Lect20 D103 W19 GM Posted

Lecture 20 – Signaling VI Additional key concepts and principles used in
signaling
Learning objectives : After reviewing this lecture you should be able to -

• Describe how different signaling pathways are tailored to control specific cellular functions.
• Explain the relationship between the rate of turnover of a signaling molecule and a cell’s ability to
rapidly respond to a signaling stimulus.
• Describe how allostery and cooperative binding enables regulation of protein function within small
concentration ranges of their ligands.
• Describe uses of positive and negative feedback in signaling.
• Explain the importance of protein scaffolds in producing sophistication, efficiency and specificity in
signaling.
• Describe ways in which cells can adjust their sensitivity to extracellular signals.
• Describe several ways in which signaling pathways achieve specificity in a “noisy” environment.
Recommended Reading: Alberts 6e; p 402-403; p 816; p 820 – 831
D103 - Cell Biology - lecture 20 © 2019 MacGregor & Sütterlin - all rights reserved Page 1
Health relevance of studying signaling pathways
• GPCR commonly influence physiological processes -

~ 50% drugs target GPCR signaling.
• ~ 500 protein kinases in human genome.
• ~ 30% of proteins encoded by human genome
undergo reversible phosphorylation.
• Abnormal phosphorylation is involved in a wide
range of human diseases from cancer to
cardiovascular disease.
• Understanding how signaling works is important to
define basis for diseases and to develop potential
therapies.
Signaling pathways we have studied in D103
Signaling pathways have evolved to suit the cellular function controlled by
that system
• Response timing – e.g. rapid response in synaptic signaling, versus slow response to morphogens
controlling cell fate during development.
• Sensitivity – e.g. high sensitivity associated with light involving signal amplification, versus minimal
amplification and relatively low sensitivity associated with synaptic signaling.
• Dynamic range – e.g. broad dynamic range associated with visual transduction, or relatively narrow
dynamic range associated with cellular differentiation.
• Persistence – e.g. transient response is suited to synaptic signaling while prolonged or permanent response
is required for cellular differentiation.
• Signal processing – e.g. conversion of a response to a gradual change in a concentration of a signaling
molecule from a step-like, to a binary switch-like response (often uses feedback).
• Integration – allows a response to be dependent upon multiple independent
inputs – e.g. combinations of different signals producing different outputs
such as cell survival, proliferation or differentiation. Cells use co-incidence
detectors to control decisions requiring multiple inputs (see right).
• Signaling systems rarely depend on a simple linear sequence of
steps to elicit a response and usually employ a branched network of
steps.
Extracellular signals can be amplified by intracellular mediators and
enzymatic cascades
• To allow cells to rapidly return to a

resting state, stimuli involving
intracellular amplification requires rapid
removal or inactivation of the
amplified intracellular mediators after
the stimulus ceases.
• Signaling molecules that are turned over rapidly (i.e. have a short lifetime) enable the cell
to quickly change the concentration of a molecule thereby effecting a rapid response to a
stimulus and rapid return to a resting state.
The speed of a cellular response is dependent on the rate of turnover of
signaling molecules
(lifetime of
molecule)
faster turnover of
signaling molecule
See lecture webpage

for Knowledge Nugget
that explains this
concept in detail
slower turnover of
signaling molecule
Signaling systems can respond continuously or abruptly to a gradually
increasing signal
• Smoothly graded (hyperbolic) responses over a wide range of signaling molecules are useful
in (e.g.) fine tuning of metabolic processes controlled by hormones.
• Sigmoidal responses are useful to reduce inappropriate responses to low level background
signal, with steep and continuous change at intermediate stimulus levels.
• “All or none” (binary) responses are especially useful for controlling choice between two cell
fates, and often involve positive feedback.
Allostery and cooperative binding of a ligand can produce a sigmoidal
response in protein activity over a narrow range of ligand concentration
Feedback systems play important roles in cellular signaling
• Diagram shows feedback loops functioning at level of gene expression.

• Similar feedback loops can also function at level of protein function – e.g. a kinase
activating a phosphatase, which in turn inhibits the initial kinase activity.
Positive feedback can turn a graded response to a signal into an binary
“switch-like” response.
Positive feedback can mediate a rapid

increase in activity of an enzyme above a
specific signaling threshold.
e.g. positive feedback via an enzyme’s

