2. Convergence, Divergence and
Crosstalk
Among Different Signaling
Pathways
Herbert, Barnabas E. MB.Sc, Hse

3. Key Concept
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

4. Key Concept
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

5. CONVERGENCE
Signals from a
VARIETY OF
UNRELATED
receptors converge to
activate a COMMON
EFFECTOR after
binding to their
individual ligand.E.g
Ras Raf.

7. MAP-kinase serine/threonine
phosphorylation Pathway activated by
Ras
• Ras-
activated
phosphorylat
ion cascade

8. CONVERGENCE
• Signals usually from RECEPTORS
• Examples:
-G-protein coupled receptors
-Receptor tyrosine kinases
-Integrins

9. CONVERGENCE
Signals transmitted form a G protein-coupled receptor, an
integrin and a receptor tyrosine kinase all converge on Ras
and are then transmitted along the MAP kinase cascade.

10. CONVERGENCE
• Lead to formation of PHOSPHOTYROSINE
DOCKING sites for SH2 domain
• Lead to TRANSCRIPTION and PROMOTION of
a SIMILAR set of growth promoting genes in
target cells.
• Signals transmitted from G-protein-coupled
receptors on integrins, and a receptor tyrosine
kinase all CONVERGE on Ras/Raf and are then
transmitted along the MAP kinase cascade.
• Integrins are receptors at sites of cell-substrate
and cell-cell contact.

11. Key Concept
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

12. Key Concept
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

13. DIVERGENCE
Signals from the same ligand diverge to
activate A VARIETY OF DIFFERENT
EFFECTORS leading to diverse cellular
responses.

14. DIVERGENCE

15. DIVERGENCE
• Effects are usually LIGAND based
• Examples
-EGF ligand
-Insulin ligand

16. KEY CONCEPTS
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

17. KEY CONCEPTS
• Convergence
• Divergence
• Crosstalk
• Signaling Pathways

19. CROSS TALK
Signals are passed
BACK AND fORTH
between DIFFERENT
PATHWAYS
Example:
Cyclic Adenosine
Monophosphate
(cAMP)

20. How does cAMP block signals
transmitted through the MAP
kinase cascade?
• Achieves this by:
-activating PKA (a cAMP dependent
kinase)
-PKA phosphorylates/inhibits Raf (a
protein that leads the MAP kinase
cascade)

21. Crosstalk between 2
major signaling pathways.
cAMP acts in some cells
via cAMP-dep.kinase,
PKA, to block
transmission of signals
from Ras to Raf which
inhibits activation of MAP
kinase cascade. Also both
PKA and kinases of MAP
kinase cascade
phosphorylate
transcription factor CREB
on same serine residue,
activating transcription
factor and allowing it to
bind to specifrc sites on
the DNA.

22. CROSSTALKS
• cAMP
-Initiator of rxn cascade for CHO
mobilization
-Can also inhibit growth of variety of cells
by blocking signals transmitted through
the MAP kinase cascade.

23. • Convergence
• Divergence
• Crosstalk
• Signaling Pathways

24. • Convergence
• Divergence
• Crosstalk
• Signaling Pathways

25. SIGNALING PATHWAYS
• Provide a mechanism for
routing information through a
cell
• Comparable to the nervous
system:
-the cell receives information
about its environment
through the activation of
various surface receptors.

26. CELL SURFACE RECEPTORS
• Acts like sensors to detect extracellular
stimuli
• Can bind only to specific ligands
• Unaffected by the presence of a large
variety of UNRELATED molecules

27. Do not forget!
“A single cell may have dozens of different
receptors sending signals to the cell
interior simultaneously!”

28. What happens
when signals
are transmitted
into cells???

29. The signals are selectively routed along a
number of different signaling pathways
that may cause the cell to:
-Divide (Mitosis)
-Change shape
-Activate a specific metabolic pathway
-Apoptosis (Commit suicide)

30. CENTRAL IDEA
In this way, the cell integrates information
arriving from different sources and mounts
an appropriate and comprehensive
response

31. How are different
stimuli able to
evoke distinct
responses, even
though they utilize
similar pathways?

32. Contrasting cellular responses are due to
differences in the protein composition of
different cell types (Different cells have
different isoforms of these various
proteins)

33. A working theory, not a satisfactory
answer!

34. In actual fact, signaling pathways in the
cell are much more complex.

35. SURMARY
Signals from a variety of UNRELATED
RECEPTORS can CONVERGE to
activate a common effector, such as
Ras/Raf; signals fro the SAME LIGAND
can DIVERGE to activate a variety of
DIFFERENT EFFECTORS; and signals
can be passed BACK AND FORTH
between pathways (Cross talk).

