, 

light-sensitive proteins, the receptor for the plantripening 

hormone ethylene, and other receptors 

for diverse environmental, hormonal, and metabolic 

signals. The mammalian mitochondrial dehydrogenase 

kinases are related in sequence to 

the bacterial histidine kinases, although the mammalian 

enzymes phosphorylate serine or threonine 

residues, not histidine. The phosphorylated 

sensor next transfers its covalently bound phos- 



phate to an aspartyl residue on a second protein 

known as a response regulator. Response regulators 

initiate cellular responses, usually by binding 

to other cytoplasmic proteins and allosterically 

regulating their activities. 

Although all two-component systems follow 

this same general pattern, their structures 

and precise reaction pathways vary enormously. 

Some two-component systems are composed 

of only one protein (sensor and response regulator 

in a single polypeptide chain). Others are 

composed of a sensor protein and two aspartylphosphorylated 

proteins, in which the first or 

the second may display response regulatory activity. 

Finally, two-component systems usually 

lack conventional protein phosphatases. 

Hydrolysis of the aspartyl-phosphate bond may 

be spontaneous or regulated by the response 

regulator itself. 

14.26 

Pharmacological 

inhibitors of protein 

kinases may be used to 

understand and treat 

disease 

Key concepts 

• Protein kinase inhibitors are useful both for 

signaling research and as drugs. 

• Protein kinase inhibitors usually bind in the ATP 

binding site. 

Many inhibitors have been developed for basic 

research purposes to explore the functions of 

protein kinases. The importance of these enzymes 

in disease processes has also made them 

targets of drug screening projects yielding inhibitors 

for many protein kinases. The majority 

of pharmacological inhibitors of protein 

kinases compete with ATP binding. Because of 

the huge number of ATP-binding proteins in a 

cell, there are inevitable concerns about inhibitor 

specificity not only with respect to the 

other protein kinases but also to the other proteins 

that bind nucleotides. This problem has 

been mitigated with variable success through 

chemical library screening, structure-based modification 

of lead compounds, and inhibitor testing 

against panels of protein kinases. 

Many inhibitors with actions on PKA or 

PKCs, for example, have effects on several other 

members of the AGC family. Although pharmacological 

inhibitors with effects on PKA 

abound, the most selective are derived from the 

naturally occurring small inhibitory protein 

known as PKI or the Walsh inhibitor. In vitro 

and cell-based screens have identified much 

more selective inhibitors for MAP2Ks in the 

ERK1/2 pathway. These inhibitors have fewer 

known protein kinase cross reactivities, probably 

due to the fact that they do not bind in the 

ATP site. Among inhibitors that have progressed 

in the clinic, compounds developed against the 

EGF receptor and certain other protein tyrosine 

kinases have had considerable success. 

14.27 

Phosphoprotein 

phosphatases reverse the 

actions of kinases and are 

independently regulated 

Key concepts 

• Phosphoprotein phosphatases reverse the actions 

of protein kinases. 

• Phosphoprotein phosphatases may 

dephosphorylate phosphoserine/threonine, 

phosphotyrosine, or all three. 

• Phosphoprotein phosphatase specificity is often 

achieved through the formation of specific protein 

complexes. 

Protein phosphorylation is reversed by phosphoprotein 

phosphatases. These enzymes display distinct 

specificities and modes of regulation. 

Phosphoprotein phosphatases can be considered 

in two broad groups based on their specificity and 

sequence relationships: protein-serine/threonine 

phosphatases and protein-tyrosine phosphatases. 

Most protein-serine/threonine phosphatases 

are regulated by association with other proteins. 

Targeted localization is the major determinant of 

substrate specificity. Phosphoprotein phosphatase 

1 (PP1) associates with a variety of regulatory 

subunits that specifically direct it to relevant organelles. 

One subunit (known as the G subunit), 

for example, specifies association with glycogen 

particles. The interaction with this subunit is itself 

regulated by phosphorylation. Small protein 

inhibitors can suppress PP1 activity. 

Phosphoprotein phosphatase 2A (PP2A) is 

composed of a catalytic subunit, a scaffolding 

subunit, and one of a large number of regulatory 

subunits. The regulatory subunit modulates activity 

and localization of the phosphatase. Some 

viruses alter the behavior of the cells they infect 

by interfering with phosphatase activity. For example, 

cells transformed with the SV40 virus 

express a viral protein known as small t anti- 

14.27 Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated 625


gen. Small t displaces the regulatory subunit 

from PP2A and alters the activity and the subcellular 

localization of the phosphatase. In addition, 

natural toxins such as okadaic acid, 

calyculin, and microcystin inhibit PP2A and PP1 

to varying extents both in vitro and in intact cells. 

Another major protein-serine/threonine 

phosphatase, called calcineurin (also known as 

phosphoprotein phosphatase 2B), is regulated 

by Ca2+-calmodulin (see 14.15 Ca2+ signaling 

serves diverse purposes in all eukaryotic cells) and 

plays essential roles in cardiac development and 

T cell activation, among other events. The major 

mechanism of action of the immunosuppressants 

cyclosporin and FK506 is to inhibit 

calcineurin. 

The protein tyrosine phosphatases (PTPs) 

are cysteine-dependent enzymes that utilize a 

conserved Cys-Xaa-Arg motif to hydrolyze phosphoester 

bonds in their substrates. The PTPs are 

encoded by over 100 genes in humans and are 

classified in four subfamilies: the phosphotyrosine-specific 

phosphatases, the Cdc25 phosphatases, 

the dual specificity phosphatases 

(DSPs), and the low molecular weight phosphatases. 

Thirty-eight of the PTPs are highly selective 

for phosphotyrosine residues within substrates. 

