Second messengers. Second messengers are diffusible small
molecules that amplify and spread intracellularly an incoming
signal cAMP amplifies GPCR-perceiving signals PI3P and DAG
amplifies GPCR and some membrane PK (e.g. EGFR-perceiving) signals
cGMP amplifies signals perceived by membrane guanylate
cyclases
Slide 3
cAMP signaling cAMP amplifyes GPCR signals cAMP is syntehsized
by adenylyl cyclase and destroyed by cAMP-phosphodiesterase
Intracellular concentration of cAMP can be rapidly changed PKA is a
major target of cAMP signaling
Slide 4
Glycogen breakdown in skeletal muscles in response to adrenalin
The cascade includes changes in protein activity and does not
include changes in gene expression, that is why it is very fast
Major participants: Adrenalin GPCR (The 2 adrenergic
receptor=ADRB2) G-protein Adenylyl cyclase PKA Phosphorylase kinase
Glycogen phosphorylase glycogen (n residues) + P i glycogen (n-1
residues) + glucose-1-phosphate
Slide 5
Enzymes that catalyze reactions with phosphate groups Kinases -
transferases G-proteins- hydrolases Phosphatases - hydrolases
Glycogen posphorylase transferase and hydrolase
Slide 6
Other signaling pathways mediated by cAMP cAMP-regulated
channels PKA-regulated channels Transcriptional factor CREB ( cAMP
response element (CRE)-binding) in nucleus!
Slide 7
Phospholipid signaling The alternative signaling pathway
involves PI(4,5)P2 degradation by Phosphoinositide- phospholipase
C(PLC ) Inositol 1,4,5-triphosphate (IP3) is soluble Diacylglycerol
(DAG) is membrane bound There are several PLC isoforms PLC is
activated by Gq PLC is activated by polypeptide growth factor
receptors, such as platelet- derived growth factor (PDGFR),
epidermal growth factor (EGFR)
Slide 8
Phospholipid signaling PLC is activated by Gq Inositol
1,4,5-triphosphate (IP3) is soluble Diacylglycaerol (DAG) is
membrane bound DAG activates PKC IP3 rises intracellular Ca 2+ IP 3
can be broken down by phosphatases or turned into inositol. DAG is
metabolized via hydrolysis to yield glycerol and fatty acids or
phosphorylation to form phosphatidic acid.
Slide 9
Ca 2+ signaling Ca 2+ is a second messenger S. Ringer found
that in the presence of Ca2+ isolated frog heart maintained
activity for hours, Locke described that absence of Ca2+ inhibited
neuromuscular transmission. Kamada and Kimoshita discovered in 1943
that introduction of Ca2+ into muscle fibers caused their
contraction. Intracytosolic concentration 50-100 nM Extracellular
concentration 1-2 mM Intracellular stores 30-300 mM
Slide 10
Without special signaling intracellular Ca2+ concentration is
low due to ion pumps Ca2+-ATPases in the plasma membrane and ER
(SERCA), and Na+/Ca2+ exchanger in the plasma membrane. When
cytoplasmic Ca2+ rises, neighbouring Ca2+ channels are activated
progressively. Their opening leads to a Ca2+ wave. This is an
example of positive feedback.
Slide 11
Calmodulin is the best studied target of Ca 2+ Calmodulin
changes conformation upon Ca 2+ binding CAMKs are the best studied
target of calmodulin Look target of CAMKs in the previous
lecture
Slide 12
cGMP The cyclic GMP signaling system consists of a single
protein - Membrane guanylate cyclase The inhibiting enzyme is
cGMP-phosphodiestrase The effector protein is PKG cGMP serves as a
second messenger in a vertebrate eye. It converts the visual
signals (photons) to nerve impulses. Sharma and Duda 2014
Slide 13
Both GPCR and MGC input in the phosphotransduction system Here,
GPCR (rodopsin) is a light acceptor. It plays an activating role
via interactions with G-protein (transducin) Transducin activates
cGMP-phosphodiesterase Reduction in cGMP concentration results in
closing of on channels MGC plays an inhibitory role via opening ion
channels and stimulation of Na/Ca influx
Slide 14
It takes about 20 ms for production of a neurotransmitting
signal upon photon/rodopsin interaction Alberts Ecb
Phototransduction
Slide 15
Receptors and their regulation We consider three types of
receptors G-protein coupled receptors (GPCRs) Ion-channel coupled
receptors Enzyme-coupled receptors
Slide 16
Ion channel-coupled receptors Convert chemical signals into
electrical signals Binding of a ligand opens the channel
Slide 17
Ion channel-coupled receptors Some ICCR are GPCR coupled with
ion channels When the GPCR binds a ligand and changes conformation,
this change is directly transmitted to the channel and results in a
change in gating and in the ionic current through the channel.
Moreau, C. J. et al. Coupling ion channels to receptors for
biomolecule sensing. Nature Nanotechnology 3, 620-625 (2008)
Slide 18
Enzyme-coupled receptors Instead of association with G-protein,
the cytoplasmatic domain of ECRs has enzymatic activity or recruits
an enzyme Growth factor receptors (including EGFR, PDGFR, HGFR and
other receptors of Tyropsine kinase Family, TKRs) are the most
important class of ECRs that regulates cell proliferation,
differentiation and growth as well as cytosceleton rearrangements
TKRs has one membrane spanning domain which is an alfa-helix.
