1. Mechanism of hormone and
neurotransmitter action
Types of membrane and and
intracelular receptors
Department of Biochemistry FM MU 2011 (E.T.)
2. Control of metabolism
– Regulation of metabolic events within particular compartment
(cellular organelle) that depends only on interactions between
molecules in the compartment;
– regulations that occur within complete cells without any regard to
extracellular signals, in which proteosynthesis and transport across
membranes that separate individual compartments have the important
roles have;
– regulations that are consequences of communication between cells
in particular tissues, organs, or the whole organism, depending on
extracellular signals – neurotransmitters, hormones, cytokines, and
other signal molecules.
Numerous metabolic pathways are controlled usually in only one or
few check-points (rate-limiting steps) by more than one different
mechanisms.
These formal levels of metabolism control mostly overlap.
2
3. Mechanism of hormone and
neurotransmitter action
Signal molecule types in neurohumoral regulations:
Signal molecule Origine
HORMONES secreted by endocrine glands, by dispersed
glandular cells (eicosanoids by many other
cellular types)
NEUROHORMONES secreted by neurons into the blood circulation
NEUROTRANSMITERS secreted by neurons at nerve endings
CYTOKINES, GROWTH secreted by various types of cells
FACTORS, IKOSANOIDS 3
4. Action of signal molecules
Action Character of action
endocrine Signal is carried by the blood, may act in the
whole body. Typically hormones
paracrine Signals act within short distances of the site of
their production
autocrine Signal act on the cells that produce them
4
5. Signal transduction
How cells receive, process, and respond to information from the
environment?
Reaction of signal molecule with receptor
Membrane receptors Intracelular receptors
Proteins and small polar Nonpolar signal molecules
signal molecules (amino (steroids, iodothyronines,
acids, peptides, biogenic retinoates)
amines, eicosanoids)
5
6. Membrane and intracellular receptors
Nonpolar signal molecule
Polar signal molecule bound to the plasma transport protein
Transport of signal molecule
Membrane receptor
Signal transduction
Intracellular receptor
Amplification
Interaction of the complex hormone-receptor
with the HRE of nuclear DNA
Biological response
(prompt effect)
Biological response
(the effect is slow, either early or late)
6
7. Main types of membrane receptors
Receptors – ion-channels (ROC, ligand gated ionophores) serve
exclusively as receptors for neurotransmitters (see lecture 7).
Receptors activating G-proteins (heterotrimeric G-proteins), the result
of specific ligand binding is mostly
- stimulation or inhibition of adenylate cyclase,
- stimulation of phospholipase C,
- stimulation of phosphodiesterase.
Receptors exhibiting intrinsic catalytic activity
- guanylate cyclase activity – receptors for natriuretic peptides,
- tyrosine kinase activity
- insulin receptor, receptors for insulin-li growth factors (IGF1,2),
- dimerizing receptor for epidermal growth factor (EGF).
Receptors cooperating with non-receptor tyrosine kinases
(e.g., Janus kinase JAK) – receptors for somatotropin (growth
hormone), prolactin, erythropoietin, interferons, interleukins and other
cytokines.
7
8. Family of heterotrimeric G-protein-coupled receptors
Common structural features : Binding site for the
agonist (also
accessory binding
All of them are seven -helical segments sites for antagonists)
that span the membrane and are connected
by intra- and extracellular hydrophilic and H2N
more divergent loops.
Intracellular domains -
binding site for the specific -COOH
G-protein type.
8
9. Heterotrimeric G-proteins
Proteins binding GDP or GTP
mostly freely membrane-bound (they can move along the inner
surface of the plasma membrane).
Subunits , a .
Subunits G and
G are
hydrophobic and
non specific
G subunit is the largest,
hydrophilic, it binds GTP
or GDP, and
It is specific for particular
More than 20 different subunits have mechanism of second
messenger production.
been identified.
9
10. The cycle of G-proteins activation
Complex
receptor-specific ligand GTP
Complex receptor-ligand-
-trimer G-GDP,G,G
GDP
Trimer G-GDP,G,G
Dimer G,G
Dimer G,G Inactive Activated
subunit G-GDP subunit G-GTP
Interaction
with the target protein
Pi
PRODUCTION OF THE
SECOND MESSENGER
10
11. Selected types of G protein -subunits
G subunit type Examples of Effect of activated G
activating receptors on the target protein
Gs (stimulatory) glucagon stimulation of
parathyrin adenylate cyclase
-adrenergic
Gi (inhibitory) somatostatin inhibition of
2-adrenergic adenylate cyclase
Gq (activating the PI vazopressin V1 stimulation of
cascade) endotelin ETA,B phospholipase C
acetylcholine M1
1-adrenergic
Gt (inhibitory) rhodopsine stimulation of
(for transducin) cGMP phosphodiesterase
11
12. Hormone receptors that activate Gs or Gi proteins
stimulates or inhibit adenylate cyclase
Adenylate cyclase, a membrane-bound enzyme, catalyzes the reaction
ATP cAMP + PPi ; the second messenger is cyclic AMP.
