Regulation of the
metabolism
PRINCIPLES OF THE REGULATION OF THE METABOLISM:
THEORETIC BASES
ENZYMES -BIOCATALYSATORS
HORMONES and NEUROTRANSMITERS (Common mechanisms of the eff
of hormones and neurotransmiters)
RECEPTORS (Type of membrane receptors and intracelular receptors)
ENZYMES
VITAMINS
METABOLIC REGULATIONS
pathobiochemistry- receptors_1 1
Definitions
Endocrine - Refers to the internal secretion of biologically
active substances.
Exocrine - Refers to secretion outside the body, for example,
through sweat glands, mammary glands, or ducts
lead to the gastrointestinal.
Hormone - Substances released by an endocrine gland and
transported through the bloodstream to another
tissue where it acts to regulate functions in the
target tissue (classic definition).
Paracrine - Hormones that act locally on cells that did not
produce them.
Autocrine - Hormones that act on cells that produced
them.
Receptors -Hormones bind to receptors molecules on cells. A
receptor must specifically recognize the hormone
from the numerous other molecules in the blood and
transmit the hormone binding information into a
cellular specific action.
pathobiochemistry- receptors_1 2
TEST
Endocrine Blood vessel
Distant target cells
Hormone secretion
into blood by
endocrine gland
Paracrine
Secretory cell Adjacent target cell
Autocrine
Target sites on same cell
Receptor
Hormone or other extra
cellular signal
pathobiochemistry- receptors_1 3
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Communication between cells
The signal transduction is the process, when signal molecules carry specific information over
membranes from the outside of the target cell into the inside, where cause relevant biological
answer.
In the higher organisms there are three main signal systems, which regulate and integrate
activity of the cells.
Except for them other signal molecules (they aren´t in these systems) also play a role in the
metabolism.
Main signal systems in the higher organisms
System Source of the signal
molecules
Signal molecules
Endocrine Endocrine glands,
scattered glandular cells
Hormones
Nervous Nervous cell Neurotransmiters,
neurohormones
Imununity Cells of the imunity
system
Cytokines
Other types Different cells Eicosanoids, growth
factors
TEST
pathobiochemistry- receptors_1 5
Effects of signal molecules
Name of the effect Character of the effect
endocrine The signal molecule is carried by blood into the target cell, which is usually
distant from the place of the synthesis.
Typically hormones
paracrine The signal molecule is secreted into the ambient surroundings of the cell(local
mediators). The signal molecule influences only the cells of the nearest
surroundings.
autocrine The cell secretes the signal molecule and it is also a target. Features ale similer
like paracrine effect.
Endocrine – the signal molecule is carried by blood into the target cell , which is usually distant from the place of the
synthesis. It is typical for hormones.
The concentration of the signal molecule in blood is very low (10−12–10−9 mol/l) – so target cell has a big affinity to the
signal molecule – the binding hormone to receptor is very strong, hormone doesn´t dissociate easy. Other feature is that it
take definite time that the concentration of the hormone in blood will rise and the concentration of the homone in blood stays
for definite time (a few minute or hour) rised.
Paracrine –The signal molecule is secreted into the ambient surroundings of the cell (local mediators). The signal
molecule influences only the cells of the nearest surroundings. The concenration of the signal molecule in the surroundings
of the cells is higher (10−9–10−6 mol/l). Affinity of the receptors to the signal molecule is lower – after decrease of the
concentratrion in the surroundings of the cell, the signal molecule is separated. Paracrine signaling is determined for fast and
localised communication between cells.
Autocrine –The cell secretes the signal molecule and it is also a target. Features ale similer like paracrine effect.
Juxtacrine – signalling between cells or cell and extracellular matrix require tight contact.
TEST
pathobiochemistry- receptors_1 6
Common mechanisms of the effect of hormones
and neurotransmiters.
Types signal molecules in the neurohumoral regulations:
Signal molecule Source
HORMONES secreted by endocrine glands, scattered glandular
cells, eicosanoids a lot of other types of cells
NEUROHORMONES secreted by neurons into the blood circulation
NEUROTRANSMITERS secreted in the synaptic ending
CYTOKINES, GROWTH
FACTORS, EICOSANOIDS
secreted by a lot of types of cells, usually not from
endocrine glands
TEST
7
Regulation of the metabolism is in the different
levels, but always on the molecular base
The Regulation of enzymatic reactions is a central
instrument of the regulation of the metabolism.
Regulation in the definite
cellular compartment
Regulation in the
complete cell
(proteome, specific
receptors, izoenzymes,
transporters, energetic
state of the cell)
Regulations followed
from the
communication
between cells
Levels of the regulation overlap.
pathobiochemistry- receptors_1 8
Regulation of the enzymatic activity
• regulation of the amount of enzyme (synthesis and
degradation)
• regulation of the activity of the enzyme
(modification of the enzyme by proteolysis, covalent
modification, allosteric regulation, interaction with the
regulatory proteins)
• availability and concentration of the substrate
(regulation of the transport)
SIGNAL TRANSDUCTION
Hormone/Receptor Interaction-Secondary Signals
pathobiochemistry- receptors_1 9
pathobiochemistry- receptors_1 10
The collective feature of all substances with modulating effects to the cells is their effect by
receptors.
Receptors are allosteric proteins, which change their conformation after the bindind ligand.
Ligands are signal molecules.
Agonists are ligands, where after the binding on the receptor cause the
transduction of signal, antagonists after the binding on the receptor defend the signal
transduction.
Receptors are localised on the outer surface of the cytoplazmatic membrane or intracellulary.
In their structure there are two main components: (1) domain binding the ligand, which ensures
the specifity of the binding with the relevant ligand; (2) efector domain, wich start a genesis of
the biological answer after the binding of the ligand.
Activated receptor can enter into the reaction with other cellular components and realise the
process of the signal transduction.
Tissues, whose cells have no molecules of the specific receptor, can´t react to the relevant
hormone.
Charakteristic feature of a transport of the signal by receptors is its amplification, when the only
one molecule of the hormone is able to cause celullar answer with 104–105 times higher
intensity.