Some of the phosphotyrosine-selective 

phosphatases are transmembrane proteins, 

whereas others are membrane associated. The 

most obvious function of the PTPs is to reverse 

the functions of tyrosine kinases; however, some 

have primary functions in transducing tyrosine 

kinase signals. For example, the protein tyrosine 

phosphatase SHP2 (also known as SHPTP2), 

binds to certain tyrosine kinase receptors 

through its SH2 domain and is itself tyrosine 

phosphorylated, thereby creating a binding site 

for the SH2 domain-containing adaptor protein, 

Grb2, which leads to activation of Ras (see 

14.32 MAPKs are central to many signaling pathways). 

The Cdc25 phosphatases recognize cyclindependent 

kinase (CDK) family members as 

substrates and play a critical role in increasing 

CDK activity at key junctures of the cell cycle 

(see Figure 14.39 and 11.4 The cell cycle is a cycle 

of CDK function). Similar to the dual specificity 

kinases, the dual specificity phosphatases are 

specific for a restricted number of substrates. A 

number of DSPs dephosphorylate MAPKs; these 

DSPs are called MAP kinase phosphatases, or 

MKPs. Several of these have been implicated 

in MAPK nuclear entry and exit. Some MKPs 

are encoded by early response genes, whose 

products are active near the initiation of the cell 

cycle (see 11.7 Entry into cell cycle and S phase is 

tightly regulated). 

Substrates of other PTP family members, 

such as the tumor suppressor PTEN, include 

phosphoinositides, which are phosphorylated 

derivatives of the glycerolipid phosphatidylinositol 

that serve as second messengers 

(see 14.16 Lipids and lipid-derived compounds are 

signaling molecules). Removal of the phosphate 

group inactivates the second messenger. It remains 

unclear whether members of this group 

work exclusively on phophoinositides or also 

on protein tyrosine phosphate. 

14.28 

Covalent modification by 

ubiquitin and ubiquitinlike 

proteins is another 

way of regulating protein 

function 

Key concepts 

• Ubiquitin and related small proteins, may be 

covalently attached to other proteins as a 

targeting signal. 

• Ubiquitin is recognized by diverse ubiquitin 

binding proteins. 

• Ubiquitination can cooperate with other covalent 

modifications. 

• Ubiquitination regulates signaling in addition to 

its role in protein degradation. 

An important mechanism for control of protein 

function is through covalent modification with 

small proteins of the ubiquitin family. Ubiquitin 

is one of a family of proteins referred to as ubiquitin-like 

(Ubl) proteins. Ubiquitin itself is highly 

conserved among species, suggesting the functional 

importance of all of its 76 residues. In addition 

to the long-established role of ubiquitin 

in initiating protein degradation, ubiquitin modification 

also has a variety of functions in signal 

transduction. 

Ubl proteins are conjugated to the substrate 

protein by an isopeptide bond between an amino 

group on the substrate, usually from a Lys side 

chain, and the C-terminal Gly residue of the 

processed Ubl protein. E1, E2, and E3 proteins 

are required to catalyze conjugation to Ubl proteins 

(see Biochem 4.3 Ubiquitin attachment to substrates 

requires multiple enzymes). Several Ubl 

proteins may be attached to one substrate, often 

by serial formation of a polyubiquitin chain. 

Mono- and polyubiquitination both change the 



protein’s behavior to induce downstream signals. 

Monoubiquitination is a significant regulatory 

modification in vesicular trafficking and DNA repair. 

For example, the monoubiquitinated form 

of the FANCD2 protein becomes associated with 

the repair protein BRCA1 at sites of DNA repair. 

Modification by the Ubl protein SUMO has 

roles in nuclear transport, transcription, and 

cell cycle progression. 

Polyubiquitin chains are formed when the 

Lys residues of ubiquitin itself, particularly K48 

and K63, are ubiquitinated. Addition of polyubiquitin 

with a K48 linkage generally directs proteins 

to the proteasome for degradation, whereas 

conjugation to polyubiquitin chains with a K63 

linkage promotes signal transmission, not proteolysis. 

Protein-bound ubiquitin is recognized by 

a variety of ubiquitin binding domains, including 

UIM (ubiquitin-interacting motif), UBA (ubiquitin 

association), and certain zinc finger domains. 

Such domains have the capacity to act as receptors 

for ubiquitin within modified proteins. 

Activation of the transcription factor NF-κB 

occurs by a mechanism dependent on modification 

both by the addition of Ubl proteins and phosphorylation. 

This fascinating example of regulation 

by ubiquitin is depicted in FIGURE 14.33. Prior to 

stimulation, NF-κB is retained in the cytoplasm 

in an inactive form by binding to its inhibitor, IκB. 

Phosphorylation of IκB by the IκB kinase (IKK) 

complex promotes its recognition by a multisubunit 

E3 ligase, which directs its ubiquitination 

and subsequent proteasomal degradation. 

Destruction of IκB allows NF-κB to move to the 

nucleus to mediate changes in transcription. 

IκB can be stabilized in response to certain 

signals through covalent attachment of the Ubl, 

SUMO. Sumoylation occurs on the same Lys 

residues that must be conjugated to ubiquitin 

to achieve IκB degradation. Thus, SUMO attachment 

stabilizes IκB and attenuates NF-κB action. 

This is one of numerous examples of 

crosstalk between Ubl conjugates. 

A key regulatory event in NF-κB signaling 

is activation of the IKK complex. IKK is itself 

regulated by ubiquitination and phosphorylation. 

The cytokine interleukin-1β (IL-1) causes 

association of adaptor proteins with its receptor 

to create a receptor activation complex. The 

interleukin-1β receptor activation complex recruits 

another adaptor complex containing 

TRAF6. A phosphorylation event releases a 

TRAF6 complex from the receptor activation 

complex into the cytoplasm. 

TRAF6 contains a RING domain, and is an 

E3 ubiquitin ligase that catalyzes formation of 

Modification with Ubl proteins plays multiple roles in IL-1β signaling 

TRAF6 

TRAF6 

TRAF6 

IKK 

IL-1β 

TRAF6 

TRAF6 

K63 ubiquitination 

TRAF6 

Nemo 

TAB2 

IKK 

TAK1 

Complex formation 

ATP 

ADP 

CYTOPLASM 

ADP 

ATP 

K63 polyubiquitin chains on the protein kinase 

TAK1. Polyubiquitinated TAK1 can then recruit 

TAB2 and TAB3, which are adaptor proteins 

with conserved zinc finger domains. These particular 

zinc finger domains bind to polyubiquitinated 

TAK1 and enhance its activity. TAK1, 

thus activated, phosphorylates and activates 

IKK, which then phosphorylates IκB, targeting 

it for degradation. Thus, ubiquitin-binding domains, 

such as the TAB2 and TAB3 zinc fingers, 

may selectively recognize K63 polyubiquitin 

chains to promote signal transmission. 

