Análisis Celular y Molecular de la Señalización Celular

TEMA 4
Transducción de la Señal
 SIGNAL TRANSDUCTION: THE CONCEPT

Signal Transduction:
• Cascades of molecular interactions that relay signals from
receptors to target molecules in the cell.
• Signal transduction usually involves multiple steps.
• Multistep pathways can amplify a signal:
• A few molecules can induce a large cellular response.
• Multistep pathways provide more opportunities for
coordination and regulation of the cellular response
 SIGNAL TRANSDUCTION: THE CONTEXT
Cells respond to external stimuli
Cell responses that involve no or limited new protein expression
(early responses)
 Cytoskeleton reorganization and changes in morphology
 Reorganization of cell-cell or cell-extracellular matrix contacts
 Metabolism changes (shift in energy sources)
 Cell migration (directional motility = chemotaxis)
 Secretion (= exocytosis)
 Uptake (= endocytosis)

Regulation by protein phosphorylation, degradation signals, ion

and second messenger concentration changes

Cell responses that involve major changes in protein expression

(delayed responses)
 Division
 Growth arrest
 Differentiation
 Apoptosis

Regulation by transcription factor activation

SIGNAL TRANSDUCTION: THE GENOMIC OUTPUT
Growth factor
Reception
Receptor

Phosphorylation
cascade

Transduction

CYTOPLASM

Inactive Active
transcription transcription
factor factor
P Response
DNA

Gene

NUCLEUS mRNA
INTRACELLULAR SIGNAL TRANSDUCTION: PATHWAY ORGANIZATION

• Different kinds of intracellular proteins

along a signaling pathway from cell-
surface receptor to the nucleus.

• A series of signaling proteins and small

intracellular mediators relay the
extracellular signal into the cell,
causing a change in gene expression.

• The signal is amplified, altered

(transduced), and distributed en route.

• Many of the steps can be modulated

by other extracellular and intracellular
signals, so that the final result of one
signal depends on other factors
affecting the cell.

• Ultimately, the signaling pathway

activates (or inactivates) target
proteins that alter cell behavior.
 INTRACELLULAR SIGNAL TRANSDUCTION: ASSOCIATING PROTEINS

Association of signaling components in membrane Rafts and caveolae

may enhance or insulate signalling pathways
 SIGNAL TRANSDUCTION: THE FINE TUNING OF THE RESPONSE

• Amplification of the signal:

• Leads to amplified response
• Specificity of the response:
• Ensures engagement of appropriate pathways
• Overall efficiency of response:
• Enhanced by scaffolding proteins
• Termination of the signal:
• Ligand-concentration dependent
• Signaling-activity dependent
THE FINE TUNING OF THE RESPONSE: AMPLIFICATION
 THE FINE TUNING OF THE RESPONSE: AMPLIFICATION

• Enzyme cascades
amplify cell response.
• At each step, the
number of activated
products is much
greater than in the
preceding step.
THE FINE TUNING OF THE RESPONSE: SPECIFICITY AND COORDINATION

• Different kinds of cells have different

collections of proteins
• These different proteins allow cells to detect
and respond to different signals
• Even the same signal can have different
effects in cells with different proteins and
pathways
• Pathway branching and “cross-talk” further
help the cell coordinate incoming signals
 THE FINE TUNING OF THE RESPONSE: SPECIFICITY AND COORDINATION

Signaling
molecule

Receptor

Relay
Activation
molecules
or inhibition

Response 1 Response 2 Response 3 Response 4 Response 5

Cell A. Pathway leads Cell B. Pathway branches, Cell C. Cross-talk occurs Cell D. Different receptor
to a single response. leading to two responses. between two pathways. leads to a different
response.
INTRACELLULAR SIGNAL TRANSDUCTION: SIGNAL INTEGRATION

Extracellular signals A and B

both activate a different series
of protein phosphorylation, each
of which leads to the
phosphorylation of protein Y but
at different sites on the protein.

Protein Y is activated only when

both of these sites are
phosphorylated, and therefore it
becomes active only when
signals A and B are
simultaneously present.