TEST
pathobiochemistry- receptors_1 11
Principle of hierarchy in some hormonal regulations
and amplification of the flow of information by signal
molecules
Neurons of the brain cortex NEUROTRANSMITERERS the highest nanograms
Neurons of nucleuses of the hypothalamus CORTICOLIBERIN micrograms
Cells of the adenohypophysis KORTICOTROPIN hundreds of micrograms
Cells of the cortex of the adrenal glands CORTISOL hundreds of micrograms
TARGET CELLS OF PERIPHERAL TISSUES
Daily production:For example.
pathobiochemistry- receptors_1 12
Signal transduction
How does the cell take over the information carried by
the chemical signal?
Reaction of the signal molecule with the receptor
Membrane receptors
Proteins and smaller signal
moleculs (peptides, amino
acids, biogenic amins,
eicosanoids)
Intracelular receptors
Nepolar signal molecules
(steroids, jodthyronins,
retinoats)
TEST
pathobiochemistry- receptors_1 13
Amplification
Membrane and intracelular receptors
Interaction of the complex
hormone- receptor with hormonsensitive
element of DNA
Nonpolar signaling molecules
bound to plasma transport protein
Intracellular receptor
Biological effect
(slower effect)
Polar signaling molecules
Biological effect
(fast effect, may be
followed by belated
action)
Transduction of signal
Membrane receptor
Transport of signal molecule
1
100
10 000
TEST
Receptors
Cell surface membrane receptors
Polypeptide hormones and catecholamines
Cytoplasmic receptors
Most steroid and thyroid hormones
Nuclear receptors
estrogens
pathobiochemistry- receptors_1 14
Secondary Messenger
or Secondary Signal
Cellular
Trafficking
Enzymes
Activated Inhibited
Nucleus
DNA Synthesis
RNA Synthesis
Protein
Synthesis
Membrane
Effects
Receptor
Effector
Plasma
Membrane
Hormone
A general model for the action of peptide hormones, catecholamines, and other
membrane-active hormones. The hormone in the extra cellular fluid binds to the receptor
and activates associated effector(s) systems, that may or may not be in the same
molecule. This activation results in generation of an intracellular signal or second
messenger that, through a variety of common and branched pathways, produces the final
effects of the hormone on metabolic enzyme activity, protein synthesis, or cellular
growth and differentiation. pathobiochemistry- receptors_1 15
pathobiochemistry- receptors_1 16
pathobiochemistry- receptors_1 17
Types of receptors
Type of receptor Ligand characteristics Receptor characteristics
Membrane Big signal molecules (peptides
and proteins)
Small, strongly hydrophilic
molecules (amonoacides and
their derivates)
Intergral membrane proteins
Intracellular Small hydrophobic molecules
(steroids, vitamin D, retinoids,
thyroidal hormones)
Proteins in cytoplasm or in
the nucleus
Main types od membrane receptors
pathobiochemistry- receptors_1 18
I. Receptors - ion channels (ROC, ligand-gated channels) only
receptors for some neurotransmitters (ion channels controlled by
neurotransmitters)
II. Receptors interacting with G-proteins (heterotrimeric)
III. Receptors with its own catalytic activity
a) guanylate-cyclase
b) proteinkinase
IV. Receptors cooperating with the non-receptor tyrosine
kinases
(eg. JAK) – receptors for somatotropin (GRH), prolactin, erythropoietin,
interferons, interleukins and other cytokines.
TEST
pathobiochemistry- receptors_1 19
pathobiochemistry- receptors_1 20
Neurotransmitters - chemical signals, enable the transfer of
of nerve impulses between neurons or between a neuron and the target cell
synaptická štěrbina
postsynaptic
membrane
receptor
synaptic vesicles
(synaptosoms)
voltage-controlled Ca2 + channel
depolarization wave
Ca2+
• Neurotransmitter binds directly to the ion channel
(ionotropic receptors) → electrical signal (neuron -neuron)
• The neurotransmitter binds to a receptor that generates second
messenger (metabotropic receptors) → chemical signal (eg.
smooth muscle)
I. Receptors – ion channels
Receptors of the type of ion channels are present in the synapses, their ligands are neurotransmitters.
Synapse
General scheme
pathobiochemistry- receptors_1 21
Membrane receptors for the neurotransmitters
Ionotropic receptors - ligand-controlled ion channels (ROC), e.g.
excitatory nicotinic acetylcholine - channel for Na+/K+,
glutamate (CNS, some afferent sensory neurons)
- channel for Na+/K+/Ca2+,
inhibitory receptor GABAA (CNS) - channel for ClMetabotropic
receptors activating G proteins, e.g.
protein Gs adrenergic 1 a 2, receptor GABAB, dopamine D1,
protein Gi adrenergic 2, dopamine D3,
muscarinic acetylcholine M2 (also opens K + channel),
protein Gq muscarinic acetylcholine M1, adrenergic 1.
TEST
pathobiochemistry- receptors_1 22
In central nervous system
inhibitory GABA (minim. 50 % all synapses)
glycine (prevails in the spinal cord)
excitatory glutamate (more than10 %)
acetylcholine (about 10 %)
dopamine(about 1 %, in striatum 15 %)
serotonine
histamine
aspartate
noradrenalin
(less than1 %, in hypotalamus 5 %)
adenosine
Neuromodulation endorphins and enkephalins,
endozepines, delta-sleep-inducing peptide
etc.
Neurotransmitters
There are more than 30 different neurotransmitters (amino acids, biogenic
amines caused their transformation, or very large peptides).
Examples:
In peripheral nervous system
– efferent neurons
excitatory acetylcholine
noradrenaline
– primary sensory afferent
excitatory glutamate
(Aβ fibers, tactile)
substantion P (peptide)
(C Aδ fiber nociceptive)
TEST
pathobiochemistry- receptors_1 23
Major neurotransmitter receptors
Acetylcholine receptors
Occurs at neuromuscular junctions of skeletal muscle and in almost all peripheral dendrites
of efferent neurons. It consists of five subunits (2a, b, g, ε) penetrating the membrane.
Acetylcholine is synthesized by the presynaptic neuron region of acetyl-CoA to choline and before release is
stored in vesicles stored close to the active zone of the presynaptic membrane. Membrane
also has the voltage-gated Ca2+ channels, which open when the action potential is expanded to the membrane.
