Prof.Dr. Hedef Dhafir El-Yassin 2013
1
Introduction
to the
Mechanism of
Action and
the Control
of Endocrine
Activity
2013
Prof. Dr. Hedef Dhafir El-Yassin
Prof.Dr. Hedef Dhafir El-Yassin 2013
2
Introduction to the Mechanism of Actions and
the Control of Endocrine Activity
Lecture 2
Sunday 24/2/2013
Objectives:
1. to understand the mechanism of action of hormones
2. to understand the basis of localization of receptors within the cell.
3. to know the four second messenger systems recognized in cells
4. to understand the control of endocrine activity
Mechanism of Action of Hormone:
Understanding mechanism of action is not only of great interest to basic
science, but critical to understanding and treating diseases of the endocrine
system, and in using hormones as drugs.
There are two fundamental mechanisms by which a hormone can change its
target cell. These mechanisms are:
Activation of enzymes and other dynamic molecules: Most
enzymes fluctuate between conformational states that are catalytically
active versus inactive. Many hormones affect their target cells by
inducing such transitions, usually causing an activation of one of more
enzymes. Because enzymes are catalysts and often serve to activate
additional enzymes, a seemingly small change induced by hormone-
receptor binding can lead to widespread consequences within the cell.
Modulation of gene expression: Stimulating transcription of a group of
genes clearly can alter a cell's phenotype by leading to a burst of
synthesis of new proteins. Similarly, if transcription of a group of
previously active genes is shut off, the corresponding proteins will soon
disappear from the cell.
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More specifically, when a receptor becomes bound to a hormone, it
undergoes a conformational change which allows it to interact
productively with other components of the cells, leading ultimately to an
alteration in the physiologic state of the cell.
Despite the molecular diversity of hormones, all hormone receptors can be
categorized into one of two types, based on their location within the cell:
Location of Receptor
Classes of Hormones
Principle Mechanism
of Action
Cell surface receptors
(plasma membrane)
Proteins and peptides,
catecholamines and
eicosanoids
(water soluble)
Generation of second
messengers which alter
the activity of other
molecules - usually
enzymes - within the cell
Intracellular receptors
(cytoplasm and/or
nucleus)
Steroids and thyroid
hormones
(lipid soluble)
Alter transcriptional
activity of responsive
genes
Quick quiz 1:
Hormone receptors are not present in:
a. Mitochondria
b. Plasma membrane
c. Cytoplasm
d. Nucleus
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1. Hormones with Cell Surface Receptors
Protein and peptide hormones, catecholamines like epinephrine, and
eicosanoids such as prostaglandins find their receptors decorating the
plasma membrane of target cells. Binding of hormone to receptor initiates a
series of events which leads to generation of so-called second messengers
within the cell (the hormone is the first messenger). The second messengers
then trigger a series of molecular interactions that alter the physiologic state
of the cell. Another term used to describe this entire process is signal
transduction.
Cell surface receptors are integral membrane proteins and, as such, have
regions that contribute to three basic domains:
Extracellular domains:
Some of the residues exposed to the outside of the
cell interact with and bind the hormone - another term for these regions is the
ligand-binding domain.
Transmembrane domains:
Hydrophobic stretches of amino acids are
"comfortable" in the lipid bilayer and serve to anchor the receptor in the membrane.
Cytoplasmic or intracellular domains:
Tails or loops of the receptor that
are within the cytoplasm react to hormone binding by interacting in some way with
other molecules, leading to
generation of second
messengers.
As shown below, some
receptors are simple, single-
pass proteins; many growth
factor receptors take this
form. Others, such as the receptor for insulin, have more than one subunit.
Another class, which includes the beta-adrenergic receptor, is threaded
through the membrane seven times.
Interaction of the hormone-bound receptor with other membrane or
cytoplasmic proteins is the key to generation of second messengers and
transduction of the hormonal signal.
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Second Messenger Systems
Nonsteroid hormones (water soluble) do not enter the cell but bind to plasma
membrane receptors, generating a chemical signal (second messenger)
inside the target cell. Second messengers activate other intracellular
chemicals to produce the target cell response.
