Signal-Transduction Pathways

Copyright © 2007 by W. H. Freeman and CompanyPal Bauer 2014/2015

„ No men is an island entire of itself; every man

Is a piece of the continent, a part of the main”

John Donne

Introduction

• Cells must respond adequately to external

stimuli to survive.

• Cells respond to stimuli via cell signaling.

• Some signal molecules enter cells; others

bind to cell-surface receptors.

Quorum sensing in bacteria

The slime mould

Dictyostelium Discoideum

The Basic Elements of Cell

Signaling Systems

• Extracellular messenger molecules

transmit messages between cells.

In autocrine signaling, the cell has receptors on its surface that respond

to the messenger

Interleukine-1, FAS-L, TNF-alfa etc.

During paracrine signaling, messenger molecules travel short distances

through extracellular space.

Somatostatine, histamine, prostaglandins, quorum sensing etc.

During endocrine signaling, messenger molecules reach their target cells

through the bloodstream.

Connexin gap junctions; ATP, GSH

Anoikis, integrins, selectins, syndecans,cadherins etc.

Chemical

Synapse -

Signal

Transduction

Extracellular vesicles, exosomes, microvesicles

Reception

Transduction

Response

Binding of epinephrine to G protein-coupled receptor (1 molecule)

Inactive G protein

Active G protein (102 molecules)

Inactive adenylyl cyclase

Active adenylyl cyclase (102)

ATP

Cyclic AMP (104)

Inactive protein kinase A

Active protein kinase A (104)

Inactive phosphorylase kinase

Active phosphorylase kinase (105)

Inactive glycogen phosphorylase

Active glycogen phosphorylase (106)

Glycogen

Glucose 1-phosphate(108 molecules)

Direct Ligand Binding Plot and the Derived Scatchard Plot

Biological Activity and Receptor

Occupancy• 50% of maximum biological

activity with ~18% of receptors occupied

• >80% of maximum biological activity with 50% of receptors occupied

• Epinephrine levels of ~10-10

M can stimulate glucogenolysis in liver cells, despite its relatively low binding affinity (Kd ~ 10-5 M)

I. The Basic Elements of Cell

Signaling Systems

• Receptors on or in target cells receive the

message.

– Some cell surface receptors generate an

intracellular second messenger through an

enzyme called an effector.

– Other surface receptors recruit proteins to

their intracellular domains.

Overview of

signaling

pathways

• Signaling pathways consist of a series

of proteins.

– Each protein in a pathway alters the

conformation of the next protein.

– Protein conformation is usually altered by

phosphorylation.

– Target proteins ultimately receive a message

to alter cell activity.

– This overall process is called signal

transduction.

A signal transduction pathway

A Survey of Extracellular

Messengers and Their Receptors

• Extracellular messengers include:

– Small molecules such as amino acids and

their derivatives (glutamate, acetylcholine,

adrenaline, dopamine, TSH).

– Gases such as NO and CO

– Steroids

– Eicosanoids, which are lipids derived from

fatty (arachidonic) acids.

– Various peptides and proteins

• Receptor types include:

– G-protein coupled receptors (GPCRs)

– Receptor protein-tyrosine kinases (RTKs)

– Ligand gated channels

– Steroid hormone receptors

– Specific receptors such as B-and T-cell

receptors

I. G Protein-Coupled Receptors

and Their Second Messengers

Signaling with G-Protein

Coupled ReceptorsReceptors (GPCRs)• integral membrane proteins with 7 transmembrane segments

• binding site for diverse ligands (hormones, odorants, tastants, light)

• >907 human GPCRs (384 olfactory receptors)

G Proteins• trimeric complexes of a, b and g subunits (20 Ga, 5 Gb, 12 Gg)

• Ga is GTPase switch protein (GDP off / GTP on)

• attached to membrane: Ga is acylated: Gg is prenylated

Effectors• adenylate cyclase, phospholipase C, phosphodiesterase, channels

• control levels of secondary messengers (cAMP, cGMP, DAG, IP3)

A GPCR and a G protein

Lipid Rafts and Signal

Transduction• microdomains on surface of

plasma membrane

• segregate proteins based on attached lipid

– acylated proteins in raft

– prenylated proteins not

• caveolin causes inward curvature forming caveolae

• localized in lipid rafts / caveolae:

– G-protein coupled receptors

– Tyr kinase receptors (some)

• not in lipid rafts:

– Ras and Gg subunit (prenylated)

Fluorescent Proteins

• Structure of green

fluorescent protein (GFP)

from jellyfish

• Chromophore is

autocatalytically formed by

cyclizing and oxidizing

SYG sequence

• Site directed mutagenesis

of GFP produced variety

of other fluorescent

proteins of different

wavelengths

Assay for Measuring Protein

Interactions• Fluorescence Resonance Energy

Transfer (FRET) uses emission of one chromophore as excitation for a second chromophore

• If proteins interact excitation of 1st

chromophore gives emission of 2nd

• Applied to signal transduction study

Mechanism of receptor-

mediated

activation/inhibition by G

proteins

G Protein-Coupled Receptors and

Their Second Messengers

• Signal Transduction by G Protein-Coupled Receptors

– Ligand binding on the extracellular domain changes the intracellular domain.

– Affinity for G proteins increases, and the receptor binds a G protein intracellularly.

– GDP is exchanged for GTP on the G protein, activating the G protein.

– One ligand-bound receptor can activate many G proteins.

G Protein-Coupled Receptors and

Their Second Messengers

• Termination of the Response

– Desensitization – by blocking active receptors

from turning on additional G proteins.

– G protein-coupled receptor kinase (GRK)

modifies GPCR via phosphorylation.

– Proteins called arrestins compete with G

proteins to bind GPCRs.

– Termination of the response is accelerated by

regulators of G protein signaling (RGSs).

G Proteins and Effector ProteinsGas activator cAMP

Gai inhibitor cAMP

Gaq phosphoinositides

G12/13 unknown

Second Messengers

*ER = endoplasmic reticulum; IP3 = inositol 1,4,5-trisphosphate;

PLC = phospholipase C; PI = phosphatidyl inositol;

DAG = diacylglycerol; PLD = phospholipase D.

G Protein-Coupled Receptors and

Their Second Messengers

• Second Messengers – cyclic AMP

– The Discovery of Cyclic AMP

• It is a second messenger, which is released into

the cytoplasm after binding of a ligand.

• Second messengers amplify the response to a

single extracellular ligand.

Formation of cAMP from ATP

Images of cAMP Transients in Cultured Aplysia Sensory Neurons.

The cell was loaded with a fluorophore that would allow for the quantification of

cAMP concentrations within the cell.

A: Free cAMP in the resting cell is < 5 X 10-8 M.

B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplasmic

cAMP to ~ 1 X 10-6 M (red), especially within fine processes with a high

surface to volume ratio. Thus, within 20 sec of stimulation, the intracellular

[cAMP] increased ~ 20-fold.

The variety of processes that can be

affected by changes in [cAMP]

G Protein-Coupled Receptors and

Their Second Messengers

• Other Aspects of cAMP Signal

Transduction Pathways

– Some PKA molecules phosphorylate nuclear

proteins.

– Phosphorylated transcription factors regulate

gene expression.

– Phosphatases halt the reaction cascade.

– cAMP is produced as long as the external

stimulus is present.

Examples of hormone-induced responses

mediated by cAMP

PKA-anchoring protein signaling

G Protein-Coupled Receptors and

Their Second Messengers

• Phosphatidylinositol-Derived Second

Messengers

– Some phospholipids of cell membranes are

converted into second messengers by

activated phospholipases.

• Phosphatidylinositol Phosphorylation

– Phosphoinositides (PI) are derivatives of

phosphatodylinositol.

Phosphatidylinositides in Signal

Transduction

Ptdlns

ATP ADP

PI-4KPtdlns4P

ATP ADP

PIP-5KPtdlns(4,5)P2

ATP

ADP

PI-3KPTEN

Pi

Ptdlns(4,3,5)P3

DAG Ins(1,4,5)P3

PLC

(PIP2)

(PIP3)

[Ca2+] ↑PKC(Active)

PKB (Active)

PI-3 Kinase Pathway

(IP3)

IP3/DAG Pathway

Activation of

Protein Kinase C

Activation of

Protein Kinase B

Cellular responses elicited by adding IP3

Phospholipid-based second messengers

G Protein-Coupled Receptors and

Their Second Messengers

• Phosphatidylinositol-specific phospholipase C-bproduces second messengers (IP3) and diacylglycerol (DAG) derived from phosphatidylinositol-inositol triphosphate

• DAG activates protein kinase C, which phosphorylates serine and threonine residues on target proteins.