37. • Paroxysmal vertigo
• Define the following and give
examples.
-Convergence
-Divergence
-Crosstalk
QUESTION ONE

38. What happens when signals
are transmitted into cells???
QUESTION TWO

39. INDISCRIMINATE FIRING ?

40. Concentration is the Key! Keep calm!! 

41. THE ROLE OF NITRIC OXIDE
AS AN INTRACELLULAR
MESSENGER
Herbert, Barnabas E.

42. History of Second Messengers
• Before 1980
-Organic compounds e.g cAMP
-Ions e.g Ca2+
• After 1980
-Inorganic gas -> Nitric Oxide (NO)

43. Nitric Oxide
- Formed from L-arginine (amino acid) in a
rxn catalyzed by the enz Nitric Oxide
Synthase (NOS)
- Discovered as second messenger by
accidental observation

44. Flash Back: Acetylcholine
- Known to act in the body to relax smooth
muscle cells of blood vessels.
- Response could not be duplicated in vitro
- Binds to receptors on the surface of
endothelial cells
- Leads to the production and release of an
AGENT that diffuse through the cell’s
plasma membrane

45. Acetylcholine
- Causes the muscle cells to relax
- The AGENT was later discovered to be
Nitric Oxide (NO)

46. NITRIC OXIDE: MOA
Step one:
Acetylcholine binds to the outer surface
of endothelial cell
Step two:
Causes a rise in cytosolic Ca2+
concentration
Step three:
Ca2+
activates NOS to synthesize NO

47. NITRIC OXIDE: MOA
Step four:
NO formed in endothelial cell diffuses
across the plasma membrane to
adjacent muscle cells
Step five:
Stimulates guanyl cyclase in smooth
muscle which synthesizes cGMP, a
2nd messenger similar in structure to
cAMP

48. NITRIC OXIDE: MOA
Step six:
cGMP leads to a decrease in cytosolic
Ca2+
concentration which leads to
smooth muscle cell relaxation
Conclusion/Discovery:
“NO acts as an activator of guanyl
cyclase”

49. Medical Relevance: Nitroglycerine
-used to treat the pain of angina that results
from an inadequate flow of blood to the heart.
-metabolized to NO which stimulates the
relaxation of the smooth muscles lining the
blood vessels of the heart
-leads to increase blood flow to the organ

50. Pharmacological Relevance: Sildenafil
- Aka Viagra
- Developed
following the
discovery of NO

51. Sildenafil: MOA
• During sexual act
-nerve endings in the penis release NO
-causes:
(a) relaxation of smooth muscle
cells in the lining of penile
blood vessels
(b) Engorgement of the organ
with blood

52. Sildenafil: MOA
• Viagra
-has no effect on the release of NO or the
activation of guanylyl cyclase
-(instead) inhibits cGMP
phosphodiesterase

53. Phosphodiesterase: MOA
• An enzyme
• Destroys cGMP
• Inhibition of this enzyme leads to:
(A) maintained, elevated levels of
cGMP ->
(B) promotes the development and
maintenance of an erection

54. Since Viagra acts to maintain
elevated levels of cGMP,
does it affect the heart as
well???

55. Viagra
-specific for one particular isoform of cGMP
phosphodiesterase (PDE5)
-version that acts in the penis
PDE3
-plays key role in the regulation of heart
muscle contraction (not inhibited by Viagra)

56. NO or N2O???

57. Do not forget!
Nitric Oxide (NO) should not be confused
with Nitrous Oxide (Laughing Gas)

60. Describe the steps
in the signaling
pathway by which
nitric oxide
mediates dilation
of blood vessels.

61. Since Viagra acts to
maintain elevated levels of
cGMP, does it affect the
heart as well??? Explain!

62. Information overload!!!

63. Five Minutes Break!!!

64. For everything there is a season, and a time
for every matter under heaven: A time to be
born, and a time to die…
Ecclesiastes 3: 1f

65. For every cell, there is a time to live and a
time to die…

66. Apoptosis

67. Apoptosis
…Programmed cell death
Discussed by:
HERBERT, B.

68. Objectives
• At the end of the discuss, students should
be able to:
– Describe the steps that occur between the
time that a TNF molecule binds to its receptor
and the eventual death of the cell.
– Describe the steps that occur between the
time a proapoptotic Bcl-2 membrane binds to
the outer mitochondrial membrane and the
death of the cell.

