Cell biology 2014 (revised 29/1 -14)
Lecture 4 & 5:
Cell Biology interactive  media  ”video” or ”interactive”
1
Cell communication
Signal molecules/proteins
Differentiate
Secrete
Proliferate
Move
Die
x
x
All diseases involve changes of normal cells. In some cases,
these changes may affect other cells of the individual
2
Events during cell communication
1. Regulated synthesis…..…..
Producer cell
.. or regulated release of a signaling molecule
1.
2. (Transport of signaling molecule to target cells)
3. Binding of the signaling molecule to a
specific receptor on/in a target cell
2.
3.
4. Activation of a transduction chain
4.
5. Target cell response
6. Termination of signal
“hormone" = to urge on/impulse
5.
Target cell
6.
3
Signaling receptor diversity
The mammalian genome
encodes for thousands
of signaling receptors
- Many of these are
targets for drugs
Tissue specific expression:
Each individual animal cell
express only some of these
receptors
4
Membrane permeability
• Hydrophobic molecules
Cholesterol
O O
Cortisol
N O
Testosterone
• “Large” uncharged polar molecules
O
• Charged
molecules
N
C
Glucose
C
O
Amino acids
Na+
Ions
Cl5
Localization of signaling receptors
Receptor in cytosol
Receptor in
nucleus
Hydrophobic
molecules
Hydrophilic
molecule
(and proteins)
Receptor on plasma membrane
Other compounds than the natural ligand may interact with a
receptor – some are used as drugs (legal & illegal)
“natural ligand” = an endogenous receptor binding molecule
hydrophobic  lipohilic  non-polar (often used as synonyms)
6
Receptor agonists and antagonists
Other compounds than the natural ligand may bind a receptor
Agonists: mimic completely, or partially, the action of the
endogenous ligand
Antagonists: bind to receptor without activating it
 block the action of the “natural” ligand
OH
Adrenalin
(natural)
OH
CHCH2NHCH3
OH
OH
Phenylephrine CHCH2NHCH3
(selective
agonist)
OH
One of the action of adrenalin is to cause a dry mouth in the
fight-or-flight reflex. Phenylephrine is used in many “cold-relief”
drugs to prevent excessive nasal mucous secretion
7
Five modes of cell communication
Surface receptor
Intra-cellular receptors
C
B
D
Bloodstream
A
E
Contact dependent signaling:
A Ligands on the cell surface
Signaling by secreted ligands:
B Paracrine
C Autocrine
D Endocrine E Neuronal/synaptic
"crinis" = secrete
Neuron
8
A Contact-dependent signaling
Target cell
Signaling cell
Receptor/ligand
Contact-dependent signaling uses ligands and receptors that are
plasma membrane-bound:
- Persistent signals (uni- or bidirectional)
- Directed toward neighboring cells
9
A Distinct types of contact-dependent cell signaling
Cell surface receptors that mediate cell-to-cell adhesion
(cadherins) and cell-to-ECM interaction (integrins) are
also involved in signaling. Important for: Development
Growth control
Survival
Cadherin
Gap junction
Integrin
Gap Junctions permit
free passage of small
molecules between
adjacent cells
Important for e.g.,
synchronous heart
contraction
10
B Paracrine signaling
Signaling cell
Adjacent
target
cells
Paracrine signaling involves secretion of a ligand that act
locally on cells with the appropriate receptors:
Local effect  "para" = near
11
C Autocrine signaling
Autocrine signaling implies
that a cell secretes a ligand
that it responds to itself
Signaling and
target cell
"autos" = self
?
12
D Endocrine signaling
Endocrine cell
secreting
Distant target
cells
Endocrine signaling involves a signal molecule (poly-peptide or
steroid hormone) produced by an endocrine cell.
"endo" = inside/within "crinis" = secrete
Each endocrine cell secrete only one type of signal molecule!
The hormone travels through the blood system:
Global signaling with long-term effect
Relatively slow responses - the signaling molecule have to travel
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through the blood systems before reaching a target cell
E Neuronal/synaptic signaling
Signaling cell
Target cell
Release of neurotransmittor
Axon
Cell body of a neuron
Synapse
"syn" = together "haptein" = hold onto
Neuronal/synaptic signaling is mediated by neurotransmitters
released at the interface between the signaling and the target
cell, called synapse. The release of neurotransmitters at the
synapse is controlled from the cell body through electrical
signals. Neurotransmitters bind cell surface receptors.