ligand ligand
receptor AMP-cyclase receptor
GS Gi
ATP
cAMP
*phosphodiesterase
AMP H2O proteinkinase A
inactive (R2C2)
phosphorylations active proteinkinase A
2 C + 2 R(cAMP)2
12
*Inhibition by caffein, theofyllin
13. They target cAMP action in the cells
activity to
physiological AKAP
substrates AKAP
Protein
cAMP
ATP
C R R C ADP
C R R Protein-P
C Protein
Proteinkinase Proteinkinase ATP
A (inactive) A (active) ADP
Protein-P
Phosphorylation of proteins.
In cytoplasma - mostly metabolic enzymes (rapid response)
In the nucleus – phosphorylation of gene specific transcription factor
CREB (cAMP response element-binding protein) (slower response)
13
14. Localization of cAMP action in specific site of
the cell
Proteins AKAPs (A kinase anchoring proteins)
Proteins binding proteinkinase A, they target its activity to physiological
substrates (they serve as a scaffold which localize PAK near to
substrate).
Similar proteins affects also the specific action of phosphatases,
phosphodiesterases.
14
16. Receptors that activate Gq protein stimulate
phospholipase C and start the phosphatidylinositol cascade
Phospholipase C
diacylglycerol
Both reaction products are the second inositol 1,4,5-
messengers: trisphosphate
Inositol 1,4,5-trisphosphate opens the Ca2+
channel in ER membrane, 16
diacylglycerol activates proteinkinase C.
17. Phosphatidylinositol cascade
specific ligand activation of
proteinkinase C
receptor phospholipase C PIP2 DG
Gq
IP3 active proteinkinase C
increase of [Ca2+]
in cytoplasm
phosphorylations
Endoplasmic reticulum
Ca2+
IP3 receptors in the membranes of ER
act as ligand gated channels for Ca2+ ions
17
18. Control of metabolism by changes of
cytoplasmic concentration of Ca2+
•Basal concentration of Ca2+ in cytoplasma 1.10-7 mol/l
• Increase to concentration to 1.10-6 rapidly and maximally
agtivates the various Ca2+-regulated cell function
• Increse of Ca2+ can be triggered
by influx of Ca2+ across the plasmatic
membrane (see e.g.smooth muscle contraction)
or by release from intracelular stores (ER,
mitochondrias) e.g. IP3 gated Ca 2+ channel in
ER, or ryanodine receptors in ER/SR of cardiac or
skeletal muscle 18
19. Regulatory protein calmoduline
Increased cytosolic Ca2+ activates various calcium-binding
regultory proteins (family of small, Ca 2+ dependent protein).
The most important is calmoduline. It is ubiquitously
expressed protein in nearly all cells.
Upon binding of Ca2+ (4 binding sites)
calmoduline undergoes conformational
changes that facilitates its interaction
with downstream signaling proteins,
e.g. kinases, phosphatases ect.
Some Ca-calmodulin-dependent
kinases are very specific, the other have
a broad substrate specifity. 19
20. Receptors with guanylate cyclase activity
After binding of ligand they convert GTP to cGMP
cGMP is the second messenger
O
It activates proteinkinase G
N
HN
Two types of receptors:
H2N N N
•membrane-associated
• soluble (cytoplasmic) O
O
O P O OH
OH
20
21. Membrane receptors with guanylate cyclase
activity
ANP
Receptors for ANP (atrial
natriuretic factor)
Mainly smooth muscle of
vessels and in kidneys
ANP is produced
GTP cGMP + PPi by cardac atrial
phosphodiesterase Protein kinase G tissue in response
H2O inactive to increase of
GMP blood volume or
pressure
active protein kinase G (PKG)
Phosphorylation of proteins 21
22. Soluble receptors with guanylate cyclase activity
NO
NH2 NH2
Receptor je dimeric complex and
hem
binds hem
Binding NO to the hem increases
catalytic acitivity guanylate cyclase
NO is generated by the action of
GTP cGMP nitroxid synthase (NOS)
phosphodiesterase NO readily permeates membranes, it
can be produced by one type of the cell
Activation of and rapidly diffuse into neighboring cell
protein kinase G types
GMP
22
23. Proteinkinase G
cGMP sensitive proteinkinase G
Widely expressed in many cells
It phosphorylates various proteins (enzymes, transportion
proteins ect.)
