Mechanisms of Hormonal Action

Bryant Miles
Multicellular organisms need to coordinate metabolic activities. Complex signaling systems have evolved
using chemicals called hormones to regulate cellular activities. Animals have an endocrine systems
consisting of glands that release hormones into the blood stream where the hormones are carried through
the body where they reversibly bind to their target receptors.
Mechanisms of Hormone Action
1. If the substrate concentration is rate limiting, then hormones may alter the concentration of the
substrate to increase or decrease the rate of flux.
2. Hormones may promote the reversible phosphorylation of the flux controlling enzymes to change
the conformation of the enzymes active site either activating or deactivating the enzyme.
3. Hormones may promote the dephosphorylation of the flux controlling enzymes.
4. Hormones can affect the concentrations of allosteric effectors.
5. Hormones can induce or repress genes to change the amount of enzyme present in the cell.
We have already studied the peptide hormones insulin and
glucagons, and the catecholamines: epinephrine and
norepinephrine. These hormones bind to receptors of the target
cells. These hormones have high affinity for their target
receptors with KD values ranging from 10-12 to 10-6 M. Only a
minute amount of the hormones are required to induce
response. The binding of the hormone stimulates a chemical
activity that is communicated into the cell. Steroids are
another type of hormone. Steroids are derived from cholesterol
and regulate metabolism, electrolyte balance, inflammatory
responses and sexual function. Steroid hormones occasionally
bind to cellular receptors to induce their effects. Steroids are
lipids. That means they are permeable to biological
membranes. Most steroids passively diffuse into the nucleus
where the bind to transcription factors directly regulating
genetic expression of genes.
Hormones provide a mechanism to maintain homeostasis, to respond to changing metabolic conditions
and to regulate cellular differentiation and genetic expression.
Signal transduction begins when a hormone is released into the blood stream. The hormone is the called
the first messenger. The hormone binds to specific cell membrane receptors. The information that the
signal molecules are present must be transmitted across the cell membrane. The binding of the hormone
to the receptor drives conformational changes that produce a response in the cytostolic side of the cell.
The response results in the change of concentration of small molecules called the second messengers in
the inside of the cell. Second messengers include cAMP, cGMP, Ca2+, inositol 1,4,5 triphosphate (IP3)
and diacylglycerol, DAG.

These second messengers can diffuse to cellular compartments, such as the nucleus where the second
messenger influences gene expression. The signal carried by the hormone becomes amplified, as one
hormone bound to the receptor can instigate the formation of 100s of second messenger molecules.
One common response to a second messenger is the activation of protein kinases which use ATP to
phosphorylate serine, threonine and tyrosine residues of the target enzymes. This phosphorylation is
reversible due to protein phosphatases which are enzymes that remove the phosphoryl groups from the
serine, threonine and tyrosine residues.

Eventually the signal needs to be terminated. Otherwise the cells would lose responsiveness to new
signals.
Three types of hormone receptors.
1. The 7-transmembrane segment (7-TMS) receptors which are integral membrane proteins with
seven transmembrane helical segments. Examples are the G-binding proteins.
2. The single transmembrane segment (1-TMS) catalytic receptors which are proteins that a single
transmembrane helix that spans the membrane. Examples are tyrosine kinases and guanylate
cyclases.
3. Oligomeric ion channels which consist of multiple protein subunits. These channels are also
called ligand gated channels because the bind of the hormone to the receptor opens the ion
channel.

7-TMS Receptors.
7-TMS receptors are involved in transmitting information initial by signals
such as photons of light, odorants, hormones and neurotransmitters. So far
7,000 7-TMS receptors have been discovered and the list is rapidly growing.
One example, is the adrenergic receptor which binds epinephrine aka
adrenaline the fight or flight hormone. The binding of epinephrine to this
receptor located on the outside of the cell induces a conformational change
that is detected inside of the cell. The binding of epinephrine activates a G
protein. The activated G protein in turn activates adenylyl cyclase which
converts ATP into cAMP and pyrophosphate.

