This document discusses various mechanisms of hormone and signaling molecule action, including: 1) Signal transduction through G-protein coupled receptors and the cyclic AMP/protein kinase A pathway. Hormones bind to GPCRs activating G-proteins that stimulate adenylate cyclase and increase cyclic AMP levels, activating protein kinase A. 2) Protein kinases phosphorylate downstream effector enzymes, altering their activity. Cyclic AMP also has long-term effects by phosphorylating gene regulatory proteins. 3) Abnormal G-protein signaling can occur through bacterial toxins that modify G-proteins, altering cyclic AMP levels and disrupting ion transport. 4) Calcium is an

1. MECHANISMS OF ACTION OF
HORMONES AND SIGNALING
MOLECULES
DR.K. RAVI BABU BSc.,MBBS.,MD BIOCHEMISTRY

2. CHAPTER AT A GLANCE
Signal transduction
Cyclic AMP and G-proteins
Protein kinases
Hormones acting through calcium
Hormones acting through PIP2 cascade
Cyclic GMP
Hormone response element

3. HORMONES
The nervous system and endocrine system -integrate the functions of the tissues
in the body.
 The nervous system transmits electrochemical signals between the brain and
peripheral tissues for coordinating the diverse body functions.
The endocrine system releases chemical mediators or hormones into the
circulation.
EH Starling -“hormone”.

4. HORMONES
Signal molecules are of different types and the process of transferring the signal
into the cell is called signal transduction.
There are two types of cells in signal transduction—the sender cell where the
signal originates and the target cell that receives the signal.
 The signal alters or modulates the activity/function of the cell.

5. HORMONES
Autocrine signaling occurs when same cell acts as
sender and recipient, e.g. growth, differentiation, immune and inflammatory
response.
Paracrine signaling is effected by local mediators which have their effect near
the site of secretion without entering the circulation.
The effect is rapid and transient.
Juxtacrine signaling occurs when the two type of cells are adjacent to each
other -gap junctions or through protein molecules on the surface of the two
cells.

6. HORMONES
Endocrine signaling is between cells which are located at a distance from each
other and the signal may be hormones or chemical messengers secreted into
circulation.
Once they reach the target cell, they bind to specific target cell receptors with
high affinity.
 Plasma carrier proteins exist for all classes of endocrine hormones.
Carrier proteins for peptide hormones prevent hormone destruction by plasma
proteases.
 Carriers for steroid and thyroid hormones allow these hydrophobic hormones to
be present in the plasma.
 Carriers for small, hydrophilic amino acid-derived hormones prevent the
infiltration through the renal glomerulus, greatly prolonging their circulating half-
life.

7. HORMONES CLASSIFICATION
“substances released from ductless or endocrine glands directly to the blood”.
It is synthesized by one type of cells and transported through blood to act on
another type of cells.
 Based on mechanism of action, the hormones may be classified into two:
I. Hormones with cell surface receptors
II. Hormones with intracellular receptors.

8. Hormones Acting through Cyclic AMP
Signal transduction pathways are like a river flowing in one direction only.
 components closer to the receptor are called “upstream”.
And closer to the response are called “downstream”.

9. Signal Transduction through G-Protein
Action is through G-protein coupled receptors (GPCRs).
Binding of different types of signal molecules to G-protein coupled receptors is a
general mechanism of signal transduction.
Action of several hormones is effected through this mechanism .
The GPCRs are transmembrane proteins with 7 helical segments spanning the
membrane.
When any ligand binds, the GPCRs activate heterotrimeric GTP binding
regulatory proteins (G-proteins).
The G-protein in turn will interact with effector proteins which may be enzymes
or ion channel proteins, which result in the desired effect.
Different types of G-proteins are present in the cells that are coupled with
different receptors and activating different effector proteins.