product on the enzyme activity
Positive feedback can be used to develop a “bi-stable” system
Bi-stable system
• Without positive feedback, activity of kinase “E” is dependent on the sustained signal kinase
activity (S).
• With positive feedback, transient stimulation by signal kinase (S) is sufficient to generate
sustained kinase E activity, even after removal of the stimulus (S).
• In this example, positive feedback allows the system to be maintained in one of two
stable states – hence the term “bi-stable”.
• e.g. use of a bi-stable switch during muscle cell differentiation allows a long-lived effect.
Negative feedback is commonly used in signaling systems
• Negative feedback counteracts the effect of a signal thereby limiting the level of a response.
• As with positive feedback, a qualitative difference in the response can be obtained dependent
on the strength of the negative feedback and delay in its activation.
• A sufficiently long delay in feedback generates an oscillatory response.
• Oscillatory responses can be interpreted by other signaling machinery depending on the frequency
and amplitude of the oscillation (see p 842 Alberts 6e).
Cells can adjust their sensitivity to a signal - examples of different ways
to adapt (desensitize) to an extracellular signal
• Adaptation allows a cell to adjust its sensitivity to a signal.

• This allows the cell to response to the same percentage of change in a signal
over a wide range of stimulus (signal) strengths.
How do signaling pathways achieve specificity in a “noisy” environment ?
• Thousands of independent signaling events occur simultaneously within the average cell.
• Many components of signaling pathways are closely related or shared.
• It is almost inevitable that signaling molecules occasionally bind to the incorrect partner
molecule, potentially generating unwanted cross-talk and interference between signaling
pathways.
• How do signaling pathways maintain specificity in such a “noisy” environment ?
+ =
simplified “cAMP pathway”, just

one of many different pathways
How signaling pathways achieve specificity in a “noisy” cytoplasmic
environment – e.g. requirement for a sustained signaling event
A feed-forward loop can detect a persistent input

signal (see also Fig 8-86 (p523) in textbook)
• Ability of signaling pathway to ignore signals that are not sustained.

• Expression of gene Z (output) requires long-term input from both gene
products A and B.
• Brief input pulse of input is either ignored or results in small output.
• Used to detect a robust and sustained signal – useful if a cell has to commit to
a ‘costly’ cellular program.
environment – affinity and specificity of signaling molecules
Multiple SH2-domain-containing proteins interact with the phosphorylated model of a SH2-domain

PDGF receptor cytoplasmic tail via different P-Tyr’s.
• Intracellular signaling complexes can form at activated receptors.

• e.g. interaction of individual proteins containing different SH2-domains with their specific
phospho-tyrosine targets.
• Molecular complementarity – binding of a signaling molecule to the correct target is determined
by precise and complex interactions between extended complementary surfaces on two
molecules.
environment – scaffolding proteins assemble groups of signaling proteins
to enhance specificity of protein interactions
• Scaffolds hold signaling proteins in proximity, effectively increasing the local concentration of the
signaling components.
• The orientation of signaling components within a scaffold also permits efficient and rapid activation
of components in a specific sequence.
• Proteins can assemble on a scaffold before (left) or after (right) the signal is received.
• Scaffold proteins can restrict a signaling event to a specific subcellular location.
environment – scaffolding proteins reduce unwanted crosstalk between
pathways
• S. cerevisae lacks cytokine receptors or

tyrosine kinase receptors.
• In S. cerevisae, GPCR can activate MAPK
pathway (via GPCR desensitization by
arrestin)
• MAPK pathway functions in five discrete
responses to environment.
• Some components (e.g. kinase A (MAPKKK))
are shared by different MAPK signaling
pathways.
• How is specificity generated if components
are shared ???
• Scaffold proteins facilitate specificity by
bringing unique combinations of signaling
molecules into proximity.
Scaffolding proteins can provide specificity of signaling, and can also
mediate negative feedback control – e.g. local concentration of a second
messenger
X
A= PKA
B = PDE
• AKAP - A-Kinase (PKA) Anchoring Protein (cell has lots of e.g. in b-adrenergic receptor mediated signaling in cardiac
these located to different organelles). muscle
• PDE - cAMP phosphodiesterase.
Note - in panel 2, catalytic subunit (C) of PKA phosphorylates other target proteins (e.g. “X” in pathway at top
right)
Local gradients of cAMP and anchored pools of PKA mediate β-adrenergic
regulation of cardiac function
• Adrenaline = ligand.
• b2-AR = receptor (GPCR).
• AC = adenylyl cyclase - GPCR effector.
• AC makes cAMP, activates PKA.
• (PDE = cAMP –> AMP).
• Ca2+ regulates contraction.
• Note how spatial organization of PKA and