Increased levels of Ca2 + in the neuron endings activates the Ca + -dependent protein kinase, which
phosphorylates synapsin and other proteins, thereby effecting fusion of the vesicles with the presynaptic plasma
membrane and release of acetylcholine into the synaptic cleft.
Acetylcholine binds the two subunits and his binding causes a conformational change and short
influx of sodium ions into the cell and potassium ions out of the cell. This causes depolarization of postsynaptic
membrane, and if the threshold is reached, potential-dependent channels are opened
for Na + and the action potential arises. Once acetylcholine secretion ceases, its concentration in the cleft
decreases and acetylcholine stops to bind to receptors. Acetylcholine is decomposed by acetylcholinesterase,
which is bound to the surface of the postsynaptic membrane.
TEST
pathobiochemistry- receptors_1 24
Receptor Nicotine Muscarinic
M1, M3 M2
Mechanism of
action
ion channel Gq Gi
The second
messenger
DG + IP3 cAMP
occurrence
• autonomic ganglia
neurons,
• neuromuscular junction,
• chromaffin cells of the
adrenal medulla
• brain,
• smooth
muscle,
•glandular
cells
• myocardium,
• brain
tubocurarine atropin
Acetylcholine receptors
Major neurotransmitter receptors
TEST
pathobiochemistry- receptors_1 25
cholinergic synapse
One nerve impulse in the neuromuscular junction releases approx. 300 vesicles, one
contains about 40,000 molecules of acetylcholine; concentration of acetylcholine in
the synaptic cleft rises to 10 000 times. The mediator is rapidly hydrolyzed by
acetylcholinesterase.
acetylcholine receptors of
postsynaptic membrane
ACETYLCHOLINE
membrane acetylcholinesterase
choline
acetate
acetyl-CoA
ATP
depolarization wave
Ca2+
Na+
choline acetyltransferase
(axonal transport)
reuptake
TEST
Acetylcholinestherase
• The hydrolysis of acetylcholine to acetate and choline
• It is a serine hydrolase
pathobiochemistry- receptors_1 26
TEST
Acetylcholinesterase inhibitors
• a) reversible: carbamates (physostigmine,
rivastigmine, neostigmine)
• b) irreversible: organophosphates (DFP, soman,
sarin)
N N
OO
NH
CH3
CH3
CH3
CH3
H
pathobiochemistry- receptors_1 27
Binding of toxic organophosphates to cholinesterase is done in two stages:
reversible can be affected by reactivators)
irreversible - formation of covalent bonds between enzyme and
organophosphate
fyzostigmin
pathobiochemistry- receptors_1 28
e.g. at the neuromuscular junction - Na + / K + ionophore: asymmetrical pentamer
of four types of homologous subunits penetrating the membrane.
conformational change of subunits,
channel is in milliseconds many times
briefly opens and closes
binding sites for local anesthetics,
psychotropic phenothiazines etc ..
binding of two molecules of
acetylcholine to 2 subunits
1 2
closed channel
synaptic
cleft
cytosol

2 2
– –
Na+
K+
massive influx of Na +
less efflux of K +
(depolarization)
Nicotinic acetylcholine receptor of the nicotine type
pathobiochemistry- receptors_1 29
Rhabdomyocytes Parasympathetic Sympathetic
(skeletal muscles) innervated cells of target tissues
postganglionic neurons of
sympathetic pathways are
almost always adrenergic
Adrenergic
receptors
N
NN
N
N
N
M1
Acetylcholine (cholinergic) receptors
in peripheral efferent neurons
N-nicotinic
M-muscarinic
pathobiochemistry- receptors_1 30
Ligands interacting with acetylcholine
receptors of nicotine types
D-tubocurarine - competitive antagonist of acetylcholine, prevents the opening
of ionophore (depolarization does not occur) paralysis of skeletal muscles
pancuronium, vecuronium ad. - muscle relaxants during prolonged operations
Succinylcholine - agonist binds more efficiently than acetylcholine and
depolarizes. The persistent depolarization leads to loss of electrical excitability of
membrane. Short term myrelaxans.
Botulotoxine – protein complex from Clostridium botulinum. Inhibits the release
of acetylcholine from the nerve endings.
Nicotine - binds to receptors in the peripheral and vegetative nervous system
which controls the internal organs. Here causes increased activity of the digestive
tract: increase of production of saliva and digestive juices and the increase in
activity of smooth muscles. Also increases the production of sweat and may cause
the contraction of pupil.
pathobiochemistry- receptors_1 31
Type Principle of action Location
M1 Gq Autonomic ganglia, CNS, exocrine gland
cells
M2 Gi heart, K+ channels opening
M3 Gq Smooth muscle
M4 Gi CNS
M5 Gq CNS
Muscarinic cholinergic receptors
The alkaloid atropin is antagonist of muscarinic receptors preventing acetylcholine binding.
pathobiochemistry- receptors_1 32
Adrenergic synapse
Neurotransmitter of most postganglionic sympathetic neurons is noradrenaline.
Some nerves can be also influenced by adrenaline.
Depolarization wave
Ca2+
Adrenergic receptors in
membranes of the target cells
dopamine--hydroxylase
and synaptic vesicles
(axonal transport)
NORADRENALINE
presynaptic
adrenergic
receptors
mitochondrial
monoamine oxidase
extracellular COMT
(catechol-
O-methyltransferase)
Partial reuptake
Varicosities of the postganglionic sympathetic axons
are analogous to the nerve terminals (string of pearls).
TEST
pathobiochemistry- receptors_1 33
• Dopamine is synthesized in the cytoplasm
• Dopamine is transported into vesicles (ATP-dependent process, against
concentration gradient).
• Final hydroxylation of dopamine to noradrenaline occurs in vesicles.