- Acts rapidly and transiently on the target cells
14
Neuroendocrine integration
Hormone secreting glands in the
brain link neuronal signals and
peripheral endocrine glands.
*Gland
Fight-or-flight reflex: the HypothalamicPituitary-Adrenal (HPA) system
The adrenal gland responds to both the
hormone (ACTH) and a nerve signal
ACTH Adrenal Cortex cortisol
Increased blood levels of lipids etc. etc
Nerve signal adrenal medulla adrenaline
 Increased blood levels of lipids & glucose
etc. etc.
Endocrine cell: a cell within an endocrine
gland that release a hormone into the
circulating blood in response to a neural
(synaptic) or hormonal stimulus
*Gland
kidney
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Signaling molecules
Molecules typically produced and released by one cell and
recognized by another cell
Signaling molecules are chemically diverse:
- Gases: nitric oxide, carbon monoxide
- Steroids: testosterone, cortisol, etc.
- Proteins: insulin, glucagon, etc.
- Amines: catecholamines, acetylcholine
Membrane
permeable
Membrane
impermeable
16
“Ryss 5a”: A mix of synthetic anabolic steroids ( muscle growth)
17
Fast versus slow signal transduction events
Signal
Altered protein
function
Altered gene
expression
DNA
mRNA
Fast (<seconds)
An altered cytoplasmic
signaling protein
Cell response
mRNA Altered
protein
Protein level
Slow (minutes to hours)
Signaling with nitric oxide gas
• Nitric oxide (NO) acts as a paracrine signal, only affecting local
area, due to its short t1/2 (1-5 seconds)
• Produced by nitric oxide synthase through the deamination
of the amino acid arginine
• Nitric oxide is a very potent vasodilator (blood vessel dilatation)
Nitroglycerin is converted
in blood to NO (used to
treat coronary artery
disease since 1878)
CH2 CH2
CH2
O
O
O
NO2 NO2
NO2
18
Three types of cells dedicated to contraction
• Skeletal muscle
• Cardiac muscle
• Smooth muscle cells:
i) surrounds hollow organs –
intestines and blood vessels
ii) arrector pili muscles
attached to hair follicles
All three muscle cell types contains filaments
consisting of actin and myosin, which may
contract and slide apart
19
Vasodilatation through nitric oxide signaling
Neuron
Blood vessel
Endothelial cell
Acetylcholine
Arginine
NO (Nitric oxide)
Diffusion to adjacent
smooth muscle cell
Smooth muscle cell
”2nd messengers”
Relaxation of smooth muscle cell
Increased blood flow
20
Cytosolic signal mediators: second messengers
1st messenger: the external signaling molecule (e.g. Nitric oxide)
2nd messenger: the molecule that transfer the signal in the cytosol
cAMP, cGMP and Ca2+ are the classical 2nd messengers
Ca2+
Ca2+
Ca2+
Ca2+
=1 mM
=10 nM
Ca2+
Adenylyl
cyclase
Ca2+
Ca2+
Ca2+
Ca2+
Guanylyl cyclase
Ca2+
Ca2+
Ca2+
Video 15.1-calcium_signaling
21
Effect of nitric oxide on smooth muscle cells
Nitric oxide
Guanylyl cyclase
22
P P P
GTP
Cyclic-GMP
phosphodiesterase
(constitutively active)
+ P P
P
Cyclic GMP
Activation of an “in-ward”
Ca2+-pump in membranes of
intra-cellular Ca2+-stores
P
Viagra
GMP
Relaxation of smooth
muscle cells and
increased blood flow
Low [Ca2+] makes contractile
filaments (actin and myosin)
slide apart
Signaling by intracellular receptors – part I
23
Hydrophobic ligand (e.g. Cortisol)
Plasma membrane
1.
2.
NLS
1. Cortisol diffuse
NLS
through the plasma
Combined receptor/
membrane
transcription factor
2. Binding displaces a
protein that masks an
3.
