This chapter explains about detail mechanism of signal transduction in a cell and shows the possible targets of pharmacological agents.

1. Signal Transduction
.
March, 2022
BY
Abreham D

2. Outlines
• Introduction to cell signaling
• Key elements in signal transduction
• Type of signaling based on distance
• Steps of signal transduction
• Signaling molecules
• Signal transduction through various receptors
• Promising drug targets

3. Introduction to cell signaling
• Cells regulate its intracellular and extracellular environment, processes the
information it gathers, and responds accordingly.
• Cell signaling is the ability of a cell to receive, process, and transmit signals
with its environment and with itself.
 It is a fundamental property of all cells in every living organism
• Cell signaling is a controlled dynamic process
• Have mechanisms for responding to physical and chemical changes in their
environment.
• This mechanism helps cells to have a communication with other cells
3

4. Cont…
• Reception of the signals depends on protein at the cell surface
• Most are receptors
• The binding activates the receptor, which in turn activates one or
more intracellular signaling pathways or systems
Then distribute it to the appropriate intracellular targets
Effector proteins

5. Key players in signal transduction
• Signaling molecules/ligands
• Receptors
• Signal transduction proteins
• Second messengers
• Effector proteins
5

6. 6
Extracellular
Intracellular
Cell Membrane
Cytoskeleton
Enzymes Transcription
regulation
Altered Metabolic
path
Alter gene
expression
Alter Cell shape
Mov’t
Receptors
Ligand
Extracellular signaling
molecules
Effectors
Intracellular signaling proteins

7. Type of signals based on distance
Cells communicate with the help of messenger molecules
• Autocrine signals – its own cell receptors
• Paracrine signals – targets near by cells
• Endocrine signals – targets far away cells
7

8. 8
1. Autocrine signaling
2. Paracrine signaling
3. Endocrine
Hormones
2 AR
Ach on NM

9. Steps of cell signaling
• The coming signal
recognized by cell surface
components call receptors
• The extracellular signal
converted to an intracellular
signal
• Response is delivered
9
Reception
Transduction
Response
e.g. Transcription
Signal transduction is the conversion of extracellular
event to intracellular biochemical cues

10. Ligands/signaling molecules
1. Hydrophilic
 Interact through extracellular
receptors
2. Hydrophobic
Interact through intracellular
receptors
1. Extracellular  NTs, Hormones
 Amino acid & derivatives  Ach, Epi,
DA, NMDA, glycine
 Steroids –steroid sex hormones,
Corticosteroids
 Peptide hormones- Insulin
2. Intracellular  cAMP and Ca2+
10
Based on chemical nature Based on location

11. Receptors
Binding sites of a signaling molecule
• Extracellular receptors
 Integral transmembrane proteins (most)
 Ligands interact with extracellular domain and other molecules may with
cytosolic domains
 May open and closed to transport ions
• Intracellular receptors  class of nuclear receptors
 Located inside the cell rather than cell membrane
 Classical examples: Hormone receptors
 IP3 receptors on Sarcoplasmic R
11

12. Receptor
• Ligand binding causes a conformational change in the receptor
 Leads to the transmission of an intracellular signal
• Receptor-ligand extent of molecular complement determined by specificity
and affinity
• A given receptor may exhibit specificity for a certain ligand or closely
related ligands exhibit effector specificity
E.g. Adrenalin and NA in alpha and Beta receptors
12

13. Types of signaling receptors
13
Signaling Receptors
G-protein
coupled receptors
Ion channels
Enzyme linked
receptors
Nuclear receptors

14. G-protein-coupled receptors (GPCRs)
• Largest family of cell-surface receptors
• 7-Trans-membrane receptors
• Coupled with G-proteins
• Extracellular ligand binding thought to change the relative orientation  contact
with G-protein
• Phosphorylation of G-protein activates/deactivates a series of proteins in a
downstream process
 Activate or inactivate secondary messengers or ion channels
14

15. 15
No Ligand binding Ligand Bind
 Sub-unit detached
GPCR activation
Gs
Gi
Gq
Examples
Gs- 2 AR in the heart
Gi – M2 in heart
Gq- H1 receptors

16. cAMP
ATP
PKA
AMP
PDE
Phosphorylation of other
proteins and enzymes
Protein Protein
P
Phosphatidylinositol-4,5
bisphosphate (PIP2)
Diacylglycerol (DAG)
Activate protein kinase C
Inositol-1,4,5-
triphosphate (IP3)
Release of
intracellular Ca+2
Regulation of gene
Response

17. cAMP
ATP
PKA
AMP
PDE
Phosphorylation of other
proteins and enzymes
Protein Protein
P
Phosphatidylinositol-4,5
bisphosphate (PIP2)
Diacylglycerol (DAG)
Activate of protein kinase C
Inositol-1,4,5-
triphosphate (IP3)
Release of
intracellular Ca+2
Regulation of gene
Response
Can GPCR be Therapeutic targets ???