Synthesis and storage of catecholamines
pathobiochemistry- receptors_1 34
Receptor 1  2
1  2
G-protein Gq Gi Gs
Second
messenger
DG + IP3 cAMP  cAMP 
Examples
of location
• GIT smooth
muscle
(sphincters)
and skin
blood vessels
(contraction)
• adrenergic and
cholinergic nervous
terminals (transmitter
releasing inhibited)
• pancreas
(glandular secretion
inhibited)
• thrombocytes
(agregation)
• myocard
(intensity
and
frequency
of
contractions
increased)
• smooth muscle in
the uterus, bronchi
(relaxation)
• GIT smooth
muscle (peristalsis)
• pancreas
(glandular
secretion inhibited
activated)
Adrenergic receptors
TEST
pathobiochemistry- receptors_1 35
-Adrenergic receptors
The typical effects of -stimulation
1 – tachycardia, inotropic effect in the myocard,
2 – bronchodilation, vasodilation in the bronchial tree,
3 – mobilization of fat stores, thermogenesis.
AMP-cyclase-receptor
g
noradrenalin / adrenalin
ATP
AMP
cAMP
fosfodiesterases
Gs
H2O
active proteinkinase Afosforylations
inactive
proteinkinase A
TEST
pathobiochemistry- receptors_1 36
Adrenergic receptors 2 a 1
2-receptors 1-receptors
adenylatecyklase fosfolipase C
PL C
cAMP decrease
Gi protein Gq protein
IP3 a diacylglycerol
Increase in [Ca2+]
Activation of PK C
The typical effects of adrenergic
2-stimulation: 1-stimulation:
glandular secretion inhibited vasoconstriction
bronchoconstriction
motility of GIT inhibited
TEST
pathobiochemistry- receptors_1 37
Inhibitory GABAA receptor
Ligand-gated channel (ROC) for chloride anions. The interaction with
g-aminomáselnou kyselinou (GABA) opens the channel.
The influx of Cl– is the cause of hyperpolarization of the postsynaptic
membrane and thus it´s depolarization (formation of an action potential)
disabled.
Cl–
–
–
–
–
–– –
1
2
g2
2
1
heteropentamer containig 3 subunits
pathobiochemistry- receptors_1 38
Cl-
GABA
CYTOSOL
Barbiturates
Benzodiazepines
(e.g. diazepam,
flunitrazepam)
Steroids
(e.g. pregnanolone,
allopregnanolone)
Endozepine (diazepam-binding inhibitor,
DBI)
Agonist: muscimol
(of Amanita muscaria)
Another binding sites of GABA receptor
TEST
pathobiochemistry- receptors_1 39
More than eleven allosteric modulation sites for substances increasing the effect
of endogenous GABA (calming down, reduction of anxiety and myorelaxation):
anesthetic, ethanol and numerous drugs such as benzodiazepines meprobamat and
also various barbiturates.
On the contrary, another ligands compete for benzodiazepine binding site or act
also as the antagonists of GABA (inverse agonists),  causing unease and
anxiety (e.g. endogenous peptides called endozepines).
In brainstern and spinal cord, glycin has a similar function to GABA in brain.
The inhibitory effect of glycinergic synapses is blocked by strychnine alkaloid,
known seizure poison.
Another binding sites of GABA receptor
pathobiochemistry- receptors_1 40
Inhibitory GABAergic synapse
g-Aminobutyric acid(GABA) is the main inhibitory neurotransmitter in
CNS. Gabaergic synapses represent about 60 % of all brain synapses.
GABA
mitochondrial
Synthesis of GABA
from glutamate
Depolarization wave
Ca2+
GABA/benzodiazepine
receptors
GABA id captured by glial cells,
degraded to sukcinate and transformed
to glutamate a glutamine
Parial reuptake
(transporters GAT 1-4)
pathobiochemistry- receptors_1 41
Receptors of the most important neurotransmitters
Receptors associated with G-proteinsIon channels
(ROC) Gs (increasing cAMP) Gi (decreasing cAMP)Gq (increasing IP3 /DG)
– adrenergic β1,β2,β3 adrenergic α2 adrenergic α1
Na+/Ca2+/K+
– glutamate ionophores
glutamate mGluR
skupiny II a III
glutamate mGluR
class I
dopamine D1,5 dopamine D3,4 dopamine D2
– serotonin 5-HT3 serotonin 5-HT4,6 serotonin 5-HT1 serotonin 5-HT2
histamine H2 histamine H3,4 histamine H1
– – tachykinin NK1
for substance P
Cl– – GABAA
– glycine
GABAB (metabotropic) – –
–
Na+/K+ – acetylcholine
nicotinic
acetylcholine
muscarinic M2,4
acetylcholine
muscarinic M1,3,5
–
–
–
–
TEST
pathobiochemistry- receptors_1 42
Common structural features:
All of them have seven hydrophobic helical
domains, penetrate the mambrane
and connect extra- and intracellular loops. H2N
-COOH
II. Receptors interacting with heterotrimeric G-proteins
Binding site for
agonist(binding sites
for antagonists are
also present)
Intracellular domains bimding
site for interaction
with G-protein of single
specific type.
Few minutes
Neurotransmitters
Hormones
Agonist-ligand causes
signale transduction
Antagonist- prevents
pathobiochemistry- receptors_1 43
Heterotrimeric G-proteins
Proteins binding GDP or GTP
Mostly freely bound to cytoplasmatic membrane – they can move
along its inner surface.
Subunits ,  a g.
Identified more than 20 types of different
G subunites.
Subunits G
and Gg are
hydrophobic
and are not
specific. Subunits G are the
biggest, bind GDP or
GTP and are specific
for every type of
transduction
mechanism.
TEST
Heterotrimeric G-proteins activation cycle by interaction
with receptor-specific ligand complex
pathobiochemistry- receptors_1 44
receptor-specific ligand
complex
BIOLOGICAL EFFECT
CONCENTRATION CHANGE OF„SECOND MESSENGER“
Trimer G-GDP,g
receptor-ligand
-trimer G-GDPg complex
Dimer g
GTP
GDP
Activated
G-GTP subunit
Interaction with target
proteinPi
Inactive
G-GDP subunit
Dimer g
pathobiochemistry- receptors_1 45
Chosen types of G-proteins
Type of G subunit Examples of
activating receptor
Effect of activated G
To target protein
(intracellular signal)
Gs (stimulating) glucagon
parathyrine
-adrenergic
Adenylatecyclase
stimulation
(cAMP, Ca2+)
Gi (inhibitory) somatostatin
2-adrenergic
Adenylatecyclase inhibition
(cAMP, K+)
Gq (activating PI
cascade)
vasopressin V1
endothelin ETA,B
acetylcholine M1
1-adrenergic
phospholipase C stimulation
(DG+IP3, Ca2+)
Gt (inhibitory)
(transducin)
rhodopsin phosphodiesterase cleaving
cGMP stimulation
TEST
Receptors activating Gs and Gi
stimulate or inhibit adenylatecyclase
pathobiochemistry- receptors_1 46
Adenylatecyclase - membrane enzyme catalysing reaction
ATP  cAMP + PPi ;
cAMP is second messanger.