Naturally occurring small molecules may 

control ubiquitin ligase activity directly. Auxin 

(indole 3-acetic acid) is a plant hormone that regulates 

development by promoting the transcription 

of a large number of genes. Rather than 

K48 ubiquitination 

Degradation 

NUCLEUS 

DNA 

FIGURE 14.33 Activation of NF-B involves steps dependent on the interaction of proteins 

attached to ubiquitin through ubiquitin-binding proteins, competition by sumoylation, 

phosphorylation, and ubiquitin-mediated protein degradation. 

14.28 Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function 627


stimulating transcription factors, however, auxin 

accelerates the degradation of several specific 

transcriptional repressors. The auxin receptor is 

in fact a ubiquitin ligase complex that targets 

the auxin-regulated transcriptional repressors 

for proteolysis. F-box proteins account for all 

of the auxin binding activity in plant extracts. 

14.29 

The Wnt pathway 

regulates cell fate during 

development and other 

processes in the adult 

Key concepts 

• Seven transmembrane-spanning receptors may 

control complex differentiation programs. 

• Wnts are lipid-modified ligands. 

• Wnts signal through multiple distinct receptors. 

• Wnts suppress degradation of -catenin, a 

multifunctional transcription factor. 

Wnt pathways function during embryonic development 

and in the adult in morphogenesis, 

body patterning, axis formation, proliferation, 

and cell motility. The classical Wnt signaling 

mechanism was uncovered largely through 

studies of Drosophila and Xenopus development, 

as well as by analyzing genetic alterations in 

cancer. 

Wnt proteins are unusual extracellular ligands. 

In addition to carbohydrate, they contain 

covalently bound palmitate that is essential for 

their biological activity. Wnts transduce signals 

by binding to multiple distinct receptors. The 

most significant are members of the Frizzled 

family of seven-transmembrane-spanning receptors. 

Wnts regulate the stability of β-catenin, 

which either is rapidly degraded or, in response 

to Wnt, is stabilized to enter the nucleus and 

induce transcription by interacting with TCF 

(T-cell factor). Genes induced include c-jun, cyclin 

D1, and many others. 

The coordinated activities of the protein kinases 

glycogen synthase kinase 3 (GSK3) and 

casein kinase 1(CK1), the scaffolding proteins 

axin and adenomatous polyposis coli (APC), and 

the protein disheveled (DSH) are key to β-catenin 

stability. In the absence of Wnt, phosphorylation 

of β-catenin by CK1 and GSK3 promotes 

its ubiquitination and subsequent destruction 

by the proteasome. Axin and APC are required 

for phosphorylation of β-catenin by GSK3. 

In contrast to most seven transmembrane- 

spanning receptors, the Frizzled family has not 

yet been shown to have significant functions 

mediated by a heterotrimeric G protein, and G 

proteins may not be central to this pathway. 

Instead a proximal step in signaling by Frizzled 

involves binding to DSH, which inactivates the 

β-catenin destruction mechanism. 

Mutations that cause changes in the 

amounts of components of the classical pathway 

are common in a wide variety of cancers. Both 

Wnts and β-catenin may be viewed as protooncogenes. 

APC is a tumor suppressor and is 

mutated in the majority of human colorectal 

cancers, for example. Either too little or too 

much axin can also disrupt Wnt signaling, and 

axin, like APC, is a tumor suppressor. 

Wnts utilize additional signaling mechanisms. 

The receptor proteins Lrp5/6 (which are 

related to the low-density lipoprotein receptor) 

are Wnt receptors and also bind axin. Wnts bind 

to tyrosine kinase receptors to influence axon 

guidance and to other proteins that inhibit their 

function. Through DSH, Wnts can regulate the 

JNK MAPK pathway and Rho family G proteins 

to control planar cell polarity. Certain Wnts increase 

intracellular calcium to activate calciumdependent 

signaling pathways. 

14.30 

Diverse signaling 

mechanisms are regulated 

by protein tyrosine kinases 

Key concepts 

• Many receptor protein tyrosine kinases are 

activated by growth factors. 

• Mutations in receptor tyrosine kinases can be 

oncogenic. 

• Ligand binding promotes receptor oligomerization 

and autophosphorylation. 

• Signaling proteins bind to the phosphotyrosine 

residues of the activated receptor. 

A large group of protein tyrosine kinases are 

receptors that span the plasma membrane and 

bind extracellular ligands, as shown in FIGURE 

14.34. The receptors are generally activated by 

growth factors whose normal physiological functions 

are to promote growth, proliferation, development, 

or maintenance of differentiated 

properties. This group includes receptors for insulin, 

epidermal growth factor (EGF), and 

platelet derived growth factor (PDGF). These 

receptors both control the activities of many 

other protein kinases of all families and directly 

regulate other classes of signaling proteins. 



Because of their physiologic roles as growth 

regulators, mutations that activate receptor tyrosine 

kinases are often oncogenic. For example, 

the oncogene erbB results from the 

mutational loss of the extracellular ligand-binding 

domain of a kinase closely related to the 

EGF receptor. This mutation causes constitutive 

activation of the protein kinase domain. 

Point mutations that affect the transmembrane 

domain can also cause oncogenic activation, as 

is found in the EGF receptor-related neu/HER2 

oncogene (see 13.8 Cell growth and proliferation 

are activated by growth factors). 

Receptor tyrosine kinases are diverse both 

in their extracellular ligand-binding domains 

and, with the exception of a conserved tyrosine 

protein kinase domain, their intracellular regulatory 

regions. These receptors usually have 

one membrane span per monomer but some, 

such as the insulin receptor, which is a disulfidebonded 

heterotetramer, have two. Ligand binding 

to receptor tyrosine kinases favors receptor 

oligomerization and enhances kinase activity 

leading to increased Tyr phosphorylation of the 

intracellular domain of the receptor and of associated 

molecules. These tyrosine-phosphorylated 

motifs create docking sites for additional 

signal transducers and adaptors. 

A comparison of the PDGF and insulin receptors 

reveals common themes and a range of 

behaviors of receptor tyrosine kinases. The two 

PDGF receptors are monomeric receptor tyrosine 

kinases. The insulin receptor exists in two 

alternatively spliced forms each of which is a 

heterotetramer of two and two subunits. In 

each case, the receptor isoforms utilize some 

unique signaling mechanisms. 