Slide 19
TKRs Ligand binding causes dimerization (oligomerization)
Dimerization results in phosphorylation of each subunit by another
subunit we speak about self-phosphorylation Self-phosphorylation of
intracellular domains causes an assembly of a transient
intracellular signaling complex Inhibition of TKRs via action of
phosphatases and endocytosis
Slide 20
TKR signaling The process of adaptor and effector proteins
binding with activated ECR is known as docking Several independent
signaling pathways might be induced by proteins of intracellular
signaling complex The most important effector proteins of the
RTK-associated signaling complex: PLC PKC Ras (small GTPase)
PI3K
Slide 21
Ras signaling cascade Ras is activated by almost all TKRs
Activation is via activation of Ras-GEF by activated TKR The
TKR-Ras specific GEF is Son of Sevenless (SOS) There are three
pathways that are activated by Ras IP3/DAG Raf/MEK/Erk MAPK
signaling pathway PI3K/Akt pathway
Slide 22
Raf/Mek/Erk MAPK signaling pathway Ras initiates the MAPK
signaling pathways when is bound with Raf In this pathway each
upstream molecule acts as a kinase to phosphorylate a subsequent
downstream molecule. An eventual result is the modulation of
transcription via the phosphorylation of a number of transcription
factors. This pathways involved in regulation of cell
proliferation, differentitation and survival It can inhibit
Fas-mediated apoptosis in T cells Controls production of IL-2 and
some chemokines Involved in Fcgamma-mediated phagocysotosis About
30 % of cancer cells carry a mutation that locks Ras in an active
form
Slide 23
Raf/Mek/Erk MAPK signaling pathway Raf =MAPKK kinase = MAPKKK
MAPK/ERK kinase = MEK=MAPK kinase= MAPKK MAPK = mitogen- activated
protein kinase = extracellular signal regulated kinase = ERK
Alberts et al., ECCB
Slide 24
PI3K pathway Phosphoinositide 3 kinases (PI3K) are composed of
two subunits, regulatory (p85) and catalytic (p110). p85 is
activated by Ras-GTP PI3K converts PtdIns(4,5)P2 into
PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 serves as a docking site (a
transient lipid anchor) for mane proteins The most important
downstream pathway is known as an Akt pathway Akt=PKB is a
serin-threonine PK Akt acts as an anti-apoptotic and cell survival
promoting factor
Slide 25
PI3K/Akt-controlled pathways Anti-apoptotic pathway via
inhibition of Bad/BAX Caspase-9 The NF-kB inhibitor IKK Cell-growth
promoting pathway via Tor
Slide 26
NF-kB NF-kB is a transcription factor NF- B regulates the
expression of cytokines, growth factors and inhibitors of
apoptosis, receptors involved in immunity including. Moreover,
pathological dysregulation of NF- B is linked to inflammatory and
autoimmune diseases as well as cancer. NF- B is not synthesized de
novo; its transcriptional activity is silenced by interactions with
inhibitory I B proteins present in the cytoplasm. NF-kB
Slide 27
Slide 28
Ubiquitin-proteasome pathway The Ubiquitin Proteasome Pathway
(UPP) is the principal mechanism for protein catabolism in the
mammalian cells. Two steps: tagging of the substrate protein by the
covalent attachment of multiple ubiquitin molecules (Conjugation);
and the subsequent degradation of the tagged protein by the 26S
proteasome, composed of the catalytic 20S core and the 19S
regulator (Degradation).
Slide 29
PI3K/Akt-controlled cell growth promoting pathway Tor (target
of rapamycin) is a serine-threonin PK Activated Tor inhibits
proteins degradation and stimulates protein synthesis
Slide 30
EGFR is a prototype ECR
Slide 31
EGFR inhibition There two major pathways for a fast EGFR
inhibition Dephosphrylase by PTPs protein tyrosine phosphatases )
Clathrin mediated endocytosis
Slide 32
Signaling cascades activating by GPCRs/TKRs
Slide 33
The Notch receptor Delta signal protein interactions Here there
is an example of contact-depedendent signaling DSL binding to the
Notch receptor triggers signaling through successive proteolytic
cleavages by ADAM protease. The intracellular Notch domain acts as
atranscriptional regulator
Slide 34
Extracellular ligands: hormones Adrenalin: produced by adrenal
glands Affect multiple organs The receptor - 2 adrenergic
receptor=ADRB2 The effect depends on a cell type and particularly
on G-protein type
Ligands that act without receptors Steroid hormones: Cortisol
Estradiol, testosterone Thyroid hormones: thyroxine Transcriptional
factors are targets of Gases NO (nitric oxide) is produced by
endothelial cells in response to neurotransmitters and affects
muscle cells. NO targets guanylyl cyclase.
Slide 38
NO signaling
Slide 39
Animals vs plants Plant PKs are structurally different from
mammalians The binding of a ligand (ethylene) switch off the
receptor Still, the effect is the same: interactions with a ligand
switch on gene expression
Slide 40
Integrative cell response The outcome depends on balancing of
different signals affecting the cell