The action of nonsteroid hormones. Images from Purves et al., Life: The Science of Biology, 4th Edition, by
Sinauer Associates
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Currently, four second messenger systems are recognized in cells, as
summarized in the table below. Note that not only do multiple hormones
utilize the same second messenger system, but a single hormone can utilize
more than one system.
Second Messenger
Examples of Hormones Which Utilize This
System
Cyclic AMP
Epinephrine and norepinephrine, glucagon,
luteinizing hormone, follicle stimulating
hormone, thyroid-stimulating hormone,
calcitonin, parathyroid hormone, antidiuretic
hormone
Protein kinase activity
Insulin, growth hormone, prolactin, oxytocin,
erythropoietin, several growth factors
Calcium and/or
phosphoinositides
Epinephrine and norepinephrine, antidiuretic
hormone, gonadotropin-releasing hormone,
thyroid-releasing hormone.
Cyclic GMP
Atrial naturetic hormone, nitric oxide
Quick quiz 2: for all the following, the second messenger is a kinase or
phosphate except:
a. Glucagons
b. Erythropoietin
c. Insulin
d. Growth hormone
In all cases, the seemingly small signal generated by hormone binding
its receptor is amplified within the cell into a cascade of actions that
changes the cell's physiologic state. Presented below are two examples of
second messenger systems commonly used by hormones.
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1. Cyclic AMP Second Messenger Systems
Cyclic adenosine monophosphate (cAMP) is a nucleotide generated from
ATP through the action of the enzyme adenylate cyclase. The intracellular
concentration of cAMP is increased or decreased by a variety of hormones
and such fluctuations affect a variety of cellular processes. One prominent
and important effect of elevated concentrations of cAMP is activation of a
cAMP-dependent protein kinase called protein kinase A.
Protein kinase A is nominally in a catalytically-inactive state, but becomes
active when it binds cAMP. Upon activation, protein kinase A phosphorylates
a number of other proteins, many of which are themselves enzymes that are
either activated or suppressed by being phosphorylated. Such changes in
enzymatic activity within the cell clearly alter its state.
Simple Example: mechanism of action of glucagon:
Glucagon binds its receptor in the plasma membrane of target cells
(e.g. hepatocytes).
Bound receptor interacts with and, through a set of G proteins, turns on
adenylate cyclase, which is also an integral membrane protein.
Activated adenylate cyclase begins to convert ATP to cyclic AMP,
resulting in an elevated intracellular concentration of cAMP.
High levels of cAMP in the cytosol make it probable that protein kinase
A will be bound by cAMP and therefore catalytically active.
Active protein kinase A "runs around the cell" adding phosphates to
other enzymes, thereby changing their conformation and modulating
their catalytic activity .
Levels of cAMP decrease due to destruction by cAMP-
phosphodiesterase and the inactivation of adenylate cyclase.
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In the above example, the hormone's action was to modify the activity of pre-
existing components in the cell. Elevations in cAMP also have important
effects on transcription of certain genes.
Quick quiz 3:
Which of the following statements concerning G
proteins is correct?
a. G proteins bind the appropriate hormone at the cell surface.
b. G proteins interact with cytoplasmic receptors
c. G proteins are second messengers
d. G proteins causes the regulatory subunits f the protein kinase to
dissociate from the catalytic subunits
e. Activated G protein may either activate or inhibit the
production o second messenger.
Quick quiz 4:The signal transducer in cholera is:
a. Cyclic nucleotide
b. Phosphoinositide
c. Inhibitory G-protein
d. Stimulatory G-protein
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2. Tyrosine Kinase Second Messenger Systems
The receptors for several protein hormones are themselves protein
kinases which are switched on by binding of hormone. The kinase
activity associated with such receptors results in phosphorylation of tyrosine
residues on other proteins. Insulin is an example of a hormone whose
receptor is a tyrosine kinase.