15. Signal Transduction through G-Protein
The extracellular messenger, the hormone (H) combines with the
specific receptor (R) on the plasma membrane
 The H-R complex activates the regulatory component of the protein
designated as G-protein or nucleotide regulatory protein.
G-proteins are so named, because they can bind GTP and GDP.
GDP-GTP exchange is mediated by the GEF (Guanine nucleotide
exchange factor).
The G- protein is a trimeric membrane protein consisting of alpha,
beta and gamma subunits .
Most of the hormone/ligand signals are transducted through GPCR.
Around 50% of medicines are acting through GPCR.

16. G-protein Activates Adenyl Cyclase
When the hormone receptor complex is formed, the activated receptor
stimulates the G-protein, which carries the excitation signal to adenylate cyclase .
The hormone is not passed through the membrane; but only the signal is
passed; hence this mechanism is called signal transduction.
The adenyl cyclase is embedded in the plasma membrane .

17. Fig. Action of hormone through G-protein

19. Subunit Activation of G-protein
The inactive G-protein is a trimer with alpha, beta and
gamma subunits. When activated, GTP binds and the
beta-gamma subunits dissociate from the alpha subunit.
Adenylate cyclase is activated by G-alpha-GTP (Fig.
50.4-2). The binding of hormone to the receptor triggers
a configurational change in the G-protein which induces
the release of bound GDP and allows GTP to bind. The
hormone has an amplified response, since several molecules
of G-alpha-GTP are formed.

20. Inactivation-G-protein
The active G-alpha-GTP is immediately inactivated by GTPase. The G-alpha-GDP
form is inactive .
 The activation is switched off when the GTP is hydrolyzed to GDP by the GTPase
activity of the alpha subunit.
This is a built-in mechanism for deactivation.
Thus GTPase acts as a molecular switch.
 The alpha subunit, which is bound to GDP, can re-associate with beta and
gamma subunits.
The GTP-GDP exchange rate decides the activity of adenyl cyclase.

22. Cyclic AMP
Adenyl cyclase or adenylate cyclase converts ATP to Camp (3’,5’-cyclic AMP), and
phosphodiesterase hydrolyzes cAMP to 5’ AMP .
Cyclic AMP is a second messenger produced in the cell in response to activation
of adenylate cyclase by active G-protein.
During hormonal stimulation, cyclic AMP level in the cell increases several times.

23. Cyclic AMP
The level of cyclic AMP in the cell is regulated by its rate of production by
adenylate cyclase (AC) and
hydrolyzis by phosphodiesterase (PDE).
The action of PDE is also regulated by hormones and drugs.
cellular level of cyclic AMP can be increased by inhibition of PDE.
 Ex. insulin activates PDE, decreasing the cellular level of cAMP while caffeine
and theophylline inhibit PDEs increasing cAMP levels.

26. Second Messenger Activates PKA
The cAMP (second messenger), in turn, activates the enzyme, PKA (Cyclic AMP
dependent protein kinase).
Cyclic AMP binds to the regulatory subunits of PKA so that the catalytic subunits
having kinase activity can phosphorylate proteins.
The cascade amplification effect is seen in this series of activation reactions.
 This PKA is a tetrameric molecule

27. Second Messenger Activates PKA
Catalytic subunit is now free to act having two regulatory (R) and two catalytic
(C) subunits (R2 C2) (fig).
This complex has no activity.
 But cAMP binds to the regulatory subunit and dissociates the tetramer into
regulatory and catalytic subunits .
Theatalytic subunit is now free to act.

28. Kinase Phosphorylates the Enzymes
The catalytic subunit then transfers a phosphate group from ATP to different
enzyme proteins (Fig. 50.4-5).
Phosphorylation usually takes place on the OH groups of serine, threonine or
tyrosine residues of the substrates.
Hence, these kinases are called Ser/Thr kinases.
 The enzymes may be activated or inactivated by this phosphorylation.
This is an example of covalent modification.

