Assignment on Secondary messengers: cyclic AMP, cyclic GMP, calcium ion, inositol 1,4,5- trisphosphate, (IP3), NO, and diacylglycerol. Detailed study of following intracellular signaling pathways: cyclic AMP signaling pathway, mitogen-activated protein kinase (MAPK) signaling, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway.

1. MESSENGERS
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 Primary messengers
 Secondary messengers

2. PRIMARY MESSENGERS SECONDARY MESSENGERS
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 Extracellular factor like
hormones, or neurotransmitter
such as epinephrine, growth
hormones and serotonin.
 Peptide hormones and
neurotransmitter are hydrophilic.
 Physically cannot cross the
phospholipids bilayer
 These are intermediate molecules
like cyclic AMP or cyclic GMP
 When hormones bind to the target
cell receptor, then the cell release
or creates these intermediates
which then stimulate cell response
 Pathway involve- SIGNAL
TRANSDUCTION PATHWAY
2
TYPES OF MESSENGERS

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HYDROPHOBIC HYDROPHILIC GASES
DIACYLGLYCEROL CYCLIC AMP NITRIC OXIDE
PHOSPHATIDYL
INOSITOL
CYCLIC GMP,IP3 HYDROGN SULPHIDE
CALCIUM IONS CARBON MONOXIDE
TYPES OF SECONDARY MESSENGERS

4. HYDROPHOBIC MESSENGERS
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 Diacylglycerol –stimulate protein kinase c activity by
greatly increasing the affinity of the enzyme for
calcium ion
 Known target protein include calmodulin the glucose
transporter, HMG-COA reductase

5. PHOSPHATIDYLINOSITOL
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 Phosphatidylinositol negative charged phospholipid
and a minor component in eukaryotic cell
membranes
 The inositol can be phosphorylated to form:
 Phosphatidylinositol-4-phosphate
 Phosphatidylinositol-4,5-biphosphate
 Phosphatidylinositol-3,4,5-triphosphate

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 Intracellular enzyme phospholipase
 Hydrolyzes PIP2 which is found in the inner of the
plasma membrane
 Two product form:
 Diacylglycerol and PIP3

7. G- PROTEIN
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 Level of middle management in the cellular
organization and are able to communicate between
receptor and the effector enzymes or ion channels
 They were called G-PROTEIN because of their
interaction with the guanine nucleotides, GTP and
GDP
 The G protein are bound to the cytoplasmic surface
of the plasma membrane
 Heterotrimeric molecules consisting 2α, β,γ

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subtype Location of receptor
Type of G
protein coupled
receptor
Basic pathways
α1 Smooth muscle Gq Increase in PLC,
Increase in
intracellular
calcium ion
contraction
α2 Presynaptic nerves Gi Decrease in
activation of AC
Decrease in cyclic
AMP
β1
β2
β3
Heart
Smooth muscle
Fat tissue
Gs
Increase in
activation of AC,
Increase in cyclic
AMP, increase
intracellular
signaling pathways

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10. GPCR SIGNALING PATHWAY
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 Ligand bind to the receptor altering its conformation
and increasing its affinity for the G protein to which
its binds
 G subunit release it GDP which is replaced by GTP
 Alpha subunit dissociate from the G complex and
bind to an effector activating the effector

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 Activated AC produce cyclic AMP
 GTPase activity of G protein hydrolyzes the bound
GTP deactivating G
 G reassociates with G reforming the trimeric G
protein and the effector ceases its activity

12. MAJOR PATHWAYS
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 ADENYL CYCLASE: CYCLIC AMP PATHWAY
 PHOSPHOLIPASE C: IP3 DAG PATHWAY
 CHANNEL REGULATION

13. ADENYLYL CYCLASE:CAMP PATHWAY
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 cAMP is a secondary messenger that is synthesized
from ATP by the action of the cAMP- dependent
protein kinase
 Which increase contractility, impulse generation,
lipolysis

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15. IP3- DAG PATHWAY
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 IP3 located on endoplasmic reticulum
 Responsible for protein and lipid synthesis
 DAG- directly activate protein kinase c and control
phosphorylation of amino acid of variety of
intracellular protein
 Causes smooth muscle contraction

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IP3-DAG PATHWAY

17. CYCLIC GMP
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 Cyclic guanosine monophosphate is a cyclic
nucleotide derived from GTP
 Common regulator of ion channel, conductance,
glycogenolysis, and cellular apoptosis
 It also relaxes smooth muscles tissue
 In blood vessels relaxation of vascular smooth
muscles lead to vasodilation and increase blood flow

