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1. SIGNAL TRANSDUCTION
AND
SECOND MESSENGERS
M Mridhuna Manoharan
M. Sc. Biotechnology

2. INTRODUCTION
 A cell is highly responsive to specific chemicals in its
environment: it may adjust its metabolism or alter gene –
expression patterns on sensing their presence.
 In multicellular organisms, these chemicals signals are crucial to
coordinating physiological responses.
 Three examples of molecular signals that stimulate a physiological
response are epinephrine, insulin, and epidermal growth factor.

3. a) When an individual organism is threatened, the adrenal
glands, located over the kidneys, release the hormone
epinephrine, which stimulates the mobilization of energy
stores and leads to improved cardiac function.
b) After a full meal, the β cells in the pancreas release insulin,
which stimulates the uptake of glucose from the bloodstream
and leads to other physiological changes.
c) The release of epidermal growth factor in response to a
wound stimulates specific cells to grow and divide.

4. In all these cases, the cell receives information that a certain
molecule within its environment is present above some threshold
concentration.
The chain of events that converts the message “this molecule is
present” into the ultimate physiological response is called signal
transduction.

5. Key Steps
Signal – transduction pathways follow a broadly similar course that
can be viewed as a molecular circuit.
1. Release of the Primary Messenger – a stimulus such as wound or
digested meal triggers the release of the signal molecule, also
called the Primary Messenger.

6. 2. Reception of the Primary Messenger - most signal molecules
do not enter cells. Instead, proteins in the cell membrane act as
receptors that bind the signal molecules and transfer the
information that the molecule has bound from the environment
to the cell’s interior. Receptors span the cell membrane and,
thus, have both extracellular and intracellular components. A
binding site on the extracellular side specifically recognizes the
signal molecule (ligand). Such binding sites are analogous to
enzyme active sites except that no catalysis takes place within
them. The interaction of the ligand and the receptor alters the
tertiary or quaternary structure of the receptor, including the
intracellular parts.

7. 3. Delivery of the Message Inside the Cell by the Second
Messenger - other small molecules, called second messengers,
are used to relay information from receptor – ligand
complexes. Second messengers are intracellular molecules that
change in concentration in response to environmental signals.
These changes in concentration constitute the next step in the
molecular information circuit. Some particularly important
second messengers are cyclic AMP and cyclic GMP, calcium
ion, inositol-1,4,5-triphosphate (IP3), and diacylglycerol.

8. 4. Activation of Effectors that directly alter the Physiological
Response - the ultimate effect of the signal pathway is to
activate (or inhibit ) the pumps, enzymes, and gene –
transcription factors that directly control metabolic pathways,
gene activation, and processes such as nerve transmission.
5. Termination of the Signal - after a cell has completed its
response to a signal, the signaling process must be terminated.

9. Epinephrine signaling
 Begins with ligand binding to a protein called the β – adrenergic
receptor ( β – AR ).
 The β – adrenergic receptor is a member of the largest class of cell
surface receptors, called the seven transmembrane – helix ( 7TM )
receptors.
 Members of this family are responsible for transmitting
information initiated by signals as diverse as hormones,
neurotransmitters, odorants, tartants, and even photons.

10. Biological functions mediated by 7TM receptors
 Hormone action
 Hormone secretion
 Neurotransmission
 Chemotaxis
 Exocytosis

11.  Control of blood pressure
 Embryogenesis
 Cell growth and differentiation
 Development
 Smell
 Taste
 Vision
 Viral infection

12.  These receptors contain seven helices that span the membrane
bilayer.
 The receptors are sometimes referred to as serpentine
receptors because the single polypeptide chain “snakes”
through the membrane seven times.
 The first member of this family to have its three - dimensional
structure determined was rhodopsin, which plays an essential
role in vision.
 7TM receptors are quite similar in structure to rhodopsin,
others have larger extracellular domains.

