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1. INTRACELLULAR SIGNALLING /
2
nd
Messengers
KAMRAN

2. G
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3. 2
nd
Messengers
•Cyclic AMP or cAMP
•DAG &IP3
•Ca
2+
•Cyclic GMP or cGMP

4. Cyclic AMP
•cAMP mediates different hormonal responses as:
•- The mobilization of stored energy (burn
carbohydrates in liver or triglycerides in fat cells by
catecholamines)
•- Conservation of water by the kidney (vasopressin)
•- Ca2+ homeostasis (parathyroid hormone), &
•- Increased rate and contractile force of heart
muscle (β-adrenomimetic catecholamines)
•Also regulates the production of adrenal and sex
steroids (in response to corticotropin or FSH)
•Relaxation of smooth muscle, and many other
endocrine and neural processes

5. •cAMP formed from ATP by the action of the enzyme
adenylyl cyclase (AC) &
•Converted to physiologically inactive 5’AMP by the
action of the enzyme phosphodiesterase (PDE)
•Several cyclic nucleotide PDE are present in the cell
•Milrinone, a selective inhibitor of type 3 PDE
expressed in cardiac muscle cells, has been used as
an adjunctive agent in treating acute heart failure
•Caffeine, theophylline, and other methylxanthines
produce their effects by blocking PDE, thus,
inhibiting cAMP degradation

6. •cAMP→activation of protein kinase A (or cAMP -
dependent protein kinase) →phosphorylation of
various cellular proteins, ion channels, and TF
•Activity or function of these target proteins alters
leading to a desired cellular response
•In addition, in some cell types, cAMP directly binds
to and affects the activity of ion channels
•Protein kinase A is composed of a cAMP-binding
regulatory (R) dimer and two catalytic (C) chains
•When cAMP binds to the R dimer, active C chains
are released to diffuse through the cytoplasm and
nucleus, where they transfer phosphate from ATP
to appropriate substrate proteins, often enzymes

7. •The specificity of the regulatory effects of cAMP
resides in the distinct protein substrates of the
kinases that are expressed in different cells
•For example, liver is rich in phosphorylase kinase
and glycogen synthase, enzymes whose reciprocal
regulation by cAMP-dependent phosphorylation
governs carbohydrate storage and release
•In addition, the active catalytic subunit of PKA
moves to the nucleus and phosphorylates the
cAMP-responsive element binding protein (CREB).
This TF, then, binds to DNA and alters transcription
of a number of genes

8. Production of cAMP by Adenlyl Cyclase
•AC - a membrane bound protein with 12
transmembrane regions
•Ten isoforms of this enzyme have been described
each having distinct regulatory properties, tailored
to specific cell / tissue need
•Stimulatory G proteins(Gs ) activate, while
inhibitory G proteins (Gi ) inactivate AC
•On binding an appropriate ligand to a stimulatory
receptor, a Gs α subunit activates one of the AC
whereas activation of inhibitory receptor lead to
inhibition of AC via a Gi α subunit

10. •The receptors are more specific to specific ligands but G
proteins mediate the stimulatory and inhibitory effects
produced by many different ligands
•In addition, cross-talk between the phospholipase C and
the AC systems results in widespread effects of PKA, as
several of the isoforms of AC are stimulated by
calmodulin
•Two bacterial toxins mediate effects on AC through G-
proteins (hence extensively used in G-protein research):
•- cholera toxin catalyzes the transfer of ADP ribose to
an arginine residue in the middle of the α subunit of G
s
inhibiting its GTPase activity, producing prolonged
stimulation of AC
•- Pertussis toxin catalyzes ADP-ribosylation of a
cysteine residue near the carboxyl terminal of the α
subunit of Gi, thus, inhibiting the function of Gi

12. IP3 & DAG AS 2
nd
MESSENGERS
•Another effector enzyme is , phospholipase C (PLC),
on the inner leaflet of the plasma membrane
•Ligands bound to GPCR can activate PLC through
the Gq proteins, while those bound to TK receptors
can do this through other cell signaling pathways
•PLC has 8 isoforms; PLC β-activated by G proteins,
while PLCγ forms-activated through TK receptors
•PLC isoforms can catalyze the hydrolysis of the
membrane lipid phosphatidylinositol 4,5-
diphosphate (PIP 2 ) to form IP3 and DAG

13. •DAG, confined to the membrane, activates one of
several isoforms of protein kinase C (PKC)
•Several tumor-promoting phorbol esters, that mimic the
structure of DAG, activate PKC directly without receptor
activation
•PKC, on activation, phosphorylates specific proteins,
including other enzymes and TF, to produce approp.
physiological effects, such as cell proliferation
•IP3,water-soluble, diffuses to ER to trigger release of
Ca2+ by binding to ligand-gated Ca
2+
channels
•The resulting ↑ in cytoplasmic Ca2+ conc. promotes the
binding of Ca2+ to the calcium-binding protein
calmodulin, which regulates activities of other enzymes,
including calcium-dependent protein kinases