Effect of PKG in smooth muscle
Phosphorylation of proteins:
• inactivation of proteins attenuating Ca2+ release from ER Ca2+
• activation of MLC phosphatase repression of actin-myosin interaction
• decrease of K+-channnels activity decrease of hyperpolarization
increased influx of Ca2+ into the cell
23
Relaxation of smooth muscle
24. NO/cGMP signaling in smooth muscle of
blood vessels
cGMP is the key second messenger for induction of vascular
smooth muscle vessels relaxation
vasodilatation and increased bloof flow
NO is produced in endothelial cells by the action of nitroxid
synthase from arginin (activation e.g. by acetylcholine) and
diffuses into adjacent smooth muscle cells
NO-synthase
L-Arg ·NO + L-citrullin
24
25. Nitroglycerin and other drugs of organic nitrate
type are donors of exogenous NO
R-O-NO2 nitrit ·NO
Glycerol trinitrate
Isosorbide nitrate Activation of soluble
guanylate cyclase
Therapy of angina pectoris
Vasodilatatory effect releases coronary spasmus and
normalizes blood perfusion.
25
26. Inhibition of phosphodiesterase potentiates
the effect of NO
Several types of
phosphodiesterase phosphodiesterase
cGMP GMP are known,
depending of the
type of cell.
H2O
The drug sildenafil (Viagra) acts as a selective inhibitor of phosphodiesterase 5
(PDE5), that is higly expressed in vascular smooth muscle.
Viagra is 80-4000 fold less potent as an inhibitor of other PDE isoforms
(including PDE3 that is expessed in heart).
During sexual stimulation NO in corpora cavernosa is released
The level of cGMP is increased
sildenafil prevents decomposition of cGMP 26
blood vessel in erectile tissue are dilated and blood flow is increased
27. Receptors with tyrosin kinase activity
Common features
• when the signal molecule binds to the receptor, it triggers conformational
change of the receptor
• this leads to activation of tyrosin kinase activity of the receptor
• the first protein substrate is the receptor itself (autophosphorylation of tyrosine
molecules in the receptor), eventually other proteins are phosphorylated
• phosphorylated tyrosines and other substrates then acts as a recognition or
anchoring site for other proteins, adaptor molecules
• adaptor proteins bind to phosphotyrosine residues by SH2 domaines (Src
homology 2 domain) and are also phosphorylated.
• adaptor proteins reacts with other molecules and signal is transmitted through
cascade of other reactions mainly phosphorylation/dephosphorylation, exchange
of quanine nucleotides, changes of conformation etc.
27
28. Insuline receptor
Dimeric structure
Binding site for insulin on - subunits
-S-S- -S-S-
Tyrosin kinase activity on -subunits
-S-S-
Binding of insuline to the receptor tyrosin kinase activity
autophosphorylation of -subunits and phosphorylation of IRS 1-4 proteins
(insulin receptor substrates 1-4)
Insulin
-S-S- -S-S-
-S-S-
P- -P P- -P activation and docking of
IRS1-4 -P PI-3-kinase on membrane
IRS1-4 activation of phosphoprotein phosphatase-1
activation of small G-protein Ras 28
29. Some signaling pathways of insuline
29
http://www.abcam.com/index.html?pageconfig=resource&rid=10602&pid=7
30. Insuline receptor substrates 1-4 are adaptor proteins.
If phosphorylated by the insuline-receptor complex, they bind to other proteins
that are activated in this way.
Examples of insulin receptor signaling pathways
Glykogen synthesis Translocation of glucoseých transporters
Phosphorylation of IRS activates Insulin receptor phosphorylates CbI
regulatory subunit PIP2-3 kinase
Complex CbI-CAP translocates to the
Catalytic subunit of PIP2-3 kinase lipid raft in the membrane
phosphorylates PIP2 to PIP3
CbI reacts with adaptor adaptor protein
PIP3 activates proteinkinase B (AKT), Crk
activation is enabled by PDK
(phosphoinositide dependent kinase) Crk is associated with C3G
activated AKT difuses to cytoplasma C3G activates TC10 (G-protein)
and phophorylates (inactivates) TC10 activates translocation of
glykogen synthase kinase transporters into the plasmatic
Synthesis of glycogen is activovated membrane
(active form is dephosphorylated form
of glycogen synthase)
30
31. Dimerizing receptor for EGF (epidermal growth factor)
containing an intrinsic tyrosine kinase activity
Dimerization of the receptor after binding of ligand
R R
RR
P- -P Ras–GTP
P- -P SoS Raf phosphorylation
phosphorylation
cascade MAP
Dimerization activates tyrosin kinase activity in cytoplazmatic domain.
Autophosphorylation of the receptor
Adaptor proteins Grb2 (SH-2 domains) bind to phosphorylated sites.