For this reason, integrator

proteins are sometimes called
coincidence detectors.
INTRACELLULAR SIGNAL TRANSDUCTION: PATHWAY ORGANIZATION
 COMPONENTS OF SURFACE RECEPTOR SIGNALLING PATHWAYS

1. Plasma membrane receptors

2. Cellular transducers of extracellular signals

3. Systems generating second messengers and changing metabolic environment

4. Intracellular second messengers

5. Calcium-binding proteins

6. Intracellular protein kinases

7. Transcription Factors
 EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS

Scaffolding proteins (protein scaffolds) are large

relay proteins to which other relay proteins are
attached.
 EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS

• Scaffolding proteins increase the signal

transduction efficiency by grouping different
proteins involved in the same pathway
• In some cases, scaffolding proteins may also
help to activate some of the relay proteins.
 EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS

• Scaffolding and
adaptor proteins are
multivalent.
• SH2 and PTB
domains bind
phospho-Tyrosine
• SH3 domains bind
Proline rich target
peptides
• Plextrin Homology
(PH) domains bind
PIP3

Assembly through specific protein binding modules/domains

EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS
EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS
 EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS

Specific signalling complexes can be formed using modular

interaction domains
EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS
 EFFICIENCY OF THE RESPONSE: SCAFFOLDING PROTEINS

Scaffold proteins can have at least four functions:

• Assembling components of a signalling pathway.
• Localizing components of a signalling pathway to a specific intracellular
compartment or location.
• Regulating positive or negative feedback signals
• Protecting active signalling intermediate proteins from deactivation by
phosphatases.
 THE FINE TUNING OF THE RESPONSE: EFFICIENCY BY SCAFFOLDING

Scaffolding can lead to the generation of complex signalling behaviours:

• Analogue (or graded) signalling, in which the signal output is
proportional to the input.
• Digital (or switch-like) signalling, in which signalling only occurs after a
certain threshold is reached and the output is always maximal.
• Sustained or transient signalling.
• Oscillatory signalling.
 GTP-BINDING PROTEINS OR G-PROTEINS

• G-proteins are enzymes belonging to the GTPase family that may bind guanine
nucleotides and function as molecular switches, alternating between two forms:
• GDP-bound form: Inactive form
• GTP-bound form : Active form, able to bind to other proteins
• They can be classified in two large groups:
• Heterotrimeric G-proteins:
• Also called large G proteins
• Made of three subunits (α, β, γ)
• Four types of subunits Gα : Gαs, Gαi, Gαq/11 and Gα12/13
• Monomeric G-proteins:
• Also called small G proteins
• They belong to the family of Ras oncoproteins
• Homologous to the α subunit of large G proteins
• They exert important roles in the intracellular signal transduction
• They are involved in many diseases, including: diabetes, blindness, allergy,
depression, cardiovascular disorders and some forms of cancer
GTP-BINDING PROTEINS OR G-PROTEINS
GTP-BINDING PROTEINS OR G-PROTEINS
 GTP-BINDING PROTEINS OR G-PROTEINS

Activation:
• Binding of ligand to GPCR receptor
provokes a conformational change that
is transmitted to the Gα subunit, allowing
to exchange GDP for GTP
• Gα subunit bound to GTP dissociates
from the dimer Gβγ and interacts with
the corresponding effector protein
• Both Gα-GTP and Gβγ may activate
different signal transduction pathways
while receptor gets ready for binding a
new ligand molecule

Termination:
• Gα subunit hydrolyzes GTP bound to GDP due to its intrinsic GTPase activity and
reassociates with Gβγ to start a new cycle.
• A gvroup of proteins (RBM proteins) may activate GTPase, accelelrating GTP
hydrolysis and terminating the signal transduced.
• In some cases, the effector protein that has interacted with Gα may be in turn an
activator of GTPases.
 GTP-BINDING PROTEINS OR G-PROTEINS

SPECIFIC MECHANISMS OF G-PROTEIN REGULATION:

• Gαs: Activates cAMP production
• Gαi: Inhibits cAMP production
• Gαq/11: Stimulates phospholipase Cb to produce inositol triphosphate (IP3) and
diacyl glycerol (DAG).
• Gα12/13: It is a GTPase of the Rho type that controls cytoskeleton structure.
• Gβγ: It may activate calcium channels
GTP-BINDING PROTEINS OR G-PROTEINS
 COMPONENTS OF SURFACE RECEPTOR SIGNALLING PATHWAYS

1. Plasma membrane receptors

2. Cellular transducers of extracellular signals

3. Systems generating second messengers and changing metabolic environment

4. Intracellular second messengers

5. Calcium-binding proteins

6. Intracellular protein kinases

7. Transcription Factors
 INTRACELLULAR SECOND MESSENGERS OF SIGNAL TRANSDUCTION

• The extracellular signal

molecule (ligand) that binds to
the receptor may be
considered as a pathway’s
“first messenger”
• Second messengers are
small, non-protein, water-
soluble molecules or ions that
spread throug the cell by
diffusion.
• Other remain bound to the
plasma membrane.
• Second messengers are
involved in signaling pathways
initiated by GPCRs and RTKs.
INTRACELLULAR SECOND MESSENGERS OF SIGNAL TRANSDUCTION