29. Kinase Phosphorylates the Enzymes
Glycogen phosphorylase and hormone sensitive lipase are controlled by cyclic
AMP.

31. There are Many G-proteins
About 30 different G-proteins are identified, each being used for different signal
transduction pathways.
The G-protein, which stimulates adenyl cyclase, is called Gs (G-stimulatory) and
the opposite group is called Gi (G-inhibitory).
 An example of inhibitory G-protein is the inhibition of adenylate kinase.
The alpha subunit of the Gs and Gi are different, but beta and gamma are the
same .
 G-proteins are also involved in toxic manifestations of cholera and pertussis.
 Mutations in gene encoding the alpha subunit of Gs-protein or abnormalities in
G-protein signaling have been found to result in the action of toxins .

33. Abnormal G-protein signaling
Cholera toxin :
Is encoded by a bacteriophage present inside the bacteria Vibrio cholerae.
The enterotoxin contains two A subunits and 5 B subunits.
 The B subunit binds to a ganglioside GM1 on the surface of intestinal mucosal
cell.
The A subunit then enters into the inner part of the membrane, which leads to
ribosylation of the alpha subunit of Gs protein.
This results in the inhibition of the inherent GTPase activity and irreversible
activation of G protein.
. Adenyl cyclase remains continuously active and keeps cyclic AMP levels high.
This prevents absorption of salts from intestine leading to watery diarrhea and
loss of water from body.
In the large intestine, chronic elevation of cAMP results in a sustained PKA
mediated phosphorylation of chloride channels (CFTRs) that normally regulate
salt and water transport.
Hyperactivity of these channels will result in loss of sodium chloride with watery
diarrhea (liquid stools), that may have fatal results.
The patient may lose as much as 1 L of water per hour

34. Abnormal G-protein signaling-
Cholera toxin :
 Adenyl cyclase remains continuously active and keeps cyclic AMP levels high.
This prevents absorption of salts from intestine leading to watery diarrhea and
loss of water from body.
In the large intestine, chronic elevation of cAMP results in a sustained PKA
mediated phosphorylation of chloride channels (CFTRs) that normally regulate
salt and water transport.
Hyperactivity of these channels will result in loss of sodium chloride with watery
diarrhea (liquid stools), that may have fatal results.
The patient may lose as much as 1 L of water per hour

35. Abnormal G-protein signaling-
Pertussis toxin :
Ribosylates the alpha subunit of Gi-protein and prevents the Gi-GDP complex
from interacting with the activated receptor.
 Hence, the action of hormones acting through Gi is inhibited.

36. Abnormal G-protein signaling-
Clostridium tetani :Effects of bacterial toxins from Clostridium tetani are exerted
through proteases, that attack proteins involved in synaptic vesicle and plasma
membrane fusion.
 The toxin has two polypeptides, one of which binds to cholinergic motor
neurons and facilitates the entry of the second polypeptide.
It is a protease that cleaves the protein necessary for vesicle fusion.
 Failure to release the neurotransmitter leads to fatal paralysis of the chest
muscles.

37. Abnormal G-protein signaling-
Gowth hormone/ acth secreting tumors of the pituitary:
Mutations in gene encoding the alpha subunit of Gs-protein (gsp gene) has been
found to result in decreased GTPase activity of the alpha subunit, leading to
continued activation of Gs alpha and adenyl cyclase.
 The resultant increase in cAMP has been found to lead to PKA-dependent
phosphorylation of cyclic AMP sensitive gene regulatory proteins.
Over-expression of cAMP inducible genes has been found to produce growth
hormone/ ACTH secreting tumors of the pituitary.

38. There are Many Protein Kinases
More than thousand protein kinases are now known.
Some important hormone responsive protein kinases are, cAMP-dependent
kinases, epidermal growth factor-dependent tyrosine kinase, insulin-dependent
tyrosine kinase.
All the known effects of cAMP in eukaryotic cells result from activation of
protein kinases, which are serine/threonine kinases

39. HSL:Hormone-sensitive lipase, CREBs:cAMP response element-binding
protein Ca2+/calmodulin-dependent protein kinase class of enzymes, kinase (JAK):
Receptor tyrosine kinases (RTKs), Janus is a family of intracellular, nonreceptor tyrosine Janus
kinase (JAK): kinases that transduce cytokine-mediated signals via the JAK-STAT pathway.,( JAK-
STAT signaling pathway: a chain of interactions between proteins in a cell, and is involved in
processes such as immunity, cell division, cell death and tumour formation.