PDGF and insulin each stimulate the kinase 

activity of their receptors, causing oligomerization 

and autophosphorylation. Seven or more 

sites are phosphorylated on the PDGF receptor, 

and each phosphotyrosine residue generates a 

binding site for one or more SH2 domain-containing 

proteins as illustrated in FIGURE 14.35. The 

PDGF receptor binds PI 3-kinase, p190 Ras GAP, 

phospholipase C-, Src (which may catalyze additional 

Tyr phosphorylation of the receptor), 

and the SHP2 tyrosine phosphatase which itself 

binds the adaptor Grb2 (see 14.32 MAPKs are central 

to many signaling pathways). With the exception 

of Src, all of these proteins are also receptor 

substrates. Thus, substrates are recruited to the 

receptor as a consequence of specific interactions 

of substrate SH2 domains with receptor phosphotyrosine 

producing changes in activities and 

distributions of numerous intracellular signal 

EGF 

receptor 

CYTOPLASM 

Receptor protein tyrosine kinase families 

KINASE 

DOMAINS 

Insulin 

receptor 

Activation of the PDGF receptor leads to many outputs 

p190 

RasGAP 

PLC-γ 

PDGF 

PDGF 

receptor 

PDGF 

receptors 

Src 

p85 p110 

PI 3-kinase 

Shp2 

Kinase 

inserts 

SOS 

FGF 

receptor 

FIGURE 14.34 The monomeric tyrosine kinase receptors consist of a 

globular extracellular domain that binds ligand, a single transmembrane 

span, and a globular intracellular region containing the protein kinase 

domain. The intracellular regions contain additional sequences preceding, 

following, and, in the case of the PDGF and FGF receptor groups, 

inserted into the protein kinase domain. These regions contain sites of 

tyrosine phosphorylation-dependent interactions. The insulin receptor 

is encoded by a single gene. The precursor is proteolyzed into and 

subunits, which are disulfide bonded to each other. Disulfide bonds also 

link two subunits, yielding an obligate heterotetramer. 

Grb2 

FIGURE 14.35 PDGF binds to its receptor and induces receptor autophosphorylation. 

The autophosphorylated receptor binds target proteins 

that contain SH2 domains. 

14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases 629


transducers. This array of signaling events leads 

to increased proliferation of connective tissue 

during development and in wound healing. 

Autophosphorylation also occurs on the insulin 

receptor to stabilize the active state and to 

generate a smaller number of binding sites, as illustrated 

in FIGURE 14.36. A key event is the Tyr 

phosphorylation of insulin receptor substrate 

(IRS) proteins, notably IRS1, on as many as a 

dozen sites. IRS1 takes over interactions with several 

signaling effectors that, in the case of PDGF, 

bind directly to the receptor. Among these targets 

is PI 3-kinase which leads to activation of 

Akt-2 and several essential metabolic actions of 

insulin (see 14.16 Lipids and lipid-derived compounds 

are signaling molecules). IRS proteins are also phosphorylated 

by serine/threonine protein kinases to 

modulate their signaling capability. 

Tyr phosphorylation often enhances the enzymatic 

activity of the associated proteins. Other 

proteins gain enhanced function primarily as a 

consequence of greater proximity to targets 

achieved by binding through their SH2 domains 

to phosphotyrosine sites either on the receptors 

or IRS adaptors. The precise actions of the 

many tyrosine kinase receptors are determined 

by the overlapping sets of signal transducers 

with which they interact, as well as by detailed 

differences in amounts of signal transducers, 

adaptor accessory proteins and receptor expression 

patterns (see Figure 14.43). 

Insulin signaling through IRS1 

Insulin 

CYTOPLASM 

Insulin 

receptor 

IRS1 

PIP 2 

p85 p110 

PI 3-kinase 

PIP3 

Akt 


FIGURE 14.36 Insulin binding to its receptor causes activation of the 

receptor tyrosine protein kinase and autophosphorylation. The receptor 

kinase also phosphorylates IRS1, a large adaptor with many potential 

phosphorylation sites. IRS1 is an essential intermediate in insulin action. 

PI 3-kinase binds to IRS1 via the SH2 domain within its p85 subunit. 

Akt and PDK1 bind to PIP 3 

produced by activated PI 3-kinase so that 

PDK1 can phosphorylate and activate Akt (see Figure 14.18). 

14.31 

Src family protein kinases 

cooperate with receptor 


Key concepts 

• Src is activated by release of intrasteric inhibition. 

• Activation of Src involves liberation of modular 

binding domains for activation-dependent 

interactions. 

• Src often associates with receptors, including 

receptor tyrosine kinases. 

The first protein tyrosine kinase to be discovered 

was Src, which was identified as the transforming 

entity in the Rous sarcoma virus. Src is the 

prototype of a number of related enzymes, the 

Src family kinases. It participates in signaling 

pathways regulated by numerous cell surface 

receptors, including those that lack their own 

kinase domain (see in 14.34 Diverse receptors recruit 

protein tyrosine kinases to the plasma membrane). 

Src is bound to the plasma membrane 

via an N-terminal myristoyl group. In the inactive 

state, Src is phosphorylated on Tyr527, C- 

terminal to its catalytic domain, by CSK 

(C-terminal Src kinase). 

The structure and regulation of Src is depicted 

in FIGURE 14.37. Phosphorylation of Tyr527 

causes it to bind to its own SH2 domain. The 

SH2 and SH3 domains suppress the kinase activity 

through interactions on the surface of the 

protein. The SH3 domain binds to an SH3 binding 

site distant from the active site. Activation 

of Src by dephosphorylation of Tyr527 causes 

its SH2 to dissociate; this causes a conformational 

change in the SH3 domain to dissociate 

it from the binding site. Viral isolates of Src are 

often truncated prior to Tyr527, which increases 

their activity. 

Conformational changes in the kinase domain 

resulting from dissociation of the SH3 promote 

Src autophosphorylation on Tyr416 in its 

activation loop and further increase protein kinase 

activity. An important consequence of the 

interaction of Src with its own SH2 and SH3 

domains is that these domains cannot bind anything 

else when in the autoinhibited state; therefore, 

other interactions are promoted when the 

SH2 and SH3 domains are released from their 

associations with the Src kinase domain. The 

heterologous interactions of the SH2 and SH3 

domains contribute to Src localization and signaling. 



Structure and regulation of Src 

INACTIVE 

ACTIVE 

KINASE 

DOMAIN 

SH3 

SH2 

P 

P 

Tyr527 

FIGURE 14.37 The structures of inactive and active Src are compared. The inactive 

protein is autoinhibited by binding to its own SH2 and SH3 domains. 