NLS on the cortisol
= DNA
receptor
NLS Target
genes
3. Receptor translocation
into the nucleus 
specific transcription
Signaling by intracellular receptors – part II
Hydrophobic ligand
Plasma membrane
2.
Inhibitor
1.
Target
genes
3.
Target
genes
1. The DNA-binding receptor/transcription factor is inactive
2. The ligand (e.g. sex hormones) diffuses into the nucleus
3. The ligand displaces the inhibitor
24
General principle of cell surface receptor signaling
Signal molecule
1. Reception
(Ligand)
Receptor
2. Signal transduction
cascade comprising:
i. molecular switches
ii. 2nd messengers
3. Response
Gene regulatory
protein
P. M.
Cytosol
Metabolic
enzyme
Etc.
25
I. Molecular switches in signal transduction
A signal that can be switched on, also needs to be switched
off (all signals are more or less transient)
1. Protein phosphorylation
The most common ‘on-off’ switch is provided by protein
phosphorylation
O
OP
Kinase + ATP
O
OH
O
Serine, threonine
or tyrosine
Phosphatase
Serine, threonine
or tyrosine
Kinase : ~1000 protein kinase genes in vertebrates. Some
have only a single substrate. Others are “multifunctional” and may have >10 substrates
26
II. Molecular switches in signal transduction
27
2. GTP binding proteins (G-proteins)
Another ‘on-off’ switch is provided by
regulatable GTP-binding and hydrolysis
GDP
GDP
Inactive
GTP
Guanine-nucleotide
Exchange Factor (GEF)
GTP >> GDP
GTP
Active
GTPase Activating
Protein (GAP)
P
Molecular_models 15.5-Ras (one PO4 makes the diff.)
Signal transduction cascades
A single cell surface receptor
may activate several signal
transduction pathways
P. M.
This involves various Gproteins, 2nd messengers and
protein kinases
Protein kinases at the
end of a cascade may
have many substrates
cGMP
cAMP
Ca2+
Kinase
P
Response:
GTP
Gene regulation
P
Metabolism
P
Etc.
28
Three main classes of cell-surface receptors
G-protein coupled
receptors
ZZZ
Receptors with intrinsic
Ion channel
enzymatic activity
coupled receptors
ZZ
Z
ZZ
Z
Ion
Ion
Ligand
Ion
29
30
G-protein coupled receptors (GPCR)
A hallmark of GPCR´s is 7 transmembrane spanning regions
ZZZ
1. Ligand binding conformational change
2. A specific G-protein is recruited and activated
3. G-proteins may regulate enzymes or ion channels
G
Down-stream effectors of various G-proteins
1.
2.
ATP
GTP
Guanylyl cyclase
Adenylyl
cyclase
Cyclic AMP
3.
Cyclic GMP
4. Ion channels
Phospholipase C
Increase in
cytosolic Ca2+
and activation of
protein kinase C
Ion
Ion
Ion
31
I. Regulation of hetero-trimeric G-proteins
GDP
GTP
32
GTP >> GDP
Complex dissociate
upon GTP binding
=GEF
GDP
Inactive
a
b
g
GTP
Active
a
+
b
RGS =GAP
P
RGS: Regulator of G-protein Signaling
a-subunit and/or b,g-subunit
can activate or suppress
different downstream targets
II. Regulation of hetero-trimeric G-proteins
No ligand (default state)
P.M.
GDP
b ga
Ligand binding causes a conformational change
P.M.
GDP
b ga
b
+
GTP
a
GDP GTP
The G-protein is recruited to the receptor, which acts as a GEF
 the a-subunit exchanges GDP for GTP
 dissociation of an active a-subunit
33
III. Regulation of hetero-trimeric G-proteins
34
The intrinsic GTP hydrolysis is slow but RGS, an a-subunit
specific GAP, catalyzes hydrolysis. This terminates the signal
P.M.
GDP
a
GTP
a
RGS
GTP
as
GDP
b ga
+ b
P
GTP
ai
Adenylyl cyclase
Alberts et al: Table 15-3 (tissue specificity)
GTP
aq
A family of asubunits with
distinct functions
Phospholipase C-b
(PLC-b)
Anim. 15.3-G-protein_signaling
Adenylyl cyclase activation by the as-subunit of G-proteins
Adenylyl cyclase
P.M.