G Proteins
G-proteins are intermediaries in signal transduction from 7-TMS receptors. They are called G-proteins
because they containing binding sites for guanosine nucleotides. In the case of the -adrenergic receptor,
the resting G-protein is a heterotrimer consisting of , and subunits. The subunit contains the
guanosine nucleotide binding site. In the resting state, the subunit has GDP bound and associates with
receptors such as the glucagon receptor or the -adrenergic receptor. The binding of the hormone to the
receptor produces allosteric conformational changes that cause the subunit to release GDP and bind
GTP. The binding of GTP is a switch which causes the subunit to dissociate from the G dimer. The
GTP bound subunit diffuses laterally through the membrane until is associates with adenylate cyclase.
The association of these two proteins activates adenylate cyclase which then starts producing cAMP. A
single hormone bound to its receptor can activate 100s of G molecules.
NH2
N
O
-

O P O P O P O
-

O-

H
H
HO

NH2
N

N
O

H
H
OH

H
O P
O-

Adenylate Cyclase

H
H
OH

N
N

NH2
N
N
O
H
O P

-O P O

H
H
OH

Phosphodiesterase
CH3
N

O-

H3C

NH2

O-

H
H

OH

H
H
OH

Second messengers need to have short half lives so that the response can be
rapidly terminated. Phosphodiesterase hydrolyzes the phosphodiester bond to
convert cAMP to AMP. This is the enzyme that is inhibited by caffeine one of
my favorite biomolecules. Caffeine increases the cellular concentration of
cAMP by inhibiting phosphodiesterase.

The G proteins ultimately need to reset themselves. The G subunit has an

intrinsic GTPase activity. The bound GTP will slowly be hydrolyzed into GDP
and Pi. This GTPase activity is like a built in clock that spontaneously resets
Caffeine
the G subunit after a short period of time. After the G subunit has
hydrolyzed GTP it tightly binds the GDP. When the G subunit had GDP bound it dissociates from
adenylate cyclase turning this enzyme off and reassociates with the G dimer to reform the heterotrimer.
This requires the hormone to be bound to the receptor to keep adenylate cyclase active.
CH3

Every 7-TMS receptor has a G type protein associated with it. They do not all function through the same
G-protein used by the glucagon and -adrenergic receptors. The typical G-proteins are heterotimers
consisting of , and subunits in the resting state. The binding of the hormone to the receptor causes
the exchange of GDP for GTP in the subunit. The G subunit then dissociates from the G dimer and
associates with an effector protein. Eventually the G subunit hydrolyzes the GTP, binds the GDP tightly,
dissociates from the effector protein and reassociates with the G dimer to reform the heterotrimer.
Glucagon and epinephrine bind to different receptors yet activate the same G protein which in turn
activates the same effector protein, adenylate kinase. Other effector proteins activated by other G proteins
are phospholipase C, phospholipase A2,
potassium channels, sodium channels,
calcium channels. There are more than 20
different G-proteins discovered to date. A
few are listed to the left.
The hormone-receptor mediated processes
regulated by G proteins may be
stimulatory as in the example of the
epinephrine, -adrenergic receptor, or
inhibitory. Each G-protein interacts with
a stimulatory G-protein denoted Gs or
with an inhibitory G protein denoted Gi.
Epinephrine also binds to a -adrenergic
receptor. The -adrenergic receptor associates with a Gi protein. The binding of epinephrine to the aadrenergic receptor causes the exchange of GDP for GTP causing the Gi to dissociate from the Gi

dimer. The inhibition comes from either the Gi subunit associated with adenylate cyclase to directly
inhibit the cyclase, or by the action of Gi which associates with the Gs subunit when it has GTP bound.
The Gi thus competes with adenylate cyclase for Gs.

Cholera Toxin
Cholera is a gram positive bacterium that causes cholera. Both the cholera bacteria and the toxin remain
localized in the intestinal epithelial cells. Cholera causes severe diarrhea in its victims which often leads
to death due to severe dehydration. The cholera toxin is protein that catalyzes the ADP-ribosylation of
Arg-201 of the Gs subunit we have been talking about. This cholera toxin uses NAD+ as a substrate to
produce the ADP-ribosylated Arg-201. The ADP-ribosylation of Arg-201 destroys the GTPase activity of
the Gs subunit. The G-protein becomes locked into the active state producing prolonged activation of
adenylate cyclase. The elevated levels of cAMP cause the intestinal epithelial cells to secrete high
volumes of fluid.