18. 18
Example:  1 Receptor activation

19. Signaling via ion channels
• Usually have only one transmembrane segment
• Ions are unequally distributed
1. Voltage gated ion channels
2. Ligand gated ion channels
19

20. Voltage gated Ion channels
20
+ + + - + - + -
++ +++
_ + - -- + - - - +
_

21. Ligand Gated Ion channels
21
Nicotinic Ach R GABA-A Receptor

22. Signal Transduction Nuclear receptor (NR)
• Ligand-Modulated Transcription Regulators located cytoplasm /Nucleus
• Hydrophobic signal molecules diffuse directly across the plasma membrane
of target cells
• Then bind to intracellular receptors that are transcription regulators.
• NRs serve both as intracellular receptors and as intracellular effectors for
the signal.
22
Steroid hormones, thyroid hormones, retinoids, and vitamin D

23. • At resting state NR are prevented by chaperons protein called HSP-90
23
HSP-90
Ligand binding
Dimerize
Recruit TF
Ligand Binding
Domain
DNA Binding Domain
Transactivation
Domain

24. How glucocorticoids have anti-inflammatory effect
• Glucocorticoids have anti-inflammatory effect by modulate the production of regulatory
proteins
• Penetrate the cell membrane, and then bind with receptors in the cytoplasm thereby
converting the receptor from an inactive form to an active form.
• Next, the receptor-steroid complex migrates to the cell nucleus, where it binds to chromatin
in DNA
 thereby altering the activity of target genes
• In most cases, activity of the target gene is increased, causing increased transcription of
mRNA that code for specific regulatory proteins
• Transcription of regulatory proteins produce Annexin A1
• Annexin A1 inhibit PLA2 enzyme
24

25. Glucocorticoid

26. Cell signal via kinase activity
26

27. Signal Transduction via Kinases/Phosphatases
• Two type of proteins for intracellular signal transduction
1. GTPase proteins – switched ON/OFF by ATP and ADP
2. Protein kinases/phosphatases switches target protein
27
Protein phosphorylation
Kinases use ATP to ON
Phosphatases hydrolyze phosphates OFF
The main approaches by which intracellular signaling takes place

28. Protein phosphorylation is a mechanism of regulation that is extremely
important in most cellular processes such as
28
To undergo these processes
Enzymes and receptors are activated and
deactivated
Via
phosphorylation/dephosphorylation
events due to specific kinases and
phosphatases
 Protein synthesis
 Cell division
 Signal transduction
 Cell growth
 Cell death (apoptosis)
 Development and aging
 Hence Protein phosphorylation reversible
Protein phosphorylation

29. Protein kinase Receptors
• This reversible mechanism occurs through protein kinases
 Add a phosphate group (PO4) to the polar group R of the amino acid
residues.
• Consequently, modifies the protein from hydrophobic a polar to hydrophilic
polar  more reactive
 Thereby allowing the protein to have conformation change when
interacting with other molecules.
• Then phosphorylated amino acid can bind molecules able to interact with
other proteins  consequently assemble and detach protein complexes
29

30. PKRs cont…
• Protein kinases are enzymes that regulate the biological activity of proteins by
phosphorylation of specific amino acids with ATP energy as source of phosphate
Inducing conformational change from its inactive state to inactive and vice versa.
30
Active
Interact with Molecules
 Signal transduction
Inactive
Many human proteins are modified by kinase activity
This kinase regulate the majority of cellular pathway especially those involved
in signal transduction.

31. Type of Protein kinase receptors
• Protein kinase is a driving force for many cellular process
• Protein kinases are classified according to the target amino
acid residues they phosphorylate
• Are grouped into two main types:
1. Serine/threonine kinases
2. Tyrosine kinases
31
In both cases ATP is utilized
to be phosphorylated

32. Serine/Threonine kinase receptors (STKs)
• Single-pass transmembrane receptors
• The protein kinase family of enzymes plays a pivotal role in signal
transduction over the cell membrane
• Involved in phosphorylation of the OH groups of Serine and
Threonine
• The autophosphorylation of most of this kinases is associated
with an increase in kinase activity
32

33. Role of Serine/Threonine kinase receptors
• STKs also known to be important for regulation of cellular metabolism
 Regulate cell proliferation,
 Programmed cell death (apoptosis)
 Cell differentiation
 Embryonic development
 Involved in cancer and metastasis.
33
Many pharmacologic agents also targets this receptor. E.g. Everolimus

34. Types of Serine threonine kinase receptors
• There are two isoforms of the monomeric receptor protein
1. Type I -primary receptor
 Seven forms
2. Type II- signal transducers
 Five forms
34
Type II receptors bind their cognate ligand on their own
Whereas type I receptors do so only when co-expressed with type II
receptor

35. • RSTKs phosphorylate serine and threonine residues on certain target cytosolic
proteins.
• Members of the TGF- superfamily of receptors are in this category.
35