AMP-cyclasereceptor receptor
GS
g
Gi
g
ligand ligand
ATP
H2O
cAMP
proteinkinase A
inactive (R2C2)
active proteinkinase A
2 C + 2 R(cAMP)2
AMP
Fosfodiesterase*
Protein phosphorylation (Thr, Ser)
x
* Inhibition by e.g. caffeine,
theophylline
(METHYLXANTHINES)
TEST
pathobiochemistry- receptors_1 47
Effects of cAMP in cells
C
C R
R
Proteinkinase
A (inactive)
R
R
C
C
Protein
Protein
Protein-P
Protein-P
ADP
ADP
ATP
ATP
Proteinkinase
A (active)
cAMP
Protein phosphorylation.
In cytoplasm- most often metabolic enzymes (fast response)
In nucleus–gene specific transcription factor CREB (cAMP response
element-binding protein) phosphorylation (pomalejší odpověď)
AKAP
AKAP
pathobiochemistry- receptors_1 48
cAMP provides many different effects in the cell.
One of the most important effect is proteinkinase A activation, which phosphorylates many others
metabolic enzymes. The effects of kinases can by aimed for certain proteins phosphorylation. That
is maintained by specific proteins binding kinases. In the case of proteinkinase A, it is about so
called AKAPs (A kinase anchoring proteins), which serve as supporting structure and localize
proteinkinase A position near by certain substrate, which is supposed to be phosphorylated and at
the same time, their spontaneous aktivity is reduced.
Proteinkinase A is heterotetrameric molecule, containing two regulatory and two catalytic
subunits. In inactive state, subunits are bound to each other. cAMP binds regulatory subunits
causing their separation from catalytic subunits, which become active and catalyze phoshate
transmission from ATP to serine or threonine residues of target proteins. Proteinkinase A catalytic
subunit also enters the nucleus where prosphorylate gen specific transcription factors so called
CREB (cyclic AMP response element-binding protein). CREB binds cAMP-responsive element in
unphosphorylated state and is poor transcription activator. After phosphorylation by proteinkinase
A, CREB bind coactivator CBP (CREB-binding protein) causing transcription amplification.
Some bacterial toxins modify G-proteins effect. Cholera is an infectious intestinal disease causing
severe life threatening diarrheas. Diarrhea is caused by enterotoxin produced by bacteria Vibrio
cholera. Choleratoxin is protein causing by its effect inhibition of GTPasa activity of Gs protein
subunit. Modificated s subunit is „frozen“ in active state continually producing cAMP. cAMP
effect is active channel for Cl− in intestinal cell membrane and its effect causes chloride ions and
water secernation to intestinal lumen. Inhibitory G-protein is the target of pertusis toxin effect,
which is produced with whooping cough by bacteria Bordetella pertusis. The result is Gi protein
inactivation and cAMP overproduction.
Besides proteinkinase A activation and proteins phosphorylation, cAMP or cGMP can bind also
ion channels influencing their permeability. Those mechanisms find its use especially in activation
of olfactory and visual perceptions.
pathobiochemistry- receptors_1
Examples of hormones effecting through
PAK activation
Hormone Localization of the effect
CRH Adenohypophysis
TSH Thyroid follicle
LH Testicular Leydig cells, yellow body (corpus
luteum)
FSH Ovaria follicle cells, testicular Sertoli cells
ACTH Adrenal cortex
ADH Kidney distale tubule cells
PGI2 Thrombocytes
Adrenaline,
noradrenaline
 - receptors in many cells
glukagon Livers
49
pathobiochemistry- receptors_1
Oba produkty jsou sekundární „poslové“:
Inositol-1,4,5-trisfosfát otevírá kanál pro Ca2+ v membráně ER,
diacylglycerol aktivuje membránovou proteinkinázu C.
III. Receptors activating Gq protein stimulate
phospholipase C triggering phosphatidylinositol
cascade
Fosfolipáza C
That kind of receptors binds q-isoform
subunit. Ligand binding results in activation
of enzyme phospholipase C. Activated form
of this enzyme hydrolyzes membrane bound
phosphatidylinositolbisphosphate (PIP2)
forming two different second messangers:
diacylglycerol (DG) and 1,4,5inositoltrisphosphate
(IP3). IP3 has its
binding site on sarko- and endoplasmatic
reticulum, where stimulates Ca2+ releasing.
Ca2+ ions activate enzymes containing
calcium-calmodulin subunit including
proteinkinase. Calcium dependent
calmodulin kinase II also influence CREB in
nucleus. DG localized in membrane activates
proteinkinase C amplyfying response by
target proteins phosphorylation.
50
pathobiochemistry- receptors_1
Both products are second „messengers“:
Inositol-1,4,5-trisphosphate opens Ca2+ channel in ER membrane,
diacylglycerol activate membrane proteinkinase C.
III. Receptors activating Gq protein stimulate
phospholipase C triggering phosphatidylinositol
cascade
Phospholipase
C
51
pathobiochemistry- receptors_1
phospholipase Creceptor
Gq
g
specific ligand
PIP2 DG
proteinkinase C activation
phosphorylation
[Ca2+] increasing
in cytoplasm
Endoplasmatic reticulum
Ca2+
IP3-receptor in ER membrane
ligand controlled Ca2+ ion channel
IP3
Phosphatidylinositol cascade
active proteinkinase C
Ca2+
TEST
52
pathobiochemistry- receptors_1
Regulation of metabolism by
cytoplasmatic Ca2+ concentration
changes
•Basal Ca2+ concentration in cytoplasm  1.10-7 mol/l
•Concentration increasing to  1.10-6 fast and effectively activates
different cellular process
•Ca2+ increasing may be caused by
Ca2+ influx through cytoplasmatic membrane (e.g.