G-protein Ras is activated by the action of SOS protein activation of
MAP-kinase cascade (Ras/MAP-cascade)
31
32. Ras
Monomeric G-protein that binds GTP and at the same time has GTPase
activity (structural analog of subunit intrimeric G-protein).
It is activated by binding GTP instead GDP
SoS
Activation of Ras is key step in transmision of signal.
Inactive Ras-GDP is converted to Ras –GTP, that activates the
next molecule of the pathway.
Inactivation of Ras: hydrolysis of GTP iniciated by other
regulating proteins
32
33. Superfamily of Ras proteins
•5 families: Ras, Rho, Arf, Rab, Ran
•They are anchored to lipid membrane by lipid anchors
(myristoyl, farnesyl)
•Monomeric G-proteins that play important role in
regulation of growing, morphogenesis, cell motility,
cytokinesis etc.
•Mutations in Ras genes induce patologic proliferation and
antiapoptosis. About 30 % ofall human tumors involve cells
expressing mutated Ras oncogenes*.
*(Ras genes are named protooncogenes) 33
34. MAP-kinase signaling pathway
(MAP kinase =Mitogen activated protein kinase)
Map-kinase cascade It regulates mainly cell growing and
Ras–GTP diferentiation.
MAP-kinase-kinase-kinase P MAPKKK, Raf
ATP
ADP phosphorylation of
MAP-kinase-kinase P MEK cytosolic or
ATP
membrane proteins
ADP
ADP
ATP
MAP-kinase P
ATP phosphorylation of
ERK ADP
regulatory proteins in
nucleus, stimulation of 34
proliferation
35. Mitogens – grow factors stimulating proliferation
Examples of mitogens:
Abbr. name Function
PDGF Platelets derived growth Mitogen for cell of connective
factor tissue and non differentiated
neuroglia
EGF Epidermal growth factor Mitogen for many cells of
ektodermal and mesodermal
origine
FGF-2 Fibroblast growth factor 2 Mitogen for many cells like
fibroblasts, endotelhelial cells,
myoblasts;
IL-2 Interleukine 2 Mitogen for T-lymphocytes
35
36. Receptors activating non-receptor tyrosine kinases
JAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription)
ligand Receptors of cytokines
dimerization – e.g.interferons,
interleukines
STAT –P
STAT STAT
–P
STAT –P
STAT
•Receptor does not have kinase activity, but is associated with tyrosinkinase JAK.
•After binding of ligand receptors dimerize (homodimers or heterodimers)
•Activated JAKs phosphorylates tyrosine residue of the receptor.
•The STAT proteins (signal transducers and activators of transcription) associate
with the receptor and are phosphorylated by JAK.
•STAT phosphates dimerize, translocate to the nucleus, bind to specific DNA
36
elements and regulate transcription.
37. Intracellular receptors of steroid hormones
(and calcitriols), iodothyronines, and retinoates
Receptors are present in cytoplazma or nucleus
Hormon-receptor complexes binds to the specific sites in
DNA and activate the transcription of specific genes
Complex hormon-receptor binds to DNA at HRE (hormon
response element)
Superfamily of steroidal and thyroidal receptors – a family of
structuraly related proteins
37
38. Example of cortisol
cortisol is transported attached on CBG (corticosteroid-binding protein) in ECF
hydrophobic molecule penetrates membrane
cortisol
CBG
active complex receptor-ligand (monomer),
hsp 90 and other proteins are released
GR
inactive receptor
for glucocorticoids (GR)
forms in the cytoplasm active complexes form dimers and
complex with hsp 90 dimer are translocated into the nucleus
38
and other proteins through nuclear pores
39. Glucocorticoid receptor (GR) – function
GRE
DNA
GR
DNA binding domain cortisol binding domain
(hydrophobic pocket)
Active complex cortisol-receptor binds onto DNA at the specific sequence
GRE (glucocorticoid response element, quite generally HRE – hormone
response element)
39
40. Initiation of transcription by cortisol
Active complex cortisol-receptor binds onto DNA at the specific sequence
GRE (glucocorticoid response element, one of the HRE – hormone response
elements).
The coactivator and specific hormone response element-binding proteins
(GREB-proteins) are also attached. This complex acquires the ability to act as
enhancer that supports initiation of transcription on the promoter by means of
mediator proteins.
cortisol-GR dimer complex
GRE enhancer
GREB protein coactivator
mediator proteins
> 1 000 bp
CTD
Pol II basal
TF IID
transcription
apparatus
promoter
GR dimer – intracellular glucocorticoid receptor (dimer)
GRE – glucocorticoid response element
GREB protein – GRE binding protein (a specific transcription factor)
40