13.  The binding of epinephrine to β – AR triggers conformational
changes in the cytoplasmic loops and the carboxyl terminus.
 Thus, the binding of a ligand from outside the cell induces a
conformational change in the part of the 7TM receptors that is
positioned inside the cell.

14. Ligand binding to 7TM Receptors leads to the Activation of
Heterotrimeric G Proteins
 Conformational change in the receptor’s cytoplasmic domain
activates a protein (G protein) because it binds guanyl
nucleotides.
 Activated G protein stimulates the activity of adenylate cyclase,
an enzyme that increases the concentration of cAMP by forming
it from ATP.
 The G protein and adenylate cyclase remain attached to the
membrane, whereas the signal originally brought by the binding
of epinephrine.

15. Fig: Activation of protein kinase A by a G – protein pathway
In the G protein's unactivated state, the guanyl nucleotide bound to
the G protein is GDP.
In this form, the G protein exists as a heterotrimer consisting α, β,
and γ subunits.
The α subunit (Gα ) binds the nucleotide. The α – subunit is a
member of the P – loop NTPase family, and the P – loop participates
in nucleotide binding.

16.  The α and γ subunits are usually anchored to the membrane by
covalently attached fatty acids.
 The role of the hormone – bound receptor is to catalyze the
exchange of GTP for bound GDP.
 The hormone – receptor complex interacts with the heterotrimeric
G – protein and opens the nucleotide – binding site so that GDP
can depart and GTP from solution can bind.
 The α – subunit simultaneously dissociates from the βγ dimer
(Gβγ).

17.  The dissociation of the G – protein heterodimer into Gα and
Gβγ units transmits the signal that the receptor has bound its
ligand.
 A single hormone receptor complex can stimulate nucleotide
exchange in many G – protein heterotrimers.
 Thus, hundreds of Gα molecules are converted from their
GDP into their GTP forms for each bound molecule of
hormone, giving an amplified response.
 Because they signal through G proteins, 7TM receptors are
often called G – protein – coupled receptors or GPCRs.

18.  Activated G – proteins transmit signals by binding to other
proteins.
 In the GTP form, the surface of the G – protein that had been
bound to the βγ dimer has changed its conformation from the GDP
form so that it no longer has a high affinity for the βγ dimer.
However, this surface is now exposed for binding to other
proteins.
 In the β – AR pathway, the new binding partner is adenylate
cyclase, the enzyme that converts ATP into Camp. This enzyme is
a membrane protein that contains 12 membrane – spanning
helices; two large cytoplasmic domains from the catalytic part of
the enzyme.

19.  The interaction of Gαs with adenylate cyclase favours a more
catalytically active conformation of the enzyme, thus
stimulating cAMP production.
 The G – protein subunit that participates in the β – AR
pathway is called Gαs.
 The binding of epinephrine to the receptor on the cell surface
increases the rate of cAMP production inside the cell.
 The generation of cAMP by adenylate cyclase provides a
second level of amplification because each activated adenylate
cyclase can convert many molecules of ATP into cAMP.
 Cyclic AMP stimulates the phosphorylation of many target
proteins by activating protein kinase A.

20.  Most effects of cAMP in eukaryotic cells are mediated by the
activation of a single protein kinase. This key enzyme is protein
kinase A.
Protein Kinase A
• Consists of two regulatory (R) chains and two catalytic (C) chains.
• In the absense of cAMP, the R₂C₂ complex is catalytically
inactive.
• The binding of cAMP to the regulatory chains releases the
catalytic chains, which are catalytically active on their own.
• Activated PKA then phosphorylates specific serine and threonine
residues in many targets to alter their activity.

21. • For instance, PKA phosphorylates two enzymes that leads to
the breakdown of glycogen, the polymeric store of glucose,
and the inhibition of further glycogen synthesis.
• PKA stimulates the expression of specific genes by
phosphorylating a transcriptional activator called the cAMP –
response element binding (CREB) protein.
• This activity of PKA illustrates the signal – transduction
pathways can extend into nucleus to alter gene expression.