14. •Multiple mechanisms damp or terminate signaling
by this pathway
•IP3 is inactivated by dephosphorylation
•DAG is either phosphorylated to yield phosphatidic
acid, which is then converted back into phospho-
lipids or it is deacylated to yield arachidonic acid
•Ca2+ is actively removed from the cytoplasm by
Ca2+ pumps

16. Cyclic GMP
•Another cyclic nucleotide of physiologic importance
is cyclic guanosine monophosphate (cGMP )
•cGMP is important in vision in both rod and cone
cells; in addition, cGMP-regulated ion channels, &
cGMP-dependent kinase → a no. of physiol. effects
•Guanylyl cyclases (GCs) are a family of enzymes that
catalyze the formation of cGMP
•GCs exist in two forms
•One form has an ECL amino terminal domain that is
a receptor, a single transmembrane domain, and a
cytoplasmic portion with GC catalytic activity

17. •Several such GCs have been characterized
•Two are receptors for atrial natriuretic peptide (ANP),
and a third binds an Escherichia coli enterotoxin and
the GI polypeptide guanylin
•The other form of GC is soluble, contains heme, and is
not bound to the membrane
•Several isoforms of the intracellular enzyme, activated
by nitric oxide (NO) and NO-containing compounds
•↑ cGMP conc. causes relaxation of vascular smooth
muscle by a kinase-mediated mechanism that results in
dephosphorylation of myosin light chains
•The actions are terminated by enzymatic degradation
of the cyclic nucleotide and by dephosphorylation of
kinase substrates

18. •A no. of useful vasodil. drugs,
such as nitro-glycerin and Na-
nitroprusside used in treating
cardiac ischemia and acute
hypertension, act by
generating or mimicking NO
•Others produce vasodilation by
inhibiting specific phospho-
diesterases, thus, interfering
with the metabolic breakdown
of cGMP
•One such drug is sildenafil,
used in treating erectile
dysfunction and pulmonary
hypertension

19. Calcium
•Ca
2+
regulates diverse physiological processes as
proliferation, neural signaling, learning, contraction,
secretion, and fertilization
•Cytosolic Ca
2+
is about 10,000 times less (10
-7
M vs
10
-3
M) than in the ECL fluid
•This large gradient is maintained by:
•- Ca
2+
transporters in the plasma membrane that
extrude calcium,
•-by Ca
2+
pumps in intracellular organelles, and
•-by cytoplasmic and organellar proteins that bind
calcium to buffer its free cytoplasmic concentration

20. •Cytosolic Ca
2+
↑ by two mechanisms:
•Plasma membrane have voltage-gated Ca
2+
ion
channels and allow Ca
2+
to come in when it
depolarizes, or these channels may be controlled by
phosphorylation by proteins PKA or PKC
•In addition, ER & other organelle has Ca
2+
channels
that, when activated, release Ca
2+
into the cytosol,
causing an ↑ in cytoplasmic calcium
•↑ cytoplasmic Ca2+ binds to and activates calcium-
binding proteins (CaBPs)
•CaBPs can have direct effects in cellular physiology,
or can activate other proteins, commonly protein
kinases, to further cell signaling pathways

21. •Many 2
nd
messengers act by ↑ the cytosolic Ca
2+

•IP3 is the major 2
nd
messenger that causes Ca
2+
release from the ER through the direct activation of
a ligand-gated channel, the IP3 receptor
•In many tissues, transient release of Ca2+ from
internal stores into the cytoplasm triggers opening
of a population of Ca2+ channels in the cell
membrane (store-operated Ca2+channels; SOCCs)
•Research has identified the physical relationships
between SOCCs and regulatory interactions of
proteins from the ER that gate these channels

22. •Two mechanisms for termination of Ca
2+
actions:
•- The IP3 from PLC can be dephosphorylated &
inactivated by cellular phosphatases
•- Cytosolic Ca
2+
can be rapidly pumped out to the
ECL space or into an ICL organelle by ATP-driven
Ca
2+
pumps in the plasma membrane, ER, SR, and
mitochondrial membranes
•Ca
2+
movement into the internal stores is through
the action of the SR or ER Ca2+ATPase , also known
as the SERCA pump

23. Calcium Binding Proteins (CaBPs)
•Different CaBPs; troponin, calmodulin, & calbindin
•Troponin-involved in contraction of skeletal muscle
•Calmodulin (Cal) contains 148 amino acid residues and
has four Ca2+ -binding domains
•On binding with Ca2+ , Calmodulin activates five
different calmodulin-dependent kinases (CaMKs)
•One kinase-myosin light-chain kinase (MLCK), phospho-
rylates myosin and cause contraction in smooth muscle
•CaMKI and CaMKII are concerned with synaptic
function, and CaMKIII with protein synthesis
•Another Cal-activated protein is calcineurin, a
phosphatase that inactivates Ca2+ channels by
dephosphorylating them