• The phosphorylated phosphoinositides form protein-binding domains, which are connected to the PH domains of participating proteins

IP3-Mediated Signal Transduction

Examples of responses mediated by

Protein Kinase C

The Role of Calcium as an

Intracellular Messenger• Cytoplasmic calcium levels are determined

by events within a membrane.

– Calcium levels are low in the cytosol (100

nM) because it is pumped out into the

extracellular space and the membrane is

highly impermeable to the ion.

– Calcium channels can be transiently opened

by action potential or calcium itself (1 mM).

– Calcium binds to calcium-binding proteins

(such as calmodulin), which affects other

proteins.

Regulation of Cytosolic [Ca2+]

• IP3-gated channels in ER release Ca2+ into cytosol

• cytosolic [Ca2+] lowers affinity of gated channels for IP3

• causes oscillation in cytosolic [Ca2+]

• cytosolic [Ca2+] measured using fluorescent Ca2+-binding dye

• Time course of cytosolic [Ca2+] with α1-adrenergic receptor stimulation by epinephrine

• high sustained Ca2+ release may be toxic

Experimental demonstration of localized

release of intracellular Ca2+

Calcium wave in a starfish egg

Calcium-induced calcium release

Examples of mammalian proteins

activated by Ca2+

Calmodulin

II: Protein-Tyrosine Phosphorylation

as a Mechanism for Signal

Transduction

• Protein-tyrosine kinases phosphorylate

tyrosine residues on target proteins.

• Protein-tyrosine kinases regulate cell

growth, division, differentiation, survival,

and migration.

• Receptor protein-tyrosine kinases

(RTKs) are cell surface receptors of the

protein-tyrosine kinase family.

2) Receptor tyrosine kinase (RTK) family of receptors

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• Receptor Dimerization

– Results from ligand binding.

– Protein kinase activity is activated.

• Tyrosine kinase phosphorylates another subunit of

the receptor (autophosphorylation).

• RTKs phosphorylate tyrosines within

phosphotyrosine motifs.

Activation of Protein Tyrosine

Kinases• Activation of a Tyr kinase by phosphorylation

Steps in the

activation of

RTK

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• Phosphotyrosine-Dependent Protein-Protein Interactions

– Phosphorylated tyrosines bind effector proteins that have SH2 domains and PTB domains.

– SH2 and PTB domain proteins include:• Adaptor proteins that bind other proteins.

• Docking proteins that supply receptors with other tyrosine phosphorylation sites.

• Signaling enzymes (kinases) that lead to changes in cell.

• Transcription factors

A diversity of signaling proteins

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• The Ras-MAP Kinase Pathway

– Ras is a G protein embedded in the

membrane by a lipid group.

– Ras is active when bound to GTP and inactive

when bound to GDP.

The structure of a G protein and the

G protein cycle

GDI-guanine dissociation inhibitorinhibitor; GEF-guanine

exchange factor; GAP- GTP-ase activator protein

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• Ras-MAP kinase pathway

– Accessory proteins play a role:

• GTPase-activating proteins (GAPs) shorten the

active time of Ras.

• Guanine nucleotide-exchange factors (GEFs)

stimulate the exchange of GDP for GTP.

• Guanine nucleotide-dissociation inhibitors

(GDIs) inhibit release of GDP.

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• Ras-MAP kinase pathway (continued)

– The Ras-MAP kinase cascade is a cascade

of enzymes resulting in activation of

transcription factors.

– Adapting the MAP kinase to transmit different

types of information:

• End result differs in different cells/situations.

• Specificity of the MAP kinase response due to

differences in the types of kinases participating

and differences in spatial organization of

components.