36. Ligands
TGF beta superfamily
• A good example of RSTKs are the receptor for transforming growth
factor (TGF-β)
• All members of the TGF- family send signals through a receptor
complex formed by two distantly related types of serine/threonine kinase
proteins.
• Small secreted signaling molecules of dimeric structure, which control
and regulate the development of many tissues
36

37. Ligands …
TGF-β superfamily
TGF-β superfamily consists of more than 45 members including
 Activins
 TGF- subfamily (1-3)
 Inhibins
 Myostatin
 Bone morphogenetic proteins (BMPs)
 Growth and differentiation factors (GDFs)
37

38. Signal transduction by RSTKs
• Protein ligands such as TGF-β activate a family of RSTKs
domain in the cytoplasmic region of the protein.
• In the basal state, these proteins exist as monomers;
 Upon binding an agonist ligand Dimerize
 Leading to phosphorylation of the kinase domain of the type I monomer,
which activates the receptor
• The activated receptor then phosphorylates a downstream
signaling molecules  gene regulatory protein termed a Smad.
38

39. Smads
• Different TGF family activate different Smad transcription
factor family
• All then associate with the common SMAD4 and translocate
to the nucleus to modify gene transcription
39
Activins and TGFs signal  via SMAD2 and SMAD3
BMPs signal  via SMAD1/5/8
R-Smads  Smad 1,2,3,5,8
Co-Smad  Smad 4
I-Smad  Smad 6, 7

40. 40
Once the protein
Phosphorylated on
Sr/ Thr residue
Phosphorylates
Smad proteins
Migrates to
the nucleus
Associates with
transcription
factors
Regulates genes
leading to
morphogenesis and
transformation.
mRNA
Transcribed

41. 41
Figure: Signal transduction by TGF-β-superfamily pathway
Smad 7
Smad 7
Smad 7
Inhibin
Inhibin

42. Signaling via Receptor Tyrosine Kinases
Signal protein family Receptor family Responses
Insulin Insulin receptor Stimulates carbohydrate
utilization and protein synthesis
Epidermal growth factor
(EGF)
EGF receptors Stimulates cell survival, growth,
proliferation
Insulin-like growth factor
(IGF1)
IGF receptor-1 Stimulates cell growth and
survival
Macrophage-colony-
stimulating factor
(MCSF)
MCSF receptor Stimulates monocyte/
macrophage proliferation and
differentiation
42

43. Example Insulin Receptor
• Classic example is Insulin signals the removal of glucose from the blood and
synthesis of glycogen.
• Ligand binding causes the receptors to dimerize
 Bringing the two cytoplasmic kinase domains together and thereby
promoting their activation
 Dimerization stimulates kinase activity by variety of mechanism
43
Example: Insulin receptor, dimerization brings the kinase domains close to each
other in an orientation  allows them to phosphorylate each other
Thereby promoting conformational changes that fully activate both kinase domains

44. Insulin receptor signaling
• Insulin receptor has  extracellularly and  subunit
intracellularly
44

45. Insulin receptor signaling
• Phosphorylated IR creates binding site for insulin receptor substrate 1 (IRS-1)
• Subsequently IRS-1 activated via phosphorylation.
• Activated IRS-1 initiates the signal transduction pathway and is the binding site
for phosphoinositide 3-kinase (PI3K)  causes activation
• Then catalyzes the conversion of membrane phospholipid, phosphatidylinositol
4-5-bisphosphate (PIP2) into phosphatidylinositol 3-4-5- triphosphate (PIP3)
45
PI3K
PIP2 PIP3
PI3K/AKT Pathway

46. Cont…
• PIP3 acts as a secondary messenger /recognition site for the activation of
phosphatidylinositol dependent protein kinase (PDK1)
• Then PDK1activates other kinases mostly Protein Kinase B, (also called
Akt).
• AKT downstream paths activate GLUT4 containing vesicles to translocate
to the Cell membrane to fuse
• GLUT 4 transporters inserted in the plasma membrane  Glucose
move in.
46

47. P
P
IRS-1
P PI3K
PDK1
Glycogen GSK3
GS
AS160
PI3K/AKT Pathway High blood Glucose
PTEN

48. The final AKT
• Activates for GLUT4 Insertion
• Phosphorylates and inhibits Glycogen synthase kinase (GSK-3)
• P-Glycogen synthase is inactive  Active GS
• AKT also phosphorylates BAD proteins to inhibit apoptosis
48
Cell Death No Cell Death

49. Insulin receptor signaling for gen expression
• Ras/MAPK pathway
49
• First, the protein Grb2 binds the P-Tyr residue in its SH2 domain .
• Grb2 is then binds SOS, catalyzes the replacement GDP with GTP on Ras G
protein
• This protein then begins a phosphorylation cascade,  activation of mitogen-
activated protein kinase (MAPK)
• The last MAPK enters the nucleus and phosphorylates various nuclear
transcription factors (such as Elk1)

50. Insulin receptor signal pathways
50
GAP
GEF

51. Summary
• Cell signaling
• Signaling molecules
• Signal transduction pathways
• Receptors
51

52. 52

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