PDE forms gradient of cAMP.
• Genetic polymorphism in D-AKAP2 (I646V)
in humans is associated with reduced
affinity of AKAP for PKA RIa.
• Results in changes in
electrocardiogram and cardiac
dysfunction.
TASKÉN K , and AANDAHL E M Physiol Rev 2004;84:137-167
Crosstalk between pathways facilitates sophisticated signaling
• Gene duplication and divergence produces specialized
proteins that have both shared and unique properties
within a signaling pathway (e.g. substrate specificity).
• Different combinations of the individual components
enables sophisticated signaling with small numbers of
components.
• Redundancy and overlap in signaling pathways offers
opportunities for evolution of novel signaling pathways.
• Simple linear pathways are rare. Many pathways branch

and converge, multiple times.
• Most pathways have positive or negative feedback
loops that can result in signals being only transient in
nature - challenging to study.
• Response of some pathways depends on both intensity
and duration of signal.
• Analysis requires advanced quantitative 4-D methods.
Page 20
D103 - Cell Biology - lecture 20 © 2019 MacGregor & Sütterlin - all rights reserved
Some common and important principles used in signaling
• Signaling pathways have evolved specific behavior to suit the cellular function controlled by that system
– e.g. response timing, sensitivity, dynamic range, persistence.
• Signaling systems can respond continuously or abruptly to a gradually increasing signal.
• The speed of a cellular response is dependent on the rate of turnover of signaling molecules.
• Allosteric interaction and cooperative binding allow a cell to produce graded activation of signaling
processes over a narrow concentration range – e.g. activation of (a) PKA by cAMP; (b) calmodulin by Ca 2+.
• Positive feedback can turn a graded response to a signal into a binary response.
• Positive feedback can be used to develop a bi-stable system.
• Negative feedback can limit the level and duration of a response.
• Adaptation allows cells to adjust their sensitivity to an extra-cellular signal, enabling them to respond to
the same percentage of change in a signal over a wide range of stimulus strengths.
• Cells have evolved several different methods to achieve specificity in response to signals in a “noisy”
environment.
• The subcellular location of a signaling event can be controlled by scaffolding proteins and via
modification of the lipid bilayer in specific subcellular compartments.
• Scaffold proteins allow a cell to control specificity and efficiency of subcellular signaling.
• Crosstalk between signaling pathways enhances sophistication of cellular response to individual signals.
End of lecture questions
16. Which factor(s) controls the specificity of an interaction between a specific protein containing
a SH2 domain and a phospho-tyrosine on a target protein ?

A. The amino acid sequences flanking the phosphotyrosine binding site in the SH2 domain.
B. The phosphorylation status of the SH2 domain.
C. The amino acid sequences flanking the tyrosine residue that is phosphorylated.
D. The kinase activity of the SH2 domain.
E. A and C.
17. Molecule “Y” is a second messenger, whose lifespan in cells is 10 min. Following stimulation
of a cell surface receptor, production of Y increases one hundred fold. An increase in
concentration of Y is required to mediate an decrease in the activity of enzyme “Z”. What best
describes the effect on this signaling pathway if the lifespan of Y was changed to 10 sec ?

A. Activity of Z will increase more rapidly.
B. Activity of Z will decrease more slowly.
C. Activity of Z will increase more slowly.
D. Activity of Z will decrease more rapidly.
18. Which of the following “feedback” systems would be suited to allow a cell to respond to
long-term but not short-term signals ?

A. A positive feedback loop.
B. A negative feedback loop.
C. A feed-forward loop.
D. An indirect positive feedback loop.
E. None of the above feedback systems is suited to allow a cell to respond to a long-term but
not short-term signal.
19. Which of the following describes a negative feedback effect mediated by a scaffolding
protein ?

A. The activity of phospholipase Cb on PKC.
B. The activity of Ras on MAP3K.
C. The activity of PKA on a cAMP PDE.
D. The activity of STATs on SOCS proteins.

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Lecture 20 – Signaling VI Additional key concepts and principles used in

Learning objectives : After reviewing this lecture you should be able to -

Recommended Reading: Alberts 6e; p 402-403; p 816; p 820 – 831

• GPCR commonly influence physiological processes -

Signaling pathways we have studied in D103

• To allow cells to rapidly return to a

See lecture webpage

• Diagram shows feedback loops functioning at level of gene expression.

Positive feedback can mediate a rapid

e.g. positive feedback via an enzyme’s

• Adaptation allows a cell to adjust its sensitivity to a signal.

simplified “cAMP pathway”, just

A feed-forward loop can detect a persistent input

• Ability of signaling pathway to ignore signals that are not sustained.

Multiple SH2-domain-containing proteins interact with the phosphorylated model of a SH2-domain

• Intracellular signaling complexes can form at activated receptors.

• S. cerevisae lacks cytokine receptors or

• Note how spatial organization of PKA and

TASKÉN K , and AANDAHL E M Physiol Rev 2004;84:137-167

• Simple linear pathways are rare. Many pathways branch

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