The hormone binds to domains exposed on the cell's surface, resulting in a
conformational change that activates kinase domains located in the
cytoplasmic regions of the receptor. In many cases, the receptor
phosphorylates itself as part of the kinase activation process. The activated
receptor phosphorylates a variety of intracellular targets, many of which are
enzymes that become activated or are inactivated upon phosphorylation.
As seen with cAMP second messenger systems, activation of receptor
tyrosine kinases leads to rapid modulation in a number of target proteins
within the cell. Some of the targets of receptor kinases are protein
phosphatases which, upon activation by receptor tyrosine kinase, become
competent to remove phosphates from other proteins and alter their activity.
Again, a seemingly small change due to hormone binding is amplified into a
multitude of effects within the cell.
Quick quiz 5:
Tyroisine kinase activity is distributed in :
a.
and
β- adrenergic receptors
b. Insulin receptors
c. Cholinergic receptors
d. All the above
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2.
Hormones with Intracellular Receptors
Receptors for steroid and thyroid hormones are located inside target cells, in
the cytoplasm or nucleus, and
function as ligand-dependent
transcription factors. The
hormone-receptor complex
binds to promoter regions of
responsive genes and stimulate
or sometimes inhibit
transcription from those genes.
Thus, the mechanism of
action of these hormones is
to modulate gene expression
in target cells. By selectively affecting transcription from a battery of genes,
the concentration of those respective proteins are altered, which clearly can
change the phenotype of the cell.
Structure of Intracellular Receptors
Steroid and thyroid hormone receptors are members of a large group of
transcription factors. All of these receptors are composed of a single
polypeptide chain that has, three distinct domains:
The amino-terminus
:
In most cases, this region is involved in activating or
stimulating transcription by interacting with other components of the transcriptional
machinery. The sequence is highly variable among different receptors.
DNA binding domain
:
Amino acids in this region are responsible for binding of
the receptor to specific sequences of DNA
.
The carboxy-terminus or ligand-binding domain
:
This is the region that
binds hormone
.
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In addition to these three core domains, two other important regions of the
receptor protein are a nuclear localization sequence, which targets the protein
to nucleus, and a dimerization domain, which is responsible for latching two
receptors together in a form capable of binding DNA.
Hormone-Receptor Binding and Interactions with DNA
Being lipids, steroid hormones enter the cell by simple diffusion across the
plasma membrane. Thyroid hormones enter the cell by facilitated diffusion.
The receptors exist either in the cytoplasm or nucleus, which is where they
meet the hormone. When hormone binds to receptor, a characteristic
series of events occurs:
Receptor activation
is the term used to describe conformational
changes in the receptor induced by binding hormone. The major
consequence of activation is that the receptor becomes competent to
bind DNA.
Activated receptors bind to hormone response elements
, which are
short specific sequences of DNA which are located in promoters of
hormone-responsive genes.
Transcription from those genes to which the receptor is bound is
affected.
Most commonly, receptor binding stimulates transcription.
The hormone-receptor complex thus functions as a transcription factor.
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Steroid Hormones
The second mechanism involves steroid hormones, which pass through the
plasma membrane and act in a two step process. Steroid hormones bind,
once inside the cell, to the nuclear membrane receptors, producing an
activated hormone-receptor complex. The activated hormone-receptor
complex binds to
DNA and
activates specific
genes,
increasing
production of
proteins.
Quick quiz 6:
Which is not true regarding steroid hormone?
a. They are lipophilic in nature
b. Their receptors are intracellular
c. They require cyclic AMP as second messenger
d. They require specific transporters to carry them in circulation
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Control of Endocrine Activity
The physiologic effects of hormones depend largely on their concentration in blood and
extracellular fluid.
The concentration of hormone as seen by target cells is determined by three factors:
1. Rate of production
:
Synthesis and secretion of hormones are the most highly
regulated aspect of endocrine control. Such control is mediated by positive and
negative feedback circuits.