GTP
as
Caffeine
P P P
ATP
Cyclic-AMP
phosphodiesterase
(constitutively active)
P
+ P P
Cyclic AMP
P
AMP
35
Cyclic AMP second messenger signaling
Cyclic AMP
Cyclic AMP activates
Protein kinase A (PKA),
which can regulate:
1. Metabolism
2. Gene transcription
Inactive PKA
Active PKA
Glycogen
phosporylase
CREB
P
Glycogen
phosporylase
1.
Glycogen
Glucose-1phosphate
P 2.
CREB Target
genes
36
Summary of the cyclic AMP signaling cascade
GTP
as
GEF (GPCR)
Cyclic AMP
Adenylyl
cyclase
PKA
ATP
Glycogen:
- Stored in muscles and liver
- Rapidly available energy source
Work/stress  adrenalin
 cAMP
 PKA    Glycogen breakdown
Alberts et al: Table 15-1 (tissue specific response)
Regulates
transcription
P
CREB
P -Regulated
DNA binding
Regulates
metabolism
P
P
Glycogen breakdown
Anim. 15.4-cAMP_signaling
37
Signal induced cleavage of phospholipids
External signals may activate distinct phospholipases that
cleave phospholipids at specific sites and thereby catalyze the
formation of various molecules with signaling properties
Fatty acid
Fatty acid
Precursors for
various signaling
substances
Phospholipase A2
Phospholipase A1
Glycerol
Soluble compounds
 release into the
cytosol
Phosphate
Variable
Phospholipase C
Phospholipase D
38
Phospholipase C activation generates two 2nd messengers
Phosphatidylinositol 4,5bisphosphate, PI (4,5)P2
Glycerol
P
P
Glycerol
OH
P
2.
1. aq-subunit activates PLC
2. PLC cleaves PIP2, generating the
two 2nd messengers DAG and IP3
Fatty acid
Fatty acid
Fatty acid
Fatty acid
Inner leaflet of
plasma membrane
GTP
aq
Phospho1. lipase C-b
(PLC-b)
Diacylglycerol
(DAG)
P
P
P
Inositol 1,4,5triphosphate, IP3
39
Role of the 2nd messengers IP3 and DAG
Inner leaflet of
plasma membrane
DAG
1. DAG recruits
1.
PKC to plasma
PKC
membrane
2. IP3 mediate release
of Ca2+ from ER
3. DAG and Ca2+
activates PKC
4. Ca2+ activates
calmodulin
to terminate
signal by
pumping Ca2+
back into ER
OH
PKC
Ca2+
3.
Ca2+
4. Ca
Ca2+
P
IP3
Ca2+
2+
Ca2+
Calmodulin
Ca2+
P
Calmodulin
Ca2+
P
Ca2+
Ca2+
2.
Calmodulin regulated
Ca2+ pump in ER
IP3 regulated
Ca2+ channel
40
41
Ca2+/calmodulin dependent protein kinase (CaMK)
Inhibitory Resting
state
Catalytic
Dephosphorylation
P
Inactive
Partially
active
Ca2+
Calmodulin
Increased
cytosolic Ca2+
Ca2+
Calmodulin
Ca2+
Ca2+
Autophosphorylation
P
Activated
Fully active
Molecular_models 15.6-calmodulin
42
Summary of G-protein signaling through PLC-β
GTP
aq
Both PKC and CaMK have
many potential (tissue
specific) substrates
GEF (GPCR)
PKC
PLC-b
DAG
OH
P
P
P
CaMK
P
Ca2+
P
P
IP3
Ca2+
Ca2+
Calmodulin
Ca2+
Other Ca2+
regulated enzymes
Ca2+
Etc!