Smooth muscle contraction)
releasing from intracellular supplies (ER,
mitochondrie) e.g. IP3 dependent Ca2+ channel in
ER or ryanodine channels in skeletal and cardiac muscle
53
pathobiochemistry- receptors_1
Regulatory protein calmodulin
Increasing Ca2+ level activates numerous Ca2+-dependent
proteins forming family of small calcium dependent proteins.
The most important is calmodulin which is present in almost all
cells.
Ca2+ binding to calmodulin (4 binding
sites) changes its conformation and
activates its interaction with another
proteins, e.g. kinases, phosphatases
and others.
Some of Ca-calmodulin-dependent
kinases are highly specific, others have
wide substrate specificity. 54
pathobiochemistry- receptors_1
Examples of hormones effecting through
phosphatidylinositol system activation
and PKC
Hormone Localization of the effect
TRH Adenohypophysis
GnRH Adenohypophysis
TSH Thyroid follicle
Angiotensin II/III Adrenal cortex
(zona glomerulosa)
Adrenaline 1- receptors
55
pathobiochemistry- receptors_1
III. A) Receptors with guanylatecyclase activity
After ligand binding transform GTP to cGMP
cGMP is second messenger
Activates proteinkinase G
Two kinds of receptors:
•membrane
•cytoplasmatic
III. Receptors with enzymatic activity
cGMP can be also second messenger. Unlike
adenylatecyclase, guanylatecyclase isn´t activated by
G-proteins.
There are two different types of guanylatecyclase:
membrane bound enzymes activated dirrectly by
extracellular ligands and soluble enzymes in cytoplasm,
reacting to small diffusible molecules. Both types of
quanylatecyclase are located in vascular smooth muscle
cells.
56
pathobiochemistry- receptors_1
Membrane receptors with guanylatecyclase activity
ANP
GTP cGMP + PPi
proteinkinase G
inactive
active proteinkinase G (PKG)
Protein phosphorylation
Receptors for ANP
Present especially in vascular
smooth muscle and in kidneys
ANP is secernated by myocytes
atria as a response to increasing
of blood volume or pressure
GMP
phosphodiesterase
H2O Membrane bound enzyme is receptor for natriuretic peptides (ANP, BNP,
urodilatin). Receptor contains extracellular domain for ligand binding, simple
transmembrane helix and intracellular guanylatecyclase domain.
Guanylatecyclase activity is initiated by ANP binding to extracellular domain.
Likewise cAMP and cGMP has effect through proteinkinase activation.
This kinase is called proteinkinase G according the convention. Natriuretic
peptides receptors are localized in vascular smooth muscle, kidneys and other
tissues. ANP is secernated by myocytes atria as a response to increasing blood
volume or pressure in right atrium causing vasculature relaxation. That leads to
decrasing of total peripheral resistance and improving of local blood flow. In
kidneys, causes dilatation of afferent and narrowing of efferent glomerular
arteriole and relaxation of mesangial cells. Glomerular capillary pressure and
glomerular filtration are increased and that leads to increased sodium and water
excretion.
TEST
57
pathobiochemistry- receptors_1 58
Cytoplasmic receptors with a guanylatecyclase activity
NH2 NH2
hem
NO
GTP cGMP
The receptor is dimeric and a binds hem
NO is bound to the hem, its bond rises a catalytic
activity of guanylatecyclase
NO is generated by nitroxide synthase
(NOS)
NO goes through by membranes easily,
it can be generated also by other cells
and to the target cell penetrates by
diffusion.
Activation of
proteincinase G
phosphodiesterase
GMP
Soluble guanylatecyclase
Soluble guanylatecyclase is in the cytoplasm of many cells. It is a dimeric molecule
with a hem. It binds NO which causes in its structure conformation changes and rises
its enzymatic activity. NO is synthetised by nitroxide synthase (NOS) from arginine. It
can be also generated in organism from some exogenous compounds (NO donors),
for example nitroglycerine, nitropruside. cGMP is degradeted by a few types soluble
or in membrane bound cGMP phosphodiesterases. Inhibitors of cGMP
phosphodiesterases cause also an increase of cGMP and prolong relaxation of
smooth muscles.
pathobiochemistry- receptors_1 59
Proteinkinase G
cGMP sensitive proteinkinase G
It is spread in a lot of tissues
It phosphorylates different proteins (enzymes, transport
proteins and so on)
Effect of PKG in smooth muscles
Phosphorylation of proteins:
• inactivation of proteins which promote releasing of Ca2+ from ER  
Ca2+
• activation MLC phosphatase  inhibition of an actin-myosin interaction
•Decrease of an activity of K+-channels which promote hyperpolarization
 decrease of an influx Ca2+ into the cell
Relaxation of smooth muscles
pathobiochemistry- receptors_1 60
Meaning of NO/cGMP signalization in
smooth muscles of vessels
cGMP is a crucial second messenger for an induction of
relaxation of smooth muscles in vessels
 vasodilatation and increase flow of blood
NO is produced in endotel cells by nitroxide synthase from
arginine (activation for example by acetylcholine) and
diffuses into adjacent cells of smooth muscle
L-Arg ·NO + L-citrulline
NO-synthase
pathobiochemistry- receptors_1 61
R-O-NO2 nitrite ·NO
Drugs like organic nitrates are a source of
exogenous NO
Glyceryl trinitrate
Isosorbide
dinitrate
Therapy of angina pectoris
Activation of soluble
guanylatecyclase
Vasodilatation effect to arteries releases coronary spasm
and normalise a perfusion
pathobiochemistry- receptors_1 62
Inhibition of phosphodiesterase potentiates
the effect of NO
cGMP GMP
phosphodiesterase
H2O
Drug sildenafil (Viagra) is selective inhibitor of phosphodiesterase 5
(PDE5) which is highly expressed in smooth muscles of vessels. Viagra
promotes the effect of NO· which is released during sexual stimulation
by inhibition of PDE5 and rises a concentration of cGMP in corpora
cavernosa. The result is a relaxation of smooth muscle in vessels and
perfusion of corpora cavernosa.