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20. CALCIUM ION
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 Great important amongest many other intracellular
secondary messengers
 Calcium ion bind with calmodulin(intracellular)
Activate
MLCK(myosin light chain kinase)

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 Smooth muscle contraction
 Calcium ion involved in release of arachidonic acid
from membrane phospholipid by activated
phospholipase and initiate the synthesis of
prostaglandin and leukotrienes
 Calcium ion synergize with PKC
 Activation of cellular function like hepatocyte,
glycogenolysis, insulin release from pancreas
 Imp. Role in contraction of smooth muscles

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REGULATION OF CALCIUM ION
IN PANCREATIC BETA CELL

23. GASEOUS SIGNALING
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 Either synthesized internally in the organism, tissue,
or cell or are received by the organism
 Induce certain physiological or biochemical changes
in the organism, tissue or cell
 Ex carbon monoxide, nitric oxide, hydrogen sulphide

24. NITRIC OXIDE
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 Known as endothelium derived relaxing factor is
biosynthesized from L-arginine, oxygen & NADPH
by various NO synthase enzymes
 The endothelium(inner lining) of blood vessel use
NO to signal the surrounding smooth muscle
 Result in vasodilation and increase blood flow

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 NO highly reactive its life time is of few sec yet it
diffuses freely across membrane
 For body to generate nitric oxide through nitrate-
nitrite-nitric oxide pathway
 Reduction of nitrate to nitrite occur in mouth by
bacteria
 Monitoring NO status by saliva testing detect the
bioconversion of plant derived nitrate into nitric
oxide

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 Production of NO is elevated in population living at
high altitudes which help these people to avoid
hypoxia by aiding in pulmonary vasodilation
 Nitroglycerin and amyl nitrite serves as vasodilator
because they converted to NO in the body
 Vasodilating antihypertensive drug minoxidil
contain an NO moiety act as an NO agonist

27. HYDROGEN SULPHIDE
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 Produced in small amount by some cell of the
mammalian body and has a number of biological
signaling function
 The gas is produced from beta- synthase and cysta
thionine gamma lyase
 Act as an relaxant of smooth muscle and as an
vasodilator

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 It also active in the brain where it increase the
response of the NMDA receptor and facilitates long
term potentiation

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MECHANISM OF
ACTION OF NITRIC
OXIDE

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CELLULAR EFFECT OF NITRIC OXIDE

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35. INTRACELLULAR SIGNALING PATHWAY
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 Cyclic AMP signaling Pathway
 Mitogen activated protein kinase signaling
 Janus kinase (JAK) /Signaling transducer and
activator of transcripton (STAT) signaling pathway

36. Mitogen activated protein kinase signaling
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 Mitogen activated protein kinases is an enzyme that
translocates the signals to the nucleus and activates
many transcriptional factor by phosphorylating
many different proteins that regulate expression of
important cell cycle and differentiation specific
protein

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38. ACTIVATION
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 Epidermal growth factor (EGF) bind to the (EGFR)
epidermal growth factor receptor in the cell
membrane g
Starting the cascade of signals
Activates MAPK(mitogen activated protein kinase)
also known (ERK)

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 Signal enter the cell nucleus and causes transcription
of DNA
 Then expressed as protein
 GRB2 growth receptor bound protein 2 is a adapter
protein which involve in signal transduction/cell
communication

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 encoded by this gene bind receptor such as the
epidermal growth factor receptor and contain one
SH2 domain and two SH3 domain
 Its two SH3 domain direct complex formation
with/other protein and bind to tyrosine
phosphorylated sequences

41. PATHWAY
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 Signal pass from activated Ras to a cascade of
protein kinases this cascade transmit signals
downstream from activated Ras protein to MAP
kinase
 Then MAP kinase translocates the signal to the
nucleus and activates trancriptional factor this whole
phenomenon called as MAP kinase pathway

42. ACTIVATION OF RAS PROTEIN
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 RAS is a monomeric GTP binding switch protein that
alternates between active on state with a bound GTP
and inactive off state with a bound GTP
 It is not directly linked to receptor
 Its activation is accelerated by a guanosine
nucleotide transfer factor

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ACTIVATION OF RAS PROTEIN

44. HOW (AMPK) GET ACTIVATED
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 It is activated by increases in the cellular AMP:ATP
ratio caused by metabolic stresses
 Muscle contraction leads to increase in AMP/ATP
level to increase in AMPK activity