The steps of a

generalized MAP

kinase cascade

Mammalian Ras activation

GRB2

PP

PP

PLC

PI3K

SH2 Domain

GAP

(DRK)

SOS

RasGTP

Downstream

pathways

GRB2

PP

PP

PLC

PI3K

GAP

Ca2+ signaling can be activated by RTKs via PLC g

Ca2+ signaling

GRB2

PP

PP

PLC

PI3K

GAP

Other signaling pathways

Cell survival

RTKs can activate PI3-Kinase

GRB2

PP

PP

PLC

PI3K

Src

Cell proliferation,

Gene expression, …

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction.

• Signaling by the Insulin Receptor

– Insulin regulates blood glucose levels by

increasing cellular uptake of glucose.

– The insulin receptor is a protein-tyrosine

kinase

• Autophosphorylated receptor associates with

insulin receptor substrate proteins (IRSs).

• IRSs bind proteins with SH2 domains, which

activate downstream signal molecules.

• SH2 domain-containing proteins are kinases that

phosphorylate a lipid, PI 3-kinase (PI3K).

The role of tyrosine-phosphorylated IRS in

activating a variety of signaling pathways

Protein-Tyrosine Phosphorylation as a

Mechanism for Signal Transduction

• Glucose Transport

– PKB regulates glucose uptake by GLUT4

transporters.

• GLUT4 transporters reside in intracellular

membrane vesicles.

• Vesicles fuse with the membrane in response to

ligand binding to the IR.

– Diabetes mellitus is caused by defects in

insulin signaling and Type 2 diabetes is

caused by gradual insensitivity to insulin.

Regulation of glucose uptake in muscle

and fat cells by insulin

Convergence, Divergence and Crosstalk

Among Different Signaling Pathways

• Signaling pathways can converge ,

diverge, and crosstalk as follows:

– Signals form unrelated receptors can

converge to activate a common effector.

– Identical signals can diverge to activate a

variety of effectors.

– Signals can be passed back and forth

between pathways as a result of crosstalk.

Examples of convergence, divergence, and

crosstalk among signal transduction

pathways

Convergence, Divergence and Crosstalk

Among Different Signaling Pathways

• Convergence – GPCRs, receptor tyrosine

kinases, and integrins bind to different

ligands but they all can lead to a docking

site for Gbr2.

Convergence of signals transmitted from a

GPCR, an integrin, and receptor tyrosine

kinase

Convergence, Divergence and Crosstalk

Among Different Signaling Pathways (3)

• Divergence – all of the examples of signal

transduction so far are evidence of

divergence of how a single stimulus sends

signals along a variety of different

pathways.

Convergence, Divergence and Crosstalk

Among Different Signaling Pathways (4)

• Crosstalk – more and more crosstalk is

found between signaling pathways:

– cAMP can block signals transmitted through

the MAP kinase cascade.

– Ca2+ and cAMP can influence each other’s

pathways.

An example of crosstalk between

two major signaling pathways

Intracellular Signaling Pathways activated by RTKs and GPCRs

Two Stages of Amplification

Adenylyl cyclase activity is modulated by the interplay

of stimulatory and inhibitory G proteins.

Hormone binding to β1- and α2-receptors activates

adenylyl cyclase, whereas hormone binding to α2-

receptors leads to inhibition of adenylyl cyclase.

The Role of NO as an

Intracellular Pathway• Nitric oxide (NO) is both an extracellular

and intercellular messenger with a variety

of functions.

• NO is produced by nitric oxide synthase.

– NO stimulates guanylyl cyclase, making

cGMP.

– cGMP decreases cytosolic calcium and

relaxes smooth muscle.

– NO also plays a role in male arousal.

Signal transduction by means of NO and

cGMP

Steroid Hormones: Features

• Cholesterol-derived

– Lipophilic and can enter target cell

• Cytoplasmic or nuclear receptors

(mostly)

• Activate DNA for protein synthesis

• Slower acting, longer half-life

• Examples

– Cortisol, estrogen, and testosterone

Steroid Hormones: Structure

Figure 7-6

Cholesterol is the parent compound for all steroid hormones.

modified by enzymes to make

steroid hormones such as

Estradiol (an estrogen)Aldosterone

Adrenal

cortex

Ovary

Cortisol

In ovaryIn adrenal cortex

Steroid Hormones: Action

Figure 7-7

1

Cell

membrane

Interstitial

fluid

Cytoplasmic

receptor

Endoplasmic

reticulum

Nucleus

Nuclear

receptor

DNA

Translation

Cell surface receptor

Rapid responses

Transcription

produces mRNA

Steroid

hormone

Blood

vessel

Protein

carrier

New

proteins

Steroid hormone receptors are in the

cytoplasm or nucleus.