The SH2 domain binds to phosphorylated Tyr527. The SH3 domain binds to a 

noncanonical SH3-binding motif on the opposite side of the kinase domain 

active site. In contrast to the steric inhibition of PKA caused by its R subunit, 

inhibition of Src by its SH2 and SH3 domains is allosteric. In the active structure 

the SH2 and SH3 domains are not bound to the kinase domain and are 

available for heterologous interactions. Structures generated from Protein Data 

Base files 1FMK and 1Y57. 

14.32 

MAPKs are central to 

many signaling pathways 

Key concepts 

• MAPKs are activated by Tyr and Thr 

phosphorylation. 

• The requirement for two phosphorylations creates 

a signaling threshold. 

• The ERK1/2 MAPK pathway is usually regulated 

through Ras. 

Mitogen-activated protein kinases (MAPKs) are 

present in all eukaryotes. They are among the 

most common multifunctional protein kinases 

mediating cellular regulatory events in response 

to many ligands and other stimuli. MAPKs are 

activated by protein kinase cascades consisting 

of at least three protein kinases acting sequentially, 

as illustrated in FIGURE 14.38. Activation 

of a MAPK is catalyzed by a MAPK kinase 

(MAP2K), which is itself activated by phosphorylation 

by a MAPK kinase kinase (MAP3K). 

MAP3Ks are activated by a variety of mechanisms 

including phosphorylation by MAP4Ks, 

oligomerization, and binding to activators such 

as small G proteins. 

MAP2Ks are activated by phosphorylation 

on two Ser/Thr residues; MAP2Ks then activate 

MAPKs by dual phosphorylation on Tyr 

and Thr residues (Figure 14.30). Each MAP2K 

phosphorylates a limited set of MAPKs and few 

or no other substrates. The great specificity of 

MAP2Ks is one means of insulating MAPKs 

from activation by inappropriate signals. Both 

Tyr and Thr phosphorylations are required for 

maximum MAPK enzymatic activity. 

Studies on the MAPK ERK2 led to an understanding 

of the events induced by phosphorylation 

that are important for increased activity. 

Conformational changes include refolding of 

the activation loop to improve substrate positioning 

and realignment of catalytic residues; 

this is most obvious in the repositioning of 

helix C, which contains a Glu involved in phosphoryl 

transfer. 

Amplification occurs moving down the cascade 

from the MAP3K to the MAP2K step because 

the MAP2Ks are much more abundant 

than the MAP3Ks. The MAP2K to MAPK step 

may also amplify the signal if the MAPK is present 

in excess of the MAP2K. In addition, the 

phosphorylation of a MAPK by a MAP2K on a 

14.32 MAPKs are central to many signaling pathways 631


Generic 

G Protein 

small or 

heterotrimer 

MAP4K 

MAP3K 

MAP2K 

MAPK 

Major 

targets: 

Transcription 

factor 

Protein 

kinase 

Output 

S. 

cerevisiae 

Gβγ 

Ste20p 

Ste11p 

Ste7p 

Fus3p 

Ste12p 

Mating 

MAPK pathways 

Mammals 

Ras Rac/Cdc42 Rac 

? 

Ste20 Ste20 

PAK/PKC ? 

family family 

Raf 

MEK1 

MEK2 

ERK1 

ERK2 

Elk-1 

Rsk 

many 

MEK4 

MEK7 

JNK1 

JNK2 

JNK3 

c-Jun 

ATF2 

many 

MEK3 

MEK6 

p38α 

p38β 

p38γ 

p38δ 

MEF2 

MAPKAPK2 

Proliferation 

Development 

Differentiation 

(and other processes) 

MEKK2 

MEKK3 

MEK5 

ERK5 

MEF2 

FIGURE 14.38 MAPK pathways can be regulated by a diverse group of upstream 

regulatory mechanisms that often include adaptors, small G proteins, and MAP4Ks. 

These molecules impinge on the activities of MAP3Ks. MAP3Ks regulate one or 

more MAP2Ks depending on localization and scaffolding. The MAP2Ks display 

great selectivity for a single MAPK type. MAPKs have overlapping and unique 

substrates and participate in signaling cascades leading to many cellular responses. 

Rsk 

Tyr and a Thr residue creates cooperative activation 

of the MAPK; this is another mechanism, 

in addition to those described for PKA and 

calmodulin, to introduce a threshold and apparently 

cooperative behavior into the pathway 

over a narrow range of input signal. This 

multistep cascade provides multiple sites for 

modulatory inputs from other pathways. 

Stabilized interactions between components 

are also important. MAP2Ks, as well as MAPK 

substrates and MAPK phosphatases, generally 

contain a basic/hydrophobic docking motif that 

interacts with acidic residues and binds in a hydrophobic 

groove on the MAPK catalytic domain. 

Additional components including scaffolds 

are necessary for the efficient activation of 

MAPK cascades in cells and usually have additional 

functions. Several scaffolds have been 

identified that bind to two or more components 

for each of the three major MAPK cascades, the 

ERK1/2, JNK1-3, and p38 α, β, γ, and δ cascades. 

The ERK1/2 pathway is regulated by most 

cell surface receptors, including receptors that 

employ tyrosine kinases, GPCRs, and others. The 

PDGF receptor, like most receptor systems, activates 

the ERK1/2 cascade through Ras. PDGF 

stimulates autophosphorylation of its receptor 

and the subsequent association of effectors with 

its cytoplasmic domain (see 14.30 Diverse signaling 

mechanisms are regulated by protein tyrosine kinases). 

In response to PDGF, ERK1/2 promotes cell 

proliferation and differentiation by phosphorylation 

of membrane enzymes, proteins involved 

in determining cell shape and motility, and also 

by concentrating in the nucleus to phosphorylate 

regulatory factors that control transcription. 

14.33 

Cyclin-dependent protein 

kinases control the cell 

cycle 

Key concepts 

• The cell cycle is regulated by cyclin-dependent 

protein kinases (CDKs). 

• Activation of CDKs involves protein binding, 

dephosphorylation, and phosphorylation. 