45. FACTORS INVOVED IN ACTIVATION
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Heat shock/hypoxia
Metabolic changes
Glucose deprivation
Exercise

46. Requirement for signal transduction
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 Signal –that is to be passed
 Receptor-to which ligand binds
 Adapter protein-form link between membrane and
bound receptors and protein is to be activated
 Protein cascade- that would lead to the activation of
transcriptional factors
 Transcriptional factors

47. LIGANDS
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 Ligands for receptor tyrosine kinase (RTKs)
including nerve growth factor, platelet derived
growth(PDGF), fibroblast growth factor, insulin,
epidermal growth factor(EDRF)

48. RECEPTORS
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 Receptor tyrosine kinases- this type of receptor
contain intrinsic protein tyrosine kinase activity in
their cytosolic domain
 These have one extracellular domain which serves as
ligand and one cytosolic domain to which adapter
molecule bind

49. ACTIVATION OF RTKs
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 Most RTKs are monomeric but ligand binding
induces dimerization of receptors
 Formation of ligand receptor complex
 Alteration and activation of receptor

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 This conformational changes facilitates binding of
ATP
 In dimeric receptor the kinases in one subunit can
phosphorylate one or more tyrosine residues
 Phosphorylates other site in the cytosolic domain
 Activated RTKs interact with adaptor protein

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52. ADAPTER PROTEIN
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 Small protein that contain sos, GRB2 domain but
have no intrinsic enzymatic or signaling activites
 These protein couple activated RTKs

53. PROTEIN INVOVED
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 RAS
 RAF
 MEK
 MAP kinase
These protein are in inactive state
they are need to be activated

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FUNCTION OF AMPK PATHWAY

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56. THERAPEUTIC POTENTIAL
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 Type 2 diabetic patient are often associated with
hypertriglyceridaemia and high cholesterol
 The potential risk factor for CV problems
 Activated AMPK could reduce this risk

57. JAK-STAT SIGNALING PATHWAY
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 Janus kinase signal transducers and activators of
transcription pathway used to transduce a multitude
of signal for development and homeostasis
 JAK activation stimulates cell proliferation,
differentiation, cell migration and apoptosis involve
in various processes such as hematopoiesis immune
development

58. JAK (Janus kinase)
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 In Mammal the JAK family comprises of 4 member
 1) JAK1
 2) JAK2
 3) JAK3
 4) TYK2

59. STAT ( signal transduction and activator of
transcriptional factor)
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 STAT are latent transcriptional factor that reside in the
cytoplasm until activated
 STAT conserve tyrosine residue near the c- terminus that
is phosphorylated by JAK
 Phosphorylated STAT enter the nucleus by mechanism
that is dependent on nucleoprotein interactor

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Dimerization of STAT
bind to the specific regulatory sequence
activate or repress transcription of target genes

61. MUTATION IN PATHWAY…..
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 Fail to regulate JAK signaling cause inflammatory
disease, erythrocytosis

62. COMPONENT
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 A receptor
 Janus kinase
 Signal transducer and activator of transcription

63. JAK ACTIVATION
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 JAK activation occur upon ligand mediated receptor
multimerization because two JAK are brought into
close allowing phosphorylation

64. ACTIVATION OF PATHWAY
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65. MECHANISM
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 JAK have tyrosine kinase activity bind to some cell
surfaces cytokine receptor, the binding of the ligand to
the receptor triggers activation of JAK
 With increased kinase activity they phosphorylate
tyrosine residues on the receptor STATs
 Possessing SH2 domain are recruited to the receptor and
are themselves tyrosine phosphorylated by JAKs

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 Activated STAT dimers accumulate in the cell
nucleus and transcription of their target genes
 STAT may also be tyrosine phosphorylated directly
by receptor tyrosine kinases such as the epidermal
growth factor receptor as well as by non receptor
tyrosine kinases such as c-src
 The receptor is activated by signal from interferon,
interleukin growth factor or other chemical
messengers

67. REFERENCES
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 Kobila .K. B ,G protein coupled receptor structure
and activation HHS public access
 Murad.F Nitric oxide: the coming of secondary
messenger rambam maimonides medical journal
 Rawling.S.J,Rosler.M.K Harrison A.D.The JAK-
STAT signaling pathway journal of cell science
2004117:128,-1283 1292/jcs.00963
 Tripathi.K.D Textbook of medical pharmacology
sixth edition jaypee brothers medical publisher page
no 22-37

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 Yan.k, Gao.N, Zhou X, the cyclic AMP signaling
pathway, molecular medicine report spandidos
publication

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