There are more
types of
phosphodiesterases,
depending in a type
of cells.
pathobiochemistry- receptors_1 63
III. B) Receptors with tyrosinkinase activity
Collective features
•binding of the signal molecule to the receptor causes conformation
changes
•tyrosinkinase activity of receptor is activated
• it caused autophosphorylation of tyrosine of receptor alternatively
other proteins (IRS)
•other proteins (adapter molecules) bind to the phosphorylated receptor
and substrates phosphorylated by the receptor
• adapter proteins bind to phosphotyrosine residue by SH2 domains (Src
homologs of 2 domains).
• adapter proteins react with other molecules and a signal is carried by a
cascade of phosphorylation/dephosphorylation reactions by changing of
guanine nucleotides, conformation changes and so on.
pathobiochemistry- receptors_1 64
Membrane receptor family with a tyrosinkinase activity is formed by receptors for growth factors and
insulin. Growth factors stimulate mitosis, cell differentiation, migration of cells and apoptosis.
Insulin stimulate an utilization of nutrients. Collective feature of receptors is intracellular tyrosinkinase
domain.
For example IGF-1 (insulin-like growth factor-1)
receptor; EGF (epidermal growth factor) receptor; PDGF (platelet-derived growth factor) receptor belong
into the subfamily with a tyrosinkinase activity.
pathobiochemistry- receptors_1 65


-S-S-
-S-S-


-S-SDimeric
structure
Binding site for insulin on - subunits
Tyrosinkinase activity on -subunits
Insulin receptor
Insulin binding to the receptor  tyrosinkinase activity
autophosphorylation of -subunits and phosphorylation of proteins IRS 1-4
(insulin receptor substrates 1-4)


-S-S-
-S-S-


-S-S-
Insulin
-P-P P-P-
IRS1-4
IRS1-4 -P
PI-3-kinase binding to the membrane
activation of phosphoproteinphosphatase-1
activation Ras – expression of genes
Binding
insulin to the
receptor
causes
internalization
of complex
hormon
receptor,
receptors are
partially
recyclated
TEST
pathobiochemistry- receptors_1 66
Insulin receptor
Insulin receptor is in membranes like a dimmer. Each monomer consist of extracellular subunit
α and integral membrane subunit β. Subunits α a β are connected by disulfide bond and
disulfide bond is also between monomers. Binding sites for insulin are on α subunits. Subunits
β include domains with own tyrosinkinase activity. After bindind insulin β subunits
phosphorylate themselves.
pathobiochemistry- receptors_1
Some of the signaling pathways of
insulin
http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7
67
pathobiochemistry- receptors_1
The substrates of the insulin receptor IRS1-4 are adapter proteins.
If the insulin-receptor complex phosphorylated these proteins, they follow more
proteins and activate them so.
Glycogen synthesis
Phosphorylation of IRS activates the
regulatory subunit of PI 3-kinase
The catalytic subunit of PI 3-kinase
phosphorylates PIP2 to PIP3
PIP3 activates protein kinase B (AKT)
activation of PKB (ACP) helps PDK
Activated Akt diffuses into the
cytoplasm and foforylates
(inactivates) glykogensynthasa kinase
Glycogen synthesis is activated (the
active form of glycogen synthase is
dephosphorylated)
Translocation of glucose transporters
Insulin receptor phosphorylates CBI
(IRS)
CBI-CAP complex translocates into lipid
raft membrane
CBI is reacted with an adapter
proteinCrk
Crk associated with C3G
C3G activates TC10 (G-protein)
Activates the translocation of
transporters into the plasma membrane
Examples mechanism of action of insulin receptor
68
pathobiochemistry- receptors_1
The receptor for epidermal growth factor EGF
Once ligand binding occurs receptor dimerization
R R
R R
-P
-PPP-
SoS
Ras–GTP
Raf
phosphorylation
cascade MAP
phosphorylation
This activates the tyrosine kinase activity in the cytoplasmatic domain.
Receptor autophosphorylation
The phosphorylated sites bind Grb2 adapter protein (SH-2 domains).
Through SOS protein is activated by G-protein Ras  aktivace MAPkinase
cascade (Ras / MAP-cascade)
Receptor with tyrosine kinase activity
69
pathobiochemistry- receptors_1
Ras is a monomeric G-protein (structural
analogue  -subunits)
SoS
Activation of Ras - a key step in signal transduction.
Inactive Ras-GDP switches to active Ras -GTP, which
activates another member of the pathway.
Inactivation of Ras - subsequent hydrolysis of GTP by using
activating protein GTPase activity of G-protein
Monomer G-protein - binds GTP and has simultaneously GTPase
activity. Activated GTP binding site GDP
70
pathobiochemistry- receptors_1
Ras superfamily proteins
5 families: Ras, Rho, Arf, Rab, Ran
Anchored to the lipid membrane by lipid anchors
(myristoyl, farnesyl)
Monomer G-proteins, which play an important role
in regulating growth, morphogenesis, cell motility,
cytokinesis like.
Mutations in the Ras genes induce proliferation and
pathologic antiapoptosis. Ras Mutations occur in
about 30% of all human cancers.
71
pathobiochemistry- receptors_1
MAP-kinase signaling pathway (Mitogen activated protein kinase)
Map-kinase cascade
Ras–GTP
MAP-kinase-kinase-kinase
MAP-kinase-kinase
MAP-kinase
Phosphorylation of
membrane proteins
or cytosolic
Phosphorylation of regulatory
proteins in the nucleus, promotion
of proliferation (e.g. activation of
transcription factors Jun, Fos)
ATP
ATP
ATP
ADP
P
ADP
ADP
ADP
ATP
P
P
MAPKKK, Raf
Especially
regulates cell growth
and differentiation.
MEK
ERK
Described 3 systems, the most famous ERK.