Intracellular Second Messengers*

Messenger Source Effect
cAMP Adenylyl cyclase Activates protein kinases
Activates protein kinases,
cGMP Guanylyl cyclase regulates ion channels,
regulates phosphodiesterases
Ion channels in ER and Activates protein kinases,
Ca2+
plasma membrane activates Ca2+-modulated proteins
IP3 PLC action on PI Activates Ca2+ channels
DAG PLC action on PI Activates protein kinase C
Membrane component and Activates Ca2+ channels,
Phosphatidic acid
product of PLD inhibits adenylyl cyclase

Ceramide PLC action on sphingomyelin Activates protein kinases

Activates guanylyl cyclase,

Nitric oxide (NO) NO synthase
relaxes smooth muscle
Cyclic ADP-ribose cADP-ribose synthase Activates Ca2+ channels
*IP3 is inositol-1,4,5-trisphosphate; PLC is phospholipase C; PLD is phospholipase D; PI is
phosphatidylinositol; DAG is diacylglycerol.
 INTRACELLULAR SECOND MESSENGERS: cAMP

• Cyclic AMP (cAMP) is one of the most widely

used second messengers
• Adenylyl cyclase, an enzyme in the plasma
membrane, converts ATP to cAMP in response to
an extracellular signal
 ADENYL CYCLASE AND CYCLIC AMP

• Adenyl cyclase is a transmembrane

protein that spans 12 times plasma
membrane
• Catalytic domain faces cytoplasm and
contains four regions from N-terminal:
• C1a, C1b, C2a y C2b.
• C1a and C2a are a dimer that contains
the catalytic site where ATP is
transformed to cAMP and
pyrophosphate
• cAMP is a second messenger that
activates a specific protein kinase
(protein kinase A, PKA).
ADENYL CYCLASE AND CYCLIC AMP
Figure 11.12
INTRACELLULAR SECOND MESSENGERS: cAMP
First messenger
(signaling molecule
such as epinephrine)
Adenylyl
cyclase
G protein

GTP
G protein-coupled
receptor
ATP
Second
cAMP messenger

Protein
kinase A

Cellular responses
 INTRACELLULAR SECOND MESSENGERS: cAMP

Table 15-1 Molecular Biology of the Cell (© Garland Science 2008)

SECOND MESSENGERS: NITRIC OXIDE
MEMBRANE PHOSPHOLIPASES AND SECOND MESSENGERS: IP3 AND DAG

• Inositol 1,4,5-triphosphate or
triphosphoinositol (InsP3 or IP3) and diacyl
glycerol are produced by hydrolysis of
phosphatidylinositol 4,5-biphosphate (PIP2)
catalyzed by phospholipase C.
• Their effect is to induce the mobilization of Ca2+
from its intracellular stores, such as the
endoplasmic reticulum.
 MEMBRANE PHOSPHOLIPASES AND SECOND MESSENGERS: IP3 AND DAG

Figure 15-37 Molecular Biology of the Cell (© Garland Science 2008)

 MEMBRANE PHOSPHOLIPASES AND SECOND MESSENGERS: IP3 AND DAG

Figure 15-38 Molecular Biology of the Cell (© Garland Science 2008)

 MEMBRANE PHOSPHOLIPASES AND SECOND MESSENGERS: IP3 AND DAG

Figure 15-39 Molecular Biology of the Cell (© Garland Science 2008)

 SECOND MESSENGERS: Ca2+

• Calcium ion (Ca2+) act as second messenger in

many pathways
• Calcium is an important second messenger
because cells can regulate its concentration
• A signal relayed by a signal transduction pathway
may trigger an increase in calcium in the cytosol
• Pathways leading to the release of calcium involve
inositol triphosphate (IP3) and diacylglycerol
(DAG) as intermediate second messengers.
 Figure 11.14-1
SECOND MESSENGERS: Ca2+
EXTRA- Signaling molecule
CELLULAR (first messenger)
FLUID
G protein

DAG
GTP
G protein-coupled PIP2
Phospholipase C
receptor
IP3
(second messenger)

IP3-gated
calcium channel

Endoplasmic Ca2
reticulum (ER)

CYTOSOL
 Figure 11.14-2
SECOND MESSENGERS: Ca2+
EXTRA- Signaling molecule
CELLULAR (first messenger)
FLUID
G protein

DAG
GTP
G protein-coupled PIP2
Phospholipase C
receptor
IP3
(second messenger)