40. Glycogen Phosphorylase is a Typical Example
Glycogen phosphorylase and hormone sensitive lipase are activated by cAMP
mediated cascade .
The termination of the effect of the hormonal action by phosphorylation is
effected by the action of protein hormone through G-protein phosphatases.
Ex. glycogen phosphorylase becomes inactive in the dephosphorylated state.
 But, glycogen synthase is active in dephosphorylated state .
Certain enzymes are activated by dephosphorylation .
Hepatic Protein Phosphatase-1 is a typical example where the enzyme

43. Protein Phosphatase-1
itself is inhibited by phosphorylation of its regulatory subunit.
 When cyclic AMP level falls, the regulatory subunit is dephosphorylated and
protein phosphatase becomes active, which in turn hydrolyzes phosphate group
from the enzyme.
Protein kinases as well as protein phosphatases are involved in the action of
different hormones.

44. The actions of cAMP in eukaryotic cells
A. Activation of protein kinase and phosphorylation of effector proteins like
enzymes and ion channels.
 These enzymes may directly phosphorylate enzymes or secondary kinases that
phosphorylate other enzyme.
i. PKA phosphorylates hormone sensitive lipase thus activating it.
 ii. Phosphorylase kinase that phosphorylates glycogen phosphorylase.
 iii. When ion channel proteins or transporters are phosphorylated, the
membrane potential is modified, thus regulating the influx of calcium.

45. The actions of cAMP in eukaryotic cells
B. cAMP also has a long lasting effect on gene expression.
 The translocation of the active PKA subunits to the nucleus induces
phosphorylation of cAMP regulated gene regulatory proteins or CREBs (cAMP
response element binding protein).
 These proteins will bind to cAMP sensitive regulatory elements (CRE) on genes,
thus controlling their expression.

46. The actions of cAMP in eukaryotic cells
C. G-protein mediated signal transduction also requires scaffold and adapter
proteins that increase the fidelity and speed of a signaling cascade.
 Anchoring proteins localize and concentrate the signaling proteins at their site
of action.
 The interaction of these proteins involve specific domains within the protein like
SH2 (Src homology type 2), PTB (phosphor tyrosine binding), etc.

47. CALCIUM-BASED SIGNAL TRANSDUCTION

48. CALCIUM-BASED SIGNAL TRANSDUCTION
Calcium is an important intracellular regulator of cell function like contraction of
muscles, secretion of hormones and neurotransmitters, cell division and
regulation of gene regulation.
 Rapid but transient increase in cytosolic calcium result from either opening of
calcium channels in the plasma membrane or calcium channels in the ER.
The released calcium can be rapidly taken-up by ER to terminate the response.

49. CALCIUM-BASED SIGNAL TRANSDUCTION
The intracellular calcium concentration is low (10-7) where
as extracellular calcium concentration is very high (10-3),
maintaining a 10,000 fold calcium gradient across the
membrane.
The inside has a negative potential therefore influx of
calcium is rapid.
Even small increase in cytosolic free calcium can have
maximal effect on calcium regulated cellular functions.

50. CALCIUM TRANSPORT
There are mainly 3 types of calcium transport systems:
a. Voltage gated calcium channels
b. Sodium/calcium antiport transporter
c. Calcium transporting ATPase.

51. CALCIUM TRANSPORT continue……
The calcium transporting ATPase transporter accumulates calcium within the
lumen of ER (sarcoplasmic reticulum) in muscle.
These calcium ions can be released into the cytoplasm by an inositol
triphosphate (IP3) gated calcium channel or by a ligand gated calcium release
channel (ryanodine receptor).