Most hydrophobic steroids are bound to

plasma protein carriers. Only unbound

hormones can diffuse into the target cell.

Translation produces new proteins

for cell processes.

Some steroid hormones also bind to

membrane receptors that use second

messenger systems to create rapid

cellular responses.

The receptor-hormone complex binds to

DNA and activates or represses one or

more genes.

Activated genes create new mRNA that

moves back to the cytoplasm.

2a

2

5

4

3

1

2a

2

3

4

5

Steroid Receptor Structure

This superfamily of ligand-activated transcription factors is also structurally related.

Three well conserved regions:

-Hormone binding domains (HBD) in carboxyl terminus

-DNA-binding domain (DBD) 5’ to ligand binding domain

A nonconserved hypervariable region, which may contribute to transcriptional activity of receptor

DBD HBDhypervariable

Not All Intracellular Receptors are

Associated with HSPs.

HSPs bind to glucocorticoid, mineralocorticoid,

androgen, progesterone, and estrogen receptors

in absence of hormone.

However, receptors for thyroid hormone, retinoic

acid, and vitamin D are not bound to HSPs.

This second group of receptors is bound to their

hormone response element (HRE) on 5’flanking

region of target genes, but are inactive until

hormone binds to them.

Following hormone binding, intracellular

receptors act as transcription factors,

binding to hormone response elements

(HREs) on the 5’ flanking region of target

genes.

HRE target gene

5’ flanking region

Steroid Receptors bind to Hormone

Response Elements (HREs) on DNA

Steroid Receptors bind to Hormone

Response Elements (HREs) on DNA

Hormone

Progesterone,

Androgen,

Glucocorticoid,

Mineralocorticoid

Consensus HREs

AGAACAnnnTGTTCT

Estrogen

Thyroid hormone

Retinoids

Vitamin D

AGGTCAnnnTGACCT

AGGTCATGACCT

Palindromic Sequences Allow

Binding of Receptors as Dimers

5’ -AGAACAnnnTGTTCT- 3’

H HNNN

A T

C G

A T

A T

G C

A TTATA EXON 1…...

Transcription

Ligand gated ion channels

Gated by ligands present outside of the

cell

In fact they are receptors

All of them are nonselective cation

channels

Mediate effects of neurotransmitters

GPCRs that Regulate Ion Channels: Muscarinic Acetylcholine Receptor

The neurotransmitter, acetylcholine (ACH) binds to two types of receptors known as the nicotinic and muscarinic acetylcholine receptors. The nicotinic receptor is itself a ligand-gated ion channel that opens on ACH binding. This receptor is located in the neuromuscular junctions of striated muscle. The muscarinic ACH receptor, is a GPCR found in cardiac muscle cells that is coupled to an inhibitory G proteinThe binding of ACH to this receptor triggers dissociation of Gai-

GTP from Gßg, which in this case, directly binds to and opens a K+

channel. The movement of K+ down its concentration gradient to the outside of the cell, increases the positive charge outside the membrane, hyperpolarizing the cell. This results in the slowing of heart rate.

Acetylcholine Receptor

b

a

ACh

g (or e)

dACh

consists of a pentamer of

protein subunits, with two

binding sites for

acetylcholine, which,

when bound, alter the

receptor's configuration

and cause an internal

pore to open.

This pore allows Na+ ions

to flow down their

electrochemical gradient

into the cell.

Nicotinic Acetylcholine Receptor

A ligand gated ion channel

the resting (closed) ion channel to acetylcholine (ACh)

produces the excited (open) state. Longer exposure leads to

desensitization and channel closure.

Acetylcholine

binding sites

ACh

Na+, Ca2+ Continued

excitation

Desensitized

(gate closed)Excited

(gate open)

Resting

(gate closed)

Outside

Inside

ACh