69. Cells die for two quite different
reasons
• Accidental death
• result of mechanical trauma or exposure to some
kind of toxic agent (necrosis)
• only type of death seen in unicellular organisms
• Deliberate death
• result of an built-in suicide mechanism known as
apoptosis or programmed cell death.

70. Necrosis
• When cells are injured
– ATP concentrations fall so low that the Na+
/K+
ATPase can no longer operate,
– ion concentrations are no longer controlled
– causes the cells to swell and then burst
– cell contents leak out
– causes the surrounding tissues to become
inflamed

71. Apoptosis
• A normal occurrence
• An orchestrated sequence of events
• Leads to death of a cell
• Eliminates cells with sustained irreparable
genomic damage
– Important because damage to genetic blue print can
result in unregulated cell division -> Cancer
• Etymology: John Kerr et al., (1972)

72. Apoptosis: Characteristics
• Shrinkage of cell volume and nucleus
• Loss of adhesion to neighbouring cells
• Formation of blebs at cell surface
• Dissection of chromatin into small
fragments
• Engulfment of the ‘corpse’ by
phagocytosis

73. Apoptosis: Working Examples
• Neurons (During embryonic development)
 grow out of CNS to innervate organs present in
the periphery of the body
 usually many more neurons grow out than are
needed for normal innervation
 Neurons that reach their destination receive
signal from the target tissue that allows them to
survive
 Neurons that fail to find their way to the target
tissue do not receive the survival signal and are
eliminated by apoptosis

74. Apoptosis: Working Example
• T Lymphocytes
– Cells of the immune system
– Recognize and kill abnormal or pathogen
infected cells
– Recognizes target cells via specific receptors
that are present on its surface

75. Apoptosis: T Lymphocytes
– Sometimes produced during embryonic
development with receptors capable of
binding tightly to proteins present on surface
of normal cells within the body.
– T Lymphocytes that have this dangerous
capability are eliminated by apoptosis.

76. Apoptosis: Medical Relevance
• Apoptosis is involved in neurodegenerative
diseases such as:
– Alzheimers’s
– Parkinson’s
– Huntington’s
Elimination of essential neurons during dz
progression gives rise to loss of memory or
decrease in motor coordination

77. Apoptosis: Triggers
Apoptosis can be triggered in three ways:
(a) binding of ligand to death domain
receptors,
(b) denial of growth factors, and
(c) cell stress.

78. Central Idea
“Apoptosis is important in maintaining
homeostasis in multicellular organisms
and failure to regulate apoptosis can
result in serious damage to an organism”

79. Apoptosis is a normal occurrence!
You can’t escape from it!

80. Apoptosis: A Worm’s Eye View!
• First revealed in studies on nematode
worm Caenorhabditis elegans
»Cells can be followed with absolute
precision during embryonic
development
»131 cells are normally destined to die
by apoptosis
»Worms carrying mutation in the CED-
3 gene proceed through development
without losing any of their cells to
apoptosis

81. Apoptosis: Caspases
• A homologous gene to CED-3 found in
humans
• Distinctive group of cysteine proteins
»i.e proteases with a key cysteine
residue in their catalytic site
»Activated at an early stage of
apoptosis
»Responsible for triggering changes
observed during cell death

82. MOA: Caspases
• Achieves apoptosis by cleaving a selected
group of proteins
• All the cells of our body contain caspases
• they are normally locked in an inactive
form by an integral inhibitory domain of
the protein
• Proteolysis cleaves the inhibitory domain
off, releasing the active caspases

83. Viruses: A Hostile Take Over!
• no protein synthesis is required to activate
the apoptotic pathway—all the
components are already present
• if a virus infects a cell and takes over all
protein synthesis, the cell can still commit
suicide and hence prevent viral replication

84. Caspases: Targets
• Focal Adhesion Kinase (FAK), PKB, and
Raf 1
– Inactivation of FAK disrupts cell adhession,
leading to detachment of apoptotic cell from
its neighbours
• Lamins
– Make up inner lining of nuclear envelope
– Cleavage of lamins leads to the disassembly
of nuclear lamina and shrinkage of the
nucleus