52. CALCIUM TRANSPORT continue……
When cytosolic calcium increases, binding regulatory proteins, activation of
several calcium binding regulatory proteins occurs.
 Calmodulin is expressed in various tissues and mediates the regulatory actions
of calcium ions.
 Calcium binding causes conformational change in calmodulin resulting in
interaction with kinases, phosphatases, NOS, etc.
 Some of these CAM kinases can phosphorylate a wide range of proteins that
alter cellular functions.
When bound to calmodulin, CAM kinase II also autophosphorylates, so that its
activity is sustained.
 Intracellular calcium acts as a mediator of hormone action either independently
or in conjunction with cAMP ( Eg. phosphorylase kinase reaction)

54. Hormones can increase the cytosolic calcium
level by the following mechanisms:
A. By altering the permeability of the membrane.
B. The action of Ca-H+-ATPase pump which extrudes calcium in
exchange for H+.
C. By releasing the intracellular calcium stores.
D. Calmodulin, the calcium dependent regulatory protein within
the cell has four calcium binding sites.
When calcium binds there is a conformational change to the
calmodulin, which has a role in regulating various kinases.
 Calmodulin is a 17 kDa protein which has structural and
functional similarity with the muscle protein troponin C.
 Eg.Adenyl cyclase, calcium-dependent protein kinases, calcium-
magnesium-ATPase, cyclic nucleotide phosphodiesterase, nitric
oxide synthase and phosphorylase kinase.

55. HORMONES ACTING THROUGH PIP2 CASCADE

56. HORMONES ACTING THROUGH PIP2 CASCADE
The major player in this type of signal transduction is phospholipase C that
hydrolyses phosphatidyl inositol in membrane lipids to 1,4,5-Inositol
triphosphate (IP3) and Diacyl Glycerol (DAG) that act as second messengers.
 PIP3 (Phosphatidyl Inositol 3,4,5- phosphate) is another second messenger
produced by the action of a phosphoinositide kinase.
 The phospholipase C may be activated either by G-proteins or calcium ions.
 DAG can also be generated by the action of phospholipase D that produces
phosphatidic acid which is hydrolyzed to DAG.

57. HORMONES ACTING THROUGH PIP2 CASCADE
continue…………..
The binding of hormones like serotonin to cell surface receptor triggers the
activation of the enzyme phospholipase-C which hydrolyzes the phosphatidyl
inositol to diacylglycerol.
 IP3 can release Ca++ from intracellular stores, such as from endoplasmic
reticulum and from sarcoplasmic reticulum .
The elevated intracellular calcium then triggers processes like smooth muscle
contraction, glycogen breakdown and exocytosis.

59. HORMONES ACTING THROUGH PIP2 CASCADE
continue…………..
PIP3 can be formed by the action of PI3-kinases that are activated through
growth factors and cytokine mediated receptor tyrosine kinases.
PIP3 which is a lipid second messenger has a role in regulation of cell motility,
membrane trafficking and cell survival signaling pathways.
The major mediator of PIP3 action is PKB (Protein kinase B) which has a role in
glucose transport, glycogen metabolism and cell death signaling pathways.
Active PKB/Akt is the major mediator of PIP3 action.

60. HORMONES ACTING THROUGH PIP2 CASCADE
continue…………..
It represses the activity of cell death signaling pathways.
The PDK (Phosphatidyl inositol dependent kinase) and IP3 kinase are also
involved in glucose transport and glycogen metabolism.
 There is “cross talk” between the various signal transduction pathways that are
coordinately regulated.

61. Diacylglycerol Pathway
Diacylglycerol (DAG), the messenger formed by the hydrolysis of PIP2 activates
protein kinase C (PKC) which in turn would phosphorylate other target proteins.
 PKC activates several serine threonine kinases that phosphorylate several
substrates including transcription factors, ion channels and transporters.
 Most effects of IP3 and DAG are found to be synergistic.
DAG also increases the affinity of protein kinase-C for calcium.
The enzymes are thus activated, even at physiological levels of calcium within
the cell.