Cell division is regulated positively and negatively 

by factors that stimulate proliferation and 

inputs that monitor cell state. The sum of these 

factors is integrated in the regulation of cyclindependent 

protein kinases (CDKs). CDKs are 

protein serine/threonine kinases that are major 

regulators of cell cycle progression. Most 

CDKs are regulated both by kinases and phosphatases 

and by association with other proteins 

called cyclins. Cyclins are synthesized and degraded 

every cell cycle. Because most CDKs are 

dependent upon cyclin binding for activation, 

the timing of the synthesis and degradation of 

individual cyclins determines when a CDK will 

function. The most notable noncycling member 

of the CDK family is Cdk5, which is highly expressed 

in terminally differentiated neurons. 

Cdk5 binds the non-cyclin protein p35 as its activating 

subunit. 

We will briefly examine the regulation of 

Cdc2, a major CDK in both mammals and yeast. 

The first step in regulation of Cdc2 is the association 

with cyclin. A second step required for 

activation of Cdc2 is phosphorylation of a Thr 



CDKs require cyclin binding for activation 

Tyr15 

Lys33 

Glu51 

Cdk2 

Cyclin A 

FIGURE 14.39 The view of the crystal structure of CDK2 bound to cyclin A 

shows residues in the ATP binding site. The enlargement on the right shows 

the interaction between Lys33 and Glu51, catalytic residues that interact with 

ATP to promote phosphoryl transfer. Tyr15 is phosphorylated in inactive forms 

of CDK2. A phosphoryl group on Tyr15 inhibits CDK activity by interfering with 

ATP binding. Structure generated from Protein Data Bank file 1JST. 

residue in its activation loop by another CDK 

type kinase. In spite of its association with cyclin, 

this form of Cdc2 is not yet active due to 

inhibitory phosphorylation of Tyr and Thr 

residues in the ATP binding pocket. Release of 

inhibition by dephosphorylation of the residues 

in the ATP pocket is catalyzed by the Cdc25 family 

of phosphoprotein phosphatases, resulting in 

activation of Cdc2. The proximity of the Tyr 

residue to catalytic residues is shown in FIGURE 

14.39. The complexity of activation of CDKs 

makes possible the imposition of cell cycle checkpoints. 

For more on CDKs and cyclins see 11.4 

The cell cycle is a cycle of CDK function. 

14.34 

Diverse receptors recruit 


to the plasma membrane 

Key concepts 

• Receptors that bind protein tyrosine kinases use 

combinations of effectors similar to those used by 

receptor tyrosine kinases. 

• These receptors often bind directly to transcription 

factors. 

Many receptors act through protein tyrosine 

kinases, but their cell surface receptors lack kinase 

activity. Instead, these receptors act by recruiting 

and activating protein tyrosine kinases 

at the plasma membrane. In this group of receptors 

are integrins, which are key molecules involved 

in cell adhesion, growth hormone 

receptors, and receptors that mediate inflammatory 

and immune responses. While their 

structures vary enormously, their mechanisms 

of action are related. 

Integrins are receptors whose major function 

is to attach cells to the extracellular matrix. They 

also mediate some interactions with proteins on 

other cells, as depicted in FIGURE 14.40. Ligands for 

integrins include a number of extracellular matrix 

proteins, such as fibronectin, as well as cell 

surface proteins that cooperate in cell-cell interactions. 

Integrin ligation provides cells with information 

about their environment that influences 

cell behavior. Ligation of integrins initiates signals 

that control cell programs, including cell cycle entry, 

proliferation, survival, differentiation, changes 

in cell shape, and motility, as well as fine-tuning 

responses to other ligands. For more details on integrins 

see 15.13 Most integrins are receptors for extracellular 

matrix proteins and 15.14 Integrin receptors 

participate in cell signaling. 

14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 633


Crk 

ECM 

Crk 

Src 

CAS 

CYTOPLASM 

FAK 

SOS 

Integrin signaling 

INTEGRINS 

Paxillin 

Talin 

Vinculin 

Grb2 

Actin 

filament 

ILK 

p85 PI 3- 

Kinase 

p110 

Vinculin 

Tensin 

Talin 

FIGURE 14.40 Integrins bind to an array of cytoplasmic proteins to regulate 

the cytoskeleton and intracellular signaling pathways. The associated cytoskeletal 

elements include actin filaments and focal adhesion proteins -actinin, vinculin, 

paxillin, and talin. Signaling molecules include the focal adhesion kinase 

FAK; the adaptors Cas, Crk and Grb2; Src and CSK (see 14.31 Src family protein 

kinases cooperate with receptor protein tyrosine kinases), PI 3-kinase (see 14.16 

Lipids and lipid-derived compounds are signaling molecules); and the Ras exchange 

factor SOS. Stimulation of GTP binding of Ras by SOS leads to activation 

of the MAPK pathway (see 14.32 MAPKs are central to many signaling 

pathways). 

Talin and α-actinin are among cytoskeletal 

proteins that interact directly with certain integrin 

subunits. These cytoskeletal proteins link 

integrins to complex cytoskeletal structures 

known as focal adhesions. 

Focal adhesions connect the cytoskeleton to 

signal transduction cascades that communicate 

states of cellular attachment to the regulation of 

cellular responses. Focal adhesion complexes contain 

the focal adhesion kinase FAK, which is activated 

by integrin ligation. Autophosphorylation 

of FAK recruits signaling proteins containing SH2 

domains, especially the p85 subunit of PI 3-kinase 

and Src family protein kinases. The signaling 

molecules associated with the integrin-bound 

cytoskeletal proteins, whether focal adhesions or 

other structural complexes, mediate the diverse 

actions of integrins. The association of cytoskeletal 

proteins with integrin receptors also causes 

functional changes to the receptors. 

Signals that act over a distance, such as hormones, 

can also employ nonreceptor tyrosine 

kinases to transmit their message inside a cell. 

Growth hormone (GH) is a protein hormone 

secreted by the anterior pituitary gland that regulates 

bone growth, fat metabolism, and other 

cellular growth phenomena. Absence of growth 

hormone results in short stature, whereas hypersecretion 

causes acromegaly, a form of gigantism. 

The GH receptor is a member of the 

cytokine receptor family, which includes receptors 

for prolactin, erythropoietin, leptin, and 

interleukins. All these receptors display similar 

biochemical functions, such as association with 

members of the JAK/TYK family of protein tyrosine 

kinases, but select for different but overlapping 

sets of cytoplasmic signaling proteins. 

Signal transduction by the GH receptor provides 

a model for receptors that lack enzymatic function 

and act as agonist-promoted scaffolds for 

intracellular signaling proteins. 