The Pertussis Toxin

Bordetella pertussis is the bacterium that causes whooping cough. Bordetella pertussis produces a toxin
which is an enzyme that catalyzes the ADP-ribosylation of a Cysteine residue of the Gi protein. The
ADP-ribosylated Gi protein cannot exchange GDP for GTP which prevents Gi from dissociating from
the Gi heterotrimer. Gi then cannot inhibit adenylate cyclase. Pertussis is a systemic infection
affecting the regulation of adenylate cyclase throughout the entire body.

Phosphatidyl Inositol Bisphosphate

Cyclic AMP is not the only second messenger produced by 7-TMS receptors and the associated Gproteins. Lets examine another second messenger cascade that is induced by many hormones to evoke a
number of cellular responses, the phosphinositide cascade. Vasopressin is an antidiuretic hormone that
binds to the vasopressin receptor which is yet another 7-TMS receptor. The binding of vasopressin to the
receptor induces the associated G protein to exchange GDP for GTP causing the Gq subunit to dissociate
from the Gq dimer. The Gq subunit with GTP bound associates with phospholipase C activating the
lipase. The activated lipase hydrolyzes the phosphodiester bond linking the phosphorylated inositol to the
diacylglycerol. The cleavage of this phosphodiester bond produces 2 second messengers, inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG). DAG diffuses laterally in the lipid membrane while IP3 is
water soluble and diffuses into the cytosol of the cell.

Inositol 1,4,5 triphosphate is a second messenger that binds to IP3-gated

channels located in the membrane of the endoplasmic reticulum and the
calcisomes. The binding of IP3 promotes the rapid release of Calcium ions.
Calcium ions are yet another second messenger which triggers muscle
contractions in muscle tissue, exocytosis and glycogen breakdown.
Inositol 1,4,5 triphosphate has a short half life. It is rapidly converted into
derivatives such as inositol or inositol 1,3,4,5 tetrakisphosphate that do not
open the calcium channels.
Lithium is used to treat bipolar disorders. Lithium inhibits the recycling of
inositol 1,3,4 triphosphate..

Diacylglycerol (DAG) is another second messenger. Diacylglycerol activates a number of protein

effectors. We will concentrate on one, protein kinase C (PKC). This is a kinase that uses ATP to
phosphorylate serine and tyrosine residues of many target proteins. Before it is activated by DAG, this
protein is free in the cytosol. A portion of the C1A subunit has a sequence that is very similar to the
sequence of the target proteins. This pseudosubstrate sequence occupies the active site of the enzyme
inactivating it. When phosphatidylinositol
diphosphate is hydrolyzed by the activated
phospholipase C, DAG is generated in the lipid
membrane. PKC binds to the DAG creating an
attachment of PKC to the membrane. When
PCK is anchored to the membrane the
psuedosubstrate sequence interacts with head
groups of the phospholipids of the membrane.
This activates the kinase activity of this
enzyme. PKC also requires Calcium ions for
activity (remember that IP3 induces the release
of Ca2+ from the endoplasmic reticulum and the
calciosomes). The two second messengers
DAG and IP3 work in tandem to activate PKC.
DAG also has a short half life. It is phosphorylated to form phosphatidic acid or it is hydrolyzed to its
glycerol and fatty acid components.

Calcium ions are another important second messenger. The binding of certain hormones and signal
molecules to receptors in the plasma membrane can cause transient increases in cytoplasmic Ca2+ levels.
Increases in cytoplasmic calcium levels activate a wide variety of enzymatic processes. Cyclic AMP
activates the opening of plasma Ca2+ channels allowing extracellular calcium to stream in. Within the cell
there are calcium reservoirs such as the endoplasmic reticulum and the calciosomes. The internal stores
are not released by cAMP. They only respond to IP3, the second messenger derived from phosphatidyl
inositol.
There are a number of intracellular calcium binding
proteins which in turn regulate many cellular processes.
The most common is calmodulin shown to the left.
Some others are paravalbumin, troponin C and the
annexin proteins. There are over 170 calcium modulated
proteins known. Most of these have the characteristic EF
hand. The conformation of these EF hand proteins
changes dramatically when calcium is bound. Shown
below are the conformational changes that occur when calmodulin binds 4 calcium ions. When
Calmodulin has 4 calcium ions bound, it is bound by target proteins which activate them. One example is
calmodulin dependent protein kinases (CaM kinases). These kinases phosphorylate many proteins that
regulate fuel metabolism, ionic permeability, neurotransmitter synthesis and neurotransmitter release.
These kinases are inactive until they bind calmodulin which has 4 calcium ions bound.