62. ROLE OF CYCLIC GMP

63. ROLE OF CYCLIC GMP
Cyclic GMP (cGMP) is another important second messenger involved in
contractile function of smooth muscles, visual signal transduction and
maintenance of blood volume.
 Cyclic GMP degradation is catalyzed by membrane bound PDEs.
i. It is formed from GTP by the action of guanyl cyclase.
 Several compounds have been found to increase the concentration of cGMP by
activating guanyl cyclase.
ii. These include drugs like nitroprusside, nitroglycerin, sodium nitrite and
atriopeptides (a group of peptides produced by atrial cardiac tissue).
 All these compounds act as potent vasodilators, by inhibiting the
phosphodiesterase

64. Mechanism of Action of Nitric Oxide NO
Mechanism of Action of Nitric Oxide NO diffuses to the adjacent smooth muscle
and activates guanylate cyclase.
Increased level of cyclic GMP activates protein kinase in smooth muscles, which
causes dephosphorylation of myosin light chains, leading to relaxation of
muscles.
 Thus NO is a vasodilator

65. ROLE OF CYCLIC GMP continue…..
iii. Cyclic GMP activates cGMP-dependent protein kinase G
(PKG), which phosphorylates important effector proteins that can
regulate calcium dependent contraction or motility by
modulating calcium influx.
 An example is smooth muscle myosin, leading to relaxation and
vasodilatation.
iv. Cyclic GMP is also involved in the rhodopsin cycle.
 The role of cGMP in the light sensing cells of retina and its
interaction with the G-protein transducin is described under
visual cycle.
v. NO (Nitric oxide) is the major activator of guanylate cyclase.
NO in turn is produced by the action of NOS (Nitric oxide
synthase) in tissues like vascular endothelial.
 NO can easily diffuse through the membrane and activate
guanylate cyclase.

66. Fig. The role of
retinaldehyde in the visual
cycle.

67. ROLE OF CYCLIC GMP continue…..
Increased level of cyclic GMP in smooth muscle triggers rapid and sustained
relaxation of the smooth muscles.
 The vasodilatation resulting from NO induced increase in cGMP has great
physiological and pharmacological significance.
The drugs that act via NO release are nitroprusside, nitrites (used in angina as
coronary vasodilators) and sildenaphil citrate (Viagra).

68. HORMONES WITH
INTRACELLULAR
RECEPTORS

69. Hormones with Intracellular Receptors :
i. The hormones in this group include the steroid
hormones and thyroid hormones.
ii. They diffuse through the plasma membrane and
bind to the receptors in the cytoplasm (Fig).

71. Hormones with Intracellular Receptors
CONTINUE……………..
ii. The hormone receptor (HR) complex is formed in
the cytoplasm.
 The complex is then translocated to the nucleus.
 Steroid hormone receptor proteins have a
molecular weight of about 80–100 kD.
Each monomer binds to a single steroid molecule at
a hydrophobic site, but on binding to genes they
dimerize (Fig).

73. Hormones with Intracellular Receptors: (HRE)CONTINUE……………..
In the nucleus, the HR binds to the hormone response
elements (HRE) or steroid response elements (SRE) (Table ).
 The SRE acts as an enhancer element and when stimulated
by the hormone, would increase the transcriptional activity
(Fig).
 The newly formed mRNA is translated to specific protein,
which brings about the metabolic effects.
Binding to the SRE sequence leads to dimerization of the
receptor.
Steroid hormones influence gene expression, so that the
rate of transcription is increased.
The stability of mRNA is also increased.
This would lead to induction of protein synthesis.
 Steroid receptors have been found to enhance initiation of
transcription by formation of complexes at promoters (Fig).

75. Hormones with Intracellular Receptors:
(HRE)CONTINUE……………..
•iv. Best examples of the effect of hormones on genes
are:
• a. The induction of synthesis of amino transferases
by glucocorticoids.
• b. Synthesis of calcium binding protein by calcitriol
(see Fig. 36.10).

77. INSULIN SIGNALING PATHWAY