IP3-gated
calcium channel

Endoplasmic Ca2
reticulum (ER)
Ca2
(second
CYTOSOL messenger)
 Figure 11.14-3
SECOND MESSENGERS: Ca2+
EXTRA- Signaling molecule
CELLULAR (first messenger)
FLUID
G protein

DAG
GTP
G protein-coupled PIP2
Phospholipase C
receptor
IP3
(second messenger)

IP3-gated
calcium channel

Various Cellular
Endoplasmic Ca2 proteins
reticulum (ER) responses
activated
Ca2
(second
CYTOSOL messenger)
SECOND MESSENGERS: Ca2+
 COMPONENTS OF SURFACE RECEPTOR SIGNALLING PATHWAYS

1. Plasma membrane receptors

2. Cellular transducers of extracellular signals

3. Systems generating second messengers and changing metabolic environment

4. Intracellular second messengers

5. Calcium-binding proteins

6. Intracellular protein kinases

7. Transcription Factors
 CALMODULIN: A Ca2+ BINDING PROTEIN

Figure 15-43 Molecular Biology of the Cell (© Garland Science 2008)

 CALMODULIN: A Ca2+ BINDING PROTEIN

Figure 15-44 Molecular Biology of the Cell (© Garland Science 2008)

 COMPONENTS OF SURFACE RECEPTOR SIGNALLING PATHWAYS

1. Plasma membrane receptors

2. Cellular transducers of extracellular signals

3. Systems generating second messengers and changing metabolic environment

4. Intracellular second messengers

5. Calcium-binding proteins

6. Intracellular protein kinases

7. Transcription Factors
PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS
 PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS

Figure 15-36 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)
SIGNAL TRANSDUCTION: METABOLIC EFFECTS
 PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS

Figure 15-36 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)
PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS
 PROTEINS KINASES: MORPHOLOGIC EFFECTS

Wild type (with shmoos) Fus3 formin

CONCLUSION

1 Mating Mating Shmoo projection

factor factor G protein-coupled forming
activates receptor Formin
receptor. P

Fus3
Actin
GTP P subunit
GDP
2 G protein binds GTP Phosphory-
and becomes activated. lation Formin Formin
cascade P
4 Fus3 phos-
phorylates
formin, Microfilament
Fus3 Fus3 activating it.
P
5 Formin initiates growth of
3 Phosphorylation cascade microfilaments that form
activates Fus3, which moves
the shmoo projections.
to plasma membrane.
 COMPONENTS OF SURFACE RECEPTOR SIGNALLING PATHWAYS

1. Plasma membrane receptors

2. Cellular transducers of extracellular signals

3. Systems generating second messengers and changing metabolic environment

4. Intracellular second messengers

5. Calcium-binding proteins

6. Intracellular protein kinases

7. Transcription Factors
PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS
PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS
PROTEINS KINASES: METABOLIC AND GENOMIC EFFECTS
 THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING

Inactivation mechanisms are essential for

regulation of cell signaling
THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING

• If ligand concentration falls, fewer receptors will be

bound and unbound receptors revert to an inactive state
 THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING

Connections
between
signaling
pathways
THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING
 THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING

• Cyclic AMP is short-lived.

It is rapidly hydrolyzed by
phosphodiesterases to
5’-AMP.

• Pyrophosphate is
hydrolyzed to inorganic
phosphates. This
reaction is the
thermodynamic driver for
the synthesis of cAMP.
 THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING

• Ca2+ signaling is terminated by:

• Pumping Ca2+ out of the cell.
• Pumping Ca2+ into ER and mitochondrion.
• Binding Ca2+ by specific proteins.
THE FINE TUNING OF THE RESPONSE: TERMINATION OF SIGNALLING
EXTRACELLULAR Plasma
FLUID membrane

Ca2
ATP pump
Mitochondrion

Nucleus

CYTOSOL

Ca2
pump
Endoplasmic
Ca2 reticulum
ATP pump (ER)

Key High [Ca2 ] Low [Ca2 ]

 INTRACELLULAR SIGNAL TRANSDUCTION: MOLECULAR SWITCHES

• The phosphate is added covalently to the signaling protein by a

protein kinase.
• A signaling protein is induced to exchange its bound GDP for
GTP.
 INTRACELLULAR SIGNAL TRANSDUCTION: MOLECULAR SWITCHES

Protein Phosphorylation and Dephosphorylation:

• In many pathways, the signal is transmitted by a

cascade of protein phosphorylations.
– Protein kinases transfer phosphates from ATP to
protein, a process called phosphorylation.

– Protein phosphatases remove the phosphates from

proteins, a process called dephosphorylation.

• This phosphorylation and dephosphorylation

system acts as a molecular switch, turning
activities on and off or up or down, as required.