FIGURE 14.41 shows the structure of growth 

hormone bound to the extracellular domain of 

its receptor. The majority of binding energy 

comes from only a small number of residues in 

the binding interface. Inside the cell, signaling 

by the GH receptor depends significantly on its 

association with the cytoplasmic tyrosine protein 

kinase Janus kinase 2 (JAK2). FIGURE 14.42 

shows that JAK2 binds to a proline-rich region 

of the receptor. Ligand binding induces receptor 

dimerization, which then promotes 

activation of JAK2 through intermolecular autophosphorylation. 

GH signaling is thus mediated primarily by 

inducing Tyr phosphorylation. In addition to 

JAK2 autophosphorylation, the receptor itself 

becomes Tyr phosphorylated. As is true for receptor 

tyrosine kinases, Tyr phosphorylation of the 

growth hormone receptor creates binding sites 

for signaling proteins that contain phosphotyrosine-binding 

domains. Primary targets are transcription 

factors known as signal transducers and 

activators of transcription, or STATs. STATs contain 

SH2 domains and bind Tyr-phosphorylated 

motifs on the growth hormone receptor. While 

receptor bound, STATs are Tyr phosphorylated 

by JAK2 and then released to travel to the nucleus 

to mediate changes in transcription. 

The growth hormone receptor and the associated 

JAK2 also activate other signaling pathways. 

For example, the adaptor Shc is Tyr 

phosphorylated by JAK2. Engagement of Shc 

leads to activation of Ras and the ERK1/2 MAPK 

pathway. Adaptors specialized for insulin-signaling 

pathways, insulin receptor substrates (IRS) 

1, 2, and 3, are also growth hormone targets, perhaps 

reflecting the ability of growth hormone 

to induce certain insulin-like metabolic actions. 

Feedback circuits are also engaged during 

GH signaling. The growth hormone receptor 

complex binds the adaptor SH2-B, which has a 



Growth hormone structure 

Growth hormone signaling is transduced by JAK2 

Growth hormone 

receptor 

hGH 

Growth 

hormone 

Dimerization 

JAK2 

JAK2 

ΔΔG (kcal/mol) 

> 1.5 

0.5 to 1.5 

-0.5 to 0.5 

< -0.5 

untested 

CYTOPLASM 

JAK2s bind and 

phosphorylate 

receptor 

STAT 

STAT 

STATs bind and 

are phosphorylated 

FIGURE 14.41 Proteins often interact over a large surface area. Growth 

hormone binding to its receptor is an example of the energy of binding 

coming primarily from a small number of the contacts between 

the two proteins, creating an interaction hot spot. The complex of 

growth hormone bound to the growth hormone receptor-binding domain 

determined by crystallography has been peeled apart in this figure 

to show the binding energy associated with residues in the binding 

interface from each protein determined by mutagenesis and binding 

studies. Fewer than half of the residues in the interface contribute 

the majority of binding energy. Reproduced with permission from T. 

Clackson and J. A. Wells. 1995. Science. 267: 383–386. © AAAS. Photos 

courtesy of Tim Clackson, ARIAD Pharmaceuticals, Inc. 

NUCLEUS 

Phosphorylated 

STATs bind DNA 

FIGURE 14.42 The growth hormone receptor binds to JAK2. Many GH signals 

are mediated by Tyr phosphorylation of the receptor by JAK2, which creates 

binding sites for signaling molecules with SH2 domains, notably STATs. STATs 

then enter the nucleus to cause changes in gene transcription. 

stimulatory effect on growth hormone signaling. 

On the other hand, suppressors of cytokine 

signaling (SOCS proteins) are among the genes 

whose transcription is induced by growth hormone. 

As the name indicates, SOCS proteins inhibit 

cytokine signaling in some if not all cases by 

inhibiting the activity of JAK2. SOCS proteins 

contain an SH2 domain that facilitates their binding 

either to phosphorylated JAK2 or cytokine 

receptors. The mechanism of signaling inhibition 

may differ among SOCS proteins because 

some require the GH receptor to interfere with 

JAK2 signaling. SOCS-1, on the other hand, binds 

directly to the JAK2 activation loop and does not 

require a receptor to inhibit JAK2 activity. This 

mechanism may be particularly important in GH 

signaling because, in contrast to the ligand-induced 

down regulation mechanisms controlling 

many receptors, the GH receptor is degraded in 

a ligand-independent manner. 

Receptors for cytokines also act by recruiting 

tyrosine kinases. The cytokines—signaling 

proteins that modulate inflammation and cell 

growth and differentiation—include interleukins, 

leukemia inhibitory factor, oncostatin M, cardiotrophin-1, 

cardiotrophin-like cytokine, and 

ciliary neurotrophic factor (CNTF). Each cytokine 

binds a unique receptor, but each receptor 

binds a transmembrane protein called gp130. 

Mechanisms of signaling by gp130 involve interactions 

with tyrosine kinases of the JAK/TYK 

types and transcription factors in the STAT family. 

This mechanism is similar to those employed 

by the growth hormone receptor. 

14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 635


CD3 

Ligand 

Receptor 

Adaptor/ 

subunit 

Transducer 

Kinase 

cascade 

Transcription 

factor 

complex 

TCR 

MHC 

Antigen 

ITAM 

CD3 

Receptor signaling pathways 

Growth 

PDGF Insulin hormone IL-1β TGF-β 

PDGF 

receptor 

SHP2/Grb2 

SOS/Ras 

MAPK 

Ternary 

complex 

factors 

Insulin 

receptor 

IRS1 

PI 3-kinase 

Akt2 

FOXO 

GH 

receptor 

STATs 

T cell receptor signaling 

Lck 

After binding of the TCR 

to the MHC-antigen 

complex, Lck 

phosphorylates ITAMS 

IL-1β 

receptor 

gp130 

STATs 

TGF-β 

type II 

receptor 

Type I 

receptor 

SMADs 

FIGURE 14.43 Major signaling cascades controlled by PDGF, insulin, TGF-, IL- 

1, and growth hormone are compared. Each receptor either contains or interacts 

with a protein kinase that associates with or recruits a transducer. The 

transducer regulates downstream effectors either directly or through an intermediate 

protein kinase cascade. The effectors shown are transcriptional regulators. 