22. Fig : Epinephrine signaling pathway

23.  Gα subunits have intrinsic GTPase activity, which is used to
hydrolyze bound GTP to GDP and Pi.
 The bound GTP act as a built – in clock that spontaneously resets
the Gα subunits after a short period of time.
 After GTP hydrolysis and the release of Pi, the GDP – bound form
of Gα then reassociates with Gβγ to reform the inactive
heterotrimeric protein.

25.  The hormone – bound activated receptor must be reset as well to
prevent the continuous activation of G proteins. This resetting is
accomplished by two processes:
1. The hormone dissociates, returning the receptor to its
initial, unactivated state.
2. The hormone – receptor complex is deactivated by the
phosphorylation of serine and threonine residues in the
carboxyl – terminal tail.
Finally, the molecule β – arrestin binds to the phosphorylated
receptor and further diminishes its ability to activate G – proteins.

27. Insulin signaling
 Insulin is released after eating a full meal, leads to the mobilization
of glucose transporters to the cell surface allow the cell to take up
the glucose that is plentiful in the bloodstream after a meal.
 Insulin is a peptide hormone that consists of two chains, linked by
three disulfide bonds.
 Its receptor has a quite different structure from that of the
epinephrine receptor β – AR.
 The insulin receptor is a dimer of two identical units. Each unit
consists of one α – chain and one β – chain linked to one another
by a single disulfide bond.

28.  Each α – subunit lies completely outside the cell, whereas each
β – subunit lies primarily inside the cell.
 The two α – subunit move together to form a binding site for a
single insulin molecule.
 The moving together of the dimeric units in the presence of an
insulin molecule sets the signaling pathway in motion.
 The closing up of an oligomeric receptor or the
oligomerization of monomeric receptors around a bound
ligand is a strategy used by many receptors to initiate a signal,
particularly receptors containing a protein kinase.

29.  Each β – subunit consists primarily of a protein kinase domain,
homologous to protein kinase A. This kinase differs from
protein kinase A in two important ways:
1. The insulin receptor kinase is a tyrosine kinase; that is it
catalyzes the transfer of a phosphoryl group from ATP to the
hydroxyl group of tyrosine, rather than serine or threonine,
as is the case for protein kinase A.

30. 2. The insulin receptor kinase is in an inactive conformation when
the domain is not covalently modified. The kinase is rendered
inactive by the position of an unstructuredloop (activation loop)
that lies in the center of the structure.

31. Fig : The insulin receptor

32. Insulin binding results in the Cross – Phosphorylation and
Activation of the Insulin Receptor
 When the two α – subunits move together to surround an
insulin molecule, the two protein kinase domains on the inside
of the cell also drawn together.
 As they come together, the flexible activation loop of one
kinase subunit is able to fit into the active site of the other
kinase subunit within the dimer.
 With the two β – subunits forced together, the kinase domains
catalyze the addition of phosphoryl groups from ATP to
tyrosine residues in the activation loops.

33.  When these tyrosine residues are phosphorylated, a striking
conformational change takes place.
 The activation loop changes conformation dramatically and the
kinase converts into an active conformation.
 Thus, insulin binding on the outside of the cell results in the
activation of a membrane – associated kinase within the cell.

34. The activated Insulin Receptor Kinase initiates a Kinase
Cascade
 The receptors for several protein hormones are themselves
protein kinases, which are switched on the membrane.
 Insulin is an example of a hormone whose receptor is a
tyrosine kinase.
 The hormone binds to domains exposed on the cell’s surface.
 Following binding of hormone, the receptor undergoes a
conformational change, phosphorylates itself, then, and
phosphorylates a variety of intracellular targets.

35.  Phosphorylation cascade follows and starts up a whole series of enzyme
phosphorylation.
 On phosphorylation, the insulin receptor tyrosine kinase is activated.
 Phosphorylated sites on the receptor acts as binding sites for insulin
receptor substrate such as IRS – 1.
 The lipid kinase phosphoinositide-3-kinase binds to phosphorylated sites
on IRS – 1 through its regulatory domain, then converts PIP₂ into PIP₃.
 Binding to PIP₃ activates PIP₃ dependent protein kinase, which
phosphorylates and activates kinases such as Akt1.
 Activated Akt1 can then diffuse throughout the cell to continue the
signal transduction pathway.