85. Caspases: Targets
• Proteins of the Cytoskeleton
– Such as those of the intermediate filaments,
actin, tubulin and gelsolin
– Cleavage and consequent inactivation of these
proteins lead to changes in cell shape
• Caspase activated Dnase (CAD)
– An endonuclease
– Activated following caspase cleavage of an
inhibitory protein
– Translocates from cytoplasm to nucleus
– Attacks DNA, severing it into fragments

86. Apoptosis: What activates it?
A. Internal stimuli (Intrinsic Pathway)
–Abnormalities in DNA
A. External stimuli (Extrinsic Pathway)
– Removal of growth factors from the
medium

87. The exTrinsic PaThway of aPoPTosis

88. Extrinsic Pathway of Apoptosis
– Removal of growth factors from the medium
– Epithelial cells of the prostate become
apoptotic when deprived of the male sex
hormone, testosterone
» Hence prostate cancer that has spread to other tissues
are often tx with drugs that interfere with testosterone
production
– Stimulis is carried by an extracellular
messenger pr called TNF

89. Tumor Necrosis Factor
• So called for its ability to kill tumor cells
• Produced by cells of the immune system
in response to adverse conditions, such
as:
• Exposure to ionizing radiation
• Elevated temperature
• Viral infection
• Toxic chemical agents such as those used in
cancer chemotherapy

90. TNF Receptor
• Present in plasma membrane as a
preassembled trimer
• Cytoplasmic domain of each receptor
subunit contains a segment of about 70
a.a called ‘death domain’ (mediates pr-pr
interactions)

91. TNF: MOA
• Evokes its response by binding to a
transmembrane receptor, TNFR1
• Member of family related to ‘death receptor’ that
mediates apoptosis
• TNF binds to the trimer receptor -> change
in conformation of the receptor’s death
domain -> recruitment of a number of pr
• Last pr to join complex are two
procaspases (8 molecules)

92. TNF: MOA
• Synthesis of caspases as proenzymes
protects the cell from accidental proteolytic
damage
• When two or more procaspases are held
in close association with one another, they
are capable of cleaving one another’s
polypeptide chain and converting the other
molwcule to the fully active caspase

93. Caspase 8
• Final mature enzyme
• Contains four polypeptide chains
• Derived from two procaspase precursors
• Described as an initiator caspase
• Initiates apoptosis by cleaving and
activating downstream or executioner
caspases.

94. Executioner Caspases
• Carry out the controlled self-destruction of
the cell

96. The inTrinsic PaThway of aPoPTosis

97. Examples of Internal Stimuli
• Irreparable genetic damage
• Extremely high concentrations of cytosolic
Ca2+
• Severe oxidative stress
• Lack of survival signals (Absence of
growth factors)

98. Bcl-2 Family of Proteins
• Regulates activation of the intrinsic
pathway
• Originally identified as a tumor-causing
oncogene
• Subdivided into two:-
– Proapoptotic : promotes apoptosis (e.g, Bad
and Bax)
– Antiapoptotic: protects cells from apoptosis (e.g
Bcl-XL, Bcl-w, and Bcl-2)

99. Don’t Forget!
“Bcl-2 acts as an oncogene by promoting
survival of potential cancer cells that would
otherwise die.”

100. MOA: Intrinsic Pathway
• Stressful stimuli:- activates proapoptotic
members of the Bcl-2 family (Bad/Bax)
Translocates from the cytosol to outer
mitochondrial membrane
Attaches to outer mitochondrial membrane

101. MOA: Intrinsic Pathway
Increases membrane permeability
Promotes release of cytochrome C (which
resides in the intermembrane space)
Moves to cytosol
Forms apoptosome (a multi protein complex
that includes procaspase-9)

102. Procaspase-9
• Activated by simply joining the multiprotein
complex
• Does not require proteolytic cleavage
• An initiator caspase; initiates executioner
caspases
Apoptosis

103. Do not forget!
“The external pathway is receptor-mediated
while the internal pathway is mitochondrial
mediated! They however CONVERGE by
activating the same executioner caspase,
which cleaves the same cellular targets.”

104. Finally!
• As cells execute the proapoptotic program
they lose contact with neighbors and start
to shrink
• Cell disintegrates into a condensed,
membrane-enclosed apoptotic body
• Apoptotic bodies are recognizd by the
presence of phosphatidylserine on their
surface

105. Phosphatidylserine
• A phospholipid that is normaly present
only on the inner leaflet of the plasma
membrane

106. During apoptosis, a phospholipid “scramblase”
moves phosphatidylserine molecules to the
outer leaflet of the plasma membrane where
they are recognized as an “eat me” signal by
specialized macrophages.