Phosphorylation by the transducer or kinase cascade activates all of the effectors 

except the FOXO proteins, which may be excluded from the nucleus by phosphorylation. 

The table only shows snapshots of much more complex signaling networks 

controlled by these ligands. Many of these and other intermediates serve 

multiple ligands. For example, IRS proteins also contribute to growth hormone 

and IL-1 signaling, and MAPK pathways are regulated by all of these ligands. 

ZAP-70 

FIGURE 14.44 The T cell receptor (TCR) is a multisubunit receptor. It is phosphorylated 

on activation motifs or ITAMs by Lck, or a related Src family protein 

kinase. The phosphorylated residues create binding sites for another tyrosine 

protein kinase ZAP-70. ZAP-70 then recruits other signaling molecules to the 

complex including phospholipase C, PI 3-kinase, and a Ras exchange factor to 

activate downstream signaling pathways. 

JAK 

JAK 

Unlike many cytokine receptors in this class, 

the CNTF receptor does not itself span the membrane. 

Instead, it is glycosyl phosphoinositol 

(GPI)-linked to the outer face of the plasma 

membrane. The GPI linkage is a covalent bond, 

and the receptor can be released into the extracellular 

fluid by a specific phospholipase. The 

freed receptor may interact with membranes of 

other cells to induce signals. 

The use of a common signal transducing 

subunit, gp130, suggests that unique mechanisms 

exist to create ligand-specific responses; 

under some circumstances competition by the 

ligand binding subunits for interaction with the 

gp130 signal transducer may influence signaling 

outcomes. FIGURE 14.43 illustrates some parallels 

in signaling pathways initiated by receptors 

with associated or intrinsic protein kinases. 

The last receptor type we will discuss takes 

the concept of the specific and common subunits 

even further. The complex multiprotein 

T cell receptor (TCR) is found uniquely on T 

lymphocytes and is responsible for the ability of 

these cells to recognize and respond to specific 

antigens. The TCR, illustrated in FIGURE 14.44, is 

composed of eight subunits that can be described 

as an assembly of four dimers, αβ, γε, δε, and ζζ. 

The specificity of antigen recognition is determined 

by the α and β subunits, which are different 

for each cell. The remaining subunits are 

invariant in TCRs. 

The CD3 complex γ, δ, and ε subunits are 

similar in sequence to one another. The ζ chain, 

unlike the other subunits, appears on certain 

other cell types and may be a component of 

other receptors, such as the Fc receptor, which 

binds a portion of certain immunoglobulins. 

A motif called the immunoreceptor tyrosine-based 

activation motif, or ITAM, which features 

closely spaced pairs of Tyr residues, is key 

to signaling by the TCR. The CD3 subunits each 

contain one ITAM and the ζ chain contains three 

ITAMs, for a total of ten motifs in each TCR. 

Engagement of the TCR causes the Src family 

kinases Lck and Fyn to phosphorylate the pairs 

of Tyr residues in the ITAMs. The ITAMs then 

bind the tandem SH2 domains of the protein tyrosine 

kinase ζ-chain-associated protein of 70 

kDa (ZAP-70), which becomes activated by Src. 

Tyr phosphorylation sites on ZAP-70 bind to 

other adaptors and signaling molecules, and Tyr 

phosphorylation by ZAP-70 activates additional 

signal transducers. The sum of these events leads 

to the downstream responses of T cells to antigen 

engagement, which include cell cycle progression 

and the elaboration of cytokines such 

as interleukin-2. 



14.35 

What’s next? 

New signaling proteins and new regulatory interactions 

seem to show up every day. The challenge 

now is to understand how cells organize 

these proteins and their individual interactions 

to create adaptable information-processing networks. 

How do cells use simple chemical reactions 

to sort and integrate multiple simultaneous 

inputs and then direct this information to diverse 

effector machinery? How do they interpret 

the inputs in the context of their growth and 

metabolic activities? In principle, three areas of 

research have to contribute to allow us to understand 

integrative cellular signaling. 

First, we need real-time, noninterfering 

biosensors to measure intracellular signaling 

reactions. Most current sensors use combinations 

of fluorescent moieties and signal-binding 

protein domains to provide fast optical 

readouts. For many pathways, several reactions 

can be monitored within cells over subsecond 

time scales. We need more, better, and faster 

sensors and sensors that can report with singlecell 

and subcellular resolution. Genetically encoded 

sensors will be complemented by synthetic 

molecules. 

Our ability to manipulate signaling networks 

is also improving dramatically but still 

falls short. We can manipulate signaling networks 

by overexpression, knockout, and knockdown 

of genes, but signaling pathways are 

wonderfully adaptive and frequently circumvent 

our best efforts to control them. We still 

need chemical regulators that can act promptly 

in cells. Structure-based design of such regulatory 

molecules will be vital. 

Last, our ability to analyze the behavior of 

signaling networks depends on our ability to 

measure and interpret signaling quantitatively. 

It is ironic but true that really complex systems 

cannot be described without explicit quantitative 

models for how they work. Computational 

modeling and simulation of signaling networks 

requires both better theoretical understanding 

of network dynamics and better algorithmic implementation. 

The goal is to understand how cells think. 

14.36 

Summary 

Signal transduction encompasses mechanisms 

used by all cells to sense and react to stimuli in 

their environment. Cells express receptors that 

recognize specific extracellular stimuli, includ- 

ing nutrients, hormones, neurotransmitters, 

and other cells. Upon receptor binding, signals 

are converted to well-defined intracellular chemical 

or physical reactions that change the activities 

and the organization of protein complexes 

within cells. The changes directed by the stimuli 

lead to altered cell behavior. The behavior of 

the cell is determined then by its intracellular 

state and the integrated information from extracellular 

stimuli so that the appropriate responses 

are achieved. 

The basic biochemical components and 

processes of signal transduction are conserved 

throughout biology. Families of proteins are 

used in a variety of ways for many different 

physiological purposes. Cells often use the same 

series of signaling proteins to regulate multiple 

processes, such as transcription, ion transport, 

locomotion, and metabolism. 

Signaling pathways are assembled into signaling 

networks to allow the cell to coordinate 

its responses to multiple inputs with its ongoing 

functions. It is now possible to discern conserved 

reaction sequences in and between 

pathways in signaling networks that are analogous 

to devices within the circuits of analog 

computers: amplifiers, logic gates, feedback and 

feed-forward controls, and memory. 

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