37.  Insulin signaling is terminated by the action of phosphates.
 Lipid phosphatases are required to remove phosphoryl groups
from inositol lipids that had been phosphorylated as part of a
signaling cascade.
 In insulin signaling, three classes of enzymes are of particular
importance: protein tyrosine phosphatases that remove phosphoryl
groups from tyrosine residues on the insulin receptor, lipid
phosphatases that hydrolyze phosphatidylinositol-3,4,5-
triphosphate to phosphatidylinositol-3,4-bisphosphate, and protein
serine phosphatases that remove phosphoryl groups from activated
protein kinases such as Akt.

38.  Many of these phosphatases are activated or recruited as part
of the response to insulin.
 Thus, the binding of the initial signal sets the stage for the
eventual termination of the response.

39. Fig : Insulin signaling pathway

40. Calcium – a Second Messenger
 Calcium ion participates in many signaling processes in addition
to the phosphoinositide cascade. Several properties of this ion
account for its widespread use as an intracellular messenger.
1. Fleeting changes in Ca²⁺ concentration are readily detected.
At steady state, intracellular levels of Ca²⁺ must be kept low
to prevent the precipitation of carboxylated and
phosphorylated compounds. Calcium ion levels are kept low
by transport systems that extrude Ca²⁺ from the cell. The
cytoplasmic level of Ca²⁺ lower than the concentration in the
extracellular medium.

41. 2. Ca²⁺ is a highly suitable intracellular messenger is that it
can bind tightly to proteins and induce conformational
changes. Calcium ions binds well to negatively charged
oxygen atom (from the side chains of glutamate and
aspartate) and uncharged oxygen atoms (main – chain
carboxyl groups and side – chain oxygen atoms from
glutamine and asparagine). The capacity of Ca²⁺ to be
coordinated to multiple ligands from six to eight oxygen
atoms – enables it to cross – link different segments of a
protein and induce significant conformational changes.

42.  Calcium ions often activates the regulatory protein Calmodulin
(CaM), a 17 kDa protein with four Ca²⁺ binding sites, serves as a
Ca²⁺ sensor in nearly all eukaryotic cells.
 It is activated by the binding of Ca²⁺, which takes place when the
cytoplasmic Ca²⁺ level is raised above about 500nM.
 Calmodulin is a member of the EF – hand protein family.
 The binding of Ca²⁺ to calmodulin induces substantial
conformational changes in the EF hands.
 These conformational changes expose hydrophobic surfaces that
can be used to bind other proteins.

43.  One especially noteworthy set of targets is several calmodulin
– dependent protein kinases (CaM kinases) that
phosphorylates many different proteins.
 These enzymes regulate fuel metabolism, ionic permeability,
neurotransmitter synthesis, and neurotransmitter release.

44. CONCLUSION
 In human beings and other multicellular organisms, specific signal
molecules are released from cells in one organ and are sensed by
cells in other organs throughout the body.
 The message “a signal molecule is present” is converted in to
specific changes in metabolism or gene expression by means of
often complex networks referred to as signal – transduction
pathways.
 These pathways amplify the initial signal and lead to changes in the
properties of specific effector molecules.

45. REFERENCES
 Berg Jeremy M, Tymoczko John L, Stryer Lubert (2013),
Biochemistry, fifth edition, W H Freeman and Company, New
York,pp.381-395
 Albert Bruce, Johnson Alexander, Lewis Julian, Morgan David,
Raff Martin, Roberts Keith, Walter Peter (2008), Molecular
Biology of Cells, fifth edition, Garland Science, New York,
pp.154-155
 Tropp Burton E (2012), Molecular biology, fourth edition, Jones
and Barlett, New York, pp. 94-99