2. Rate of delivery
:
An example of this effect is blood flow to a target organ or group
of target cells:
TRANSPORT OF HORMONES: hormones must be transported at least some distance to
their target organs. The primary transport medium is the plasma, although the lymphatic
system and the cerebrospinal fluid are also important. Since delivery of the hormone to its
target tissue is required before a hormone can exert its effects, the presence or absence of
specific transport mechanisms play a major role in mediating hormonal action.
A) The water-soluble hormones (peptide hormones, catecholamines) are transported
in plasma in solution and require no specific transport mechanism. Because of this,
the water-soluble hormones are generally short-lived. These properties allow for
rapid shifts in circulating hormone concentrations, which is necessary with the
pulsatile tropic hormones or the catecholamines. This is consistent with the
rapid onset of action of the water-soluble hormones.
B) The lipid-soluble hormones (thyroid hormone, steroids) circulate in the plasma
bound to specific carrier proteins. Many of the proteins have a high affinity for
specific hormone, such as thyroxine-binding globulin (TBG), sex hormone-
binding globulin (SHBG), and cortisol-binding globulin (CBG). Non-specific, low-
affinity] binding of these hormones to albumin also occurs. Carrier proteins
act as reservoirs of hormone. Since it is generally believed that only the free hor-
mone can enter cells, a dynamic equilibrium must exist between the bound and
free hormone. Thus, alterations in the amount of binding protein available, or in
the affinity of the hormone for the binding protein, can markedly alter the total
circulating pool of hormone without affecting the free concentration of hormone.
Carrier proteins act as buffers to both blunt sudden increases in hormone
concentration and to diminish degradation of the hormone once it is secreted. Thus, the
half-life of hormones that utilize carrier proteins is longer than those that are not protein-
bound. Indeed, carrier proteins have a profound effect on the clearance rate of hormones;
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the greater the capacity for high affinity binding of the hormone in the plasma, the slower
the clearance rate. Also, the carrier proteins allow slow, tonic delivery of the hormone to
its target tissue. This is consistent with the slower onset of action of the lipid-soluble
hormones.
3. Rate of degradation and elimination
:
Hormones, like all biomolecules, have
characteristic rates of decay, and are metabolized and excreted from the body
through several routes.
HORMONE METABOLISM: Clearance of hormones from the circulation plays a critical role
in the modulation of hormone levels in response to varied physiologic and pathologic
processes. The time required to reach a new steady-state concentration in response to
changes in hormone release is dependent upon the half-life of the hormone in the serum.
Thus, an increase in hormone release or administration will have a much more marked effect
if the hormone is cleared rapidly from the circulation as opposed to one that is cleared more
slowly.
Most peptide hormones have a plasma half-life measured in minutes, consistent with the
rapid actions and pulsatile nature of the secretion of these hormones. This rapid clearance
is achieved by the lack of protein binding in the plasma, degradation or internalization of the
hormone at its site of action, and ready clearance of the hormone by the kidney. Binding to
serum proteins markedly decreases hormone clearance, as is observed with the steroid
hormones and the iodothyronines. Metabolism of the steroid hormones occurs primarily in
the liver by reductions, conjugations, oxidations, and hydroxylations, which serve to
inactivate the hormone and increase their water-solubility, facilitating their excretion in the
urine and the bile. Metabolic transformation also may serve to activate an inactive hormone
precursor, such as the deiodination of thyroxine to form T
3
. Hormone metabolism is not as
tightly regulated as is hormone synthesis and release. However, alterations in the metabolic
pathways may be clinically important.
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Conclusions:
1. There are two fundamental mechanisms by which a hormone can
change its target cell.
a. activation of enzymes
b. modulation of gene expesion
2. all hormone receptors can be categorized into one of two types, based
on their location within the cell either to
a. Cell surface receptors (plasma membrane) or
b. Intracellular receptors (cytoplasm and/or nucleus)
3. not only do multiple hormones utilize the same second messenger
system, but a single hormone can utilize more than one system.
4. The concentration of hormone as seen by target cells is determined by
three factors
a. Rate of production
b. Rate of delivery
c. Rate of elimination