72
pathobiochemistry- receptors_1
Abbreviation Name Function
PDGF Derived Growth
Factor
platelet
mitogen for connective tissue cells
and undifferentiated neuroglia
EGF Epidermal growth
factor
mitogen series of cells of
mesodermal origin and ectodermal
FGF-2 Fibroblast growth
factor 2
mitogen for a variety of cells such
as fibroblasts, endothelial cells,
myoblasts; induces embryonic
mesoderm
IL-2 Interleukin 2 mitogen for T-lymphocytes
The mitogens - growth factors supporting the proliferation
Examples of mitogen:
73
pathobiochemistry- receptors_1
Receptors with a serine / threonine kinase activity
The ligand is e.g. transforming growth factor-  (TGF- )
After ligand binding receptors dimerize and activate the serine /
threonine kinase activity in their respective domains
One active subunit phosphorylates partner subunit in the catalytic
site
Phosphorylated partner subunit phosphorylates the cytoplasmic
proteins SMAD
Smad proteins are activated by phosphorylation and forms dimers
with other SMAD proteins
The translocation to the nucleus, where they interact with other
regulation proteins
P
P
P
P
Migration to
nucleus
SMAD
SMAD
74
pathobiochemistry- receptors_1
IV. Receptors activating non-receptor tyrosine kinase
ligand
dimerization
–PSTAT
STATSTAT
JAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription)
Receptor has kinase activity, but is associated with the tyrosine kinase JAK.
After ligand binding receptors dimerize (homodimers or heterodimers)
Activated somehow phosphorylate tyrosine residues on the receptor.
The sites phosphorylated adapter proteins bind STAT (using SH2 domains)
STAT are phosphorylated and dimerize.
STAT dimers translocate to the nucleus, where they act as transcription factors
–P STAT
–PSTAT
TEST
75
pathobiochemistry- receptors_1
JAK-STAT receptors - a family of receptors for
cytokines * (e.g.interferons, interleukins)
Diverse cytokine effects are caused by the existence of large
amounts of STAT proteins
Receptors for various cytokines bind the various states which
produces heterodimers in different combinations
Thus, it is possible that different cytokines affect different
genes
Receptors cooperating with the JAK-STAT also have
prolactin, erythropoietin ad.
* Cytokines - small signaling proteins involved in the immune response significantly. They are
produced by immune cells (macrophages, T-cells etc.) and are capable of inducing such as rapid
division and differentiation of certain cell types involved in the fight against pathogens and other
features of the immune defense
76
pathobiochemistry- receptors_1
Control Membrane Receptors
Regulation by changing the number of
receptors (down regulation, up-
regulation)
Control properties
receptor
(desensitization)
For example: desensitization of -adrenergic receptor
Upon binding of the ligand to the receptor activates BARK (β-adrenergic
receptor kinase)
The cytoplasmic portion of the receptor molecule, the phosphorylation
The phosphorylated site binding arrestin protein that inhibits the ability to
activate G-protein
77
pathobiochemistry- receptors_1
Intracellular receptors
Steroid hormones, calcitriol, and iodo thyronine Retinome
Receptors are located in the cytoplasm or in the nucleus
The hormone-receptor complexes bind to specific DNA sites
and serve as transcription factors
Hormone-receptor complex to the DNA binding site HRE
(hormone response element)
Superfamily of steroid and thyroid receptors - a family of
structurally related proteins.
Activation of transcription is a slower process, a response delay
TEST
78
pathobiochemistry- receptors_1
Example cortisol (glucocorticoid GK, their receptors GR)
hydrophobic molecule penetrates the cell hormone
inactive receptor
glucocorticoid (GR)
exists in cytoplasm as a complex with
a protein dimer
hsp 90 (chaperone) and other proteins
active monomeric complex cortisol-receptor,
separated hsp 90 proteins with others
active complex and dimerizes
nukleoporus translocated
into the nucleus
GR
kortisol
CBG
cortisol in the extracellular space transmitted CBG (corticosteroid-binding globulin)
79
pathobiochemistry- receptors_1
Dimeric complex cortisol receptor in the nucleus continues to site-specific
dsDNA sequences of GRE (glucocorticoid response element), i.e. on the HRE
(hormone response element) specific to glucocorticoids.
DNA binding domain
GR
receptor
DNA
GRE
Binding domain for cortisol
(hydrophobic pocket)
80
pathobiochemistry- receptors_1
GR dimer – intracellular glucocorticoid receptor (dimer)
GRE – glucocorticoid response element
GREB protein – GRE binding protein (specific transcription factor)
TF IID Pol II
CTD
> 1 000 bp
Mediator proteins
enhancer
koactivatorGREB protein
GRE
dimeric complex cortisol-GR
promotor
basal
Transcriptional apparatus
Active complex cortisol receptor binds to DNA in a sequence-specific site
GRE (glucocorticoid response element, one of the many HRE - hormone response
elements). Complex itself, however, hormone-receptor binding to DNA and affect
transcription is not capable. Connects to a specific coactivator proteins
GREB(glucocorticoid response element-binding proteins). This complex via
mediator proteins builds upon the basal transcriptional apparatus to the promoter
and initiates transcription.
Initiation of transcription cortisol
81
pathobiochemistry- receptors_1
Overview of the most important neurotransmitter receptors
Cholinergic synapsis
Nicotine MuscarinicReceptor
Mechanism of
action
Second messenger
Location
Block receptor
Ion channel
Tubokurarine Atropine
•Brain
•Smooth muscle
•Glandular cells
•Myocardium
•Brain
•autonomic ganglia neurons
•neuromuscular junction
•chromaffin cells of the adrenal medulla
DG + IP3 cAMP
M1, M3, M5 M2, M4
Gq Gi
TEST
82
pathobiochemistry- receptors_1
Adrenergic synapses
Second
messenger
Examples
of location
•smooth muscles of
the GIT (sphincters)
and skin blood vessels
(contraction)
•adrenergic and
cholinergic nerve endings
(inhibition of transmitter
release)
•pancreas (exocrine
secretion inhibition)
•platelets (aggregation)
•myocardium
(increase in
strength and
frequency of
contractions)
•smooth muscle of
the uterus, bronchus
(relaxation)
•smooth muscle of
GIT (peristalsis)
•pancreas (exocrine
secretion activation)
The action of the neurotransmitters in the autonomic nervous system
System Parasympaticus Sympaticus
Mediator Acetylcholine Acetylcholine Noradrenaline
Receptor
Location
nicotine muscarinenicotine muscarine
•dendrites
ganglion
neurons
•membrane
s of target
cells
•sweat
glands
•membranes of
target cells
•chromafinní buňky dřeně
nadledvin
•dendrity neuronů ganglií
TEST
83