STOP
Termination
of Ca2+ signal
Enzyme linked receptors
Many variants on this theme – here we focus on:
Receptor tyrosine kinases
Receptor serine/threonine kinases
Single pass transmembrane receptors. Ligand binding cause
dimer formation and consequent “auto”-phosphorylation
Tyr P
Homo-dimers
Alberts et al: Table 15-4 (tissue specific RTK’s)
Jenkinson : RTK - dimerization
Hetero-dimers
Ser/Thr P
43
Signaling through Receptor Tyrosine Kinases
Single pass
transmembrane
protein
Ligand binding causes
receptor dimerization
P. M.
Kinase
domain
Kinase
domain
Kinase
domain
Kinase
domain
Tyr
Tyr
Tyr
Tyr
P Tyr Tyr P
P Tyr Tyr P
Tyr
Tyr
P Tyr Tyr P
Inactive receptor
monomers
Cis- prefix means "on this side"
Trans- prefix means "across"
Active receptor
dimer
Trans-phosphorylation
of tyrosine residues
44
SH2-proteins binds at specific phospho-tyrosines
45
Regions containing phospho-Tyr may serve as specific docking sites for
SH2 domain-containing signaling proteins (SH = Src Homology domain)
Phosphatidylinositol (PI)
3
P
Monomeric G-protein
Kinase
domain
Kinase
domain
GDP
Ras
PI-3 Kinase P Tyr Tyr P
P Tyr Tyr P SH2 SH3
These can
P Tyr Tyr P
be enzymes….
………….or they
act as adaptors for
signaling proteins
GTP
Ras
Ras GEF
(Sos)
Fig. 15-55
Phosphorylation cascade downstream of Ras
P. M.
GTP
Ras
Raf
1. Altered protein function
Mek
Erk
(MAPK)
Cytosolic
target
proteins
2. Altered gene expression
P
Mek
P
Erk
P
Erk
1.
P
2.
P
Target
genes
46
Termination of RTK/Ras/MAPK pathway
1. Receptor and ligand internalization 2. Ras GTP hydrolysis
GDP
Ras
Fusion with
endosome
GTP
Ras
Ras GAP
Note: Signaling
receptors are
rarely recycled
Fusion with
primary lysosome
 degradation
Anim. 13.3-receptor_endocytosis
3. Dephosphorylation
Erk
(MAPK)
P
Erk
Phosphatase
(Note: vesicle fusion with endosome)
47
Extracellular space
Fatty acid
Fatty acid
I. PI-kinases act at specific positions of the inositol ring
Glycerol
Phosphate
Cytosol
Phosphatidylinositol (PI)
Inositol
PI – phosphorylation cycles on inositol ring position 4 & 5
P
5
P
P
P
4
Inositol
3
PI kinase
P
P
PIP kinase
PI(4)P
PI(4,5)P2
48
II. PI-3 kinase completes a PH-domain binding site
Phosphatidylinositol (PI)
1.
P
PI-3 Kinase
PI(4,5)P2
P
PI(3,4,5)P3
PTEN
P
P
3.
P
3
P
3 P
P
2.
PI-3 kinase
1. Activated receptor recruits and activates PI-3 kinase
2. PI-3 kinase phosphorylates PI(4,5)P2 to generate PI(3,4,5)P3,
which will serve as a docking-site for a family of signaling
proteins with a “PH-domain” (PH= Pleckstrin Homology)
3. PTEN removes phosphorylation on position 3 on PI(3,4,5)P3
to terminate signal
49
III. PKB/Akt activation downstream of PI-3 kinase
P
P
P
3 P
P
P
3 P
P
PH-domains
1.
P
P
P
3
P
PDK1
P
P
P
3
P
PKB/Akt
P
2.
PKB/Akt
P
PDK1
PKB/Akt
1. PI(3,4,5)P3 brings PDK1 and PKB/Akt
into proximity through their PH-domains
2. PDK1 phosphorylates PKB/Akt
50
thereby mediating its activation
IV. Different signaling pathways – same target
GDP
a
b g
GTP
a
+ b
P
PI-3 K
Both G-protein- and RTK signaling may result in
generation of PI(3,4,5)P3
3
P
P P
PI-3 K
There are two distinct PI-3 kinases which differ in
their regulatory domains
Thus, a PI-3 kinase may be recruited to the plasma membrane
51
via a bg-subunit binding domain or a SH2 domain
I. Transcriptional regulation by TGF-b / BMP
Type II receptor: Ser/Thr kinase
TGF-b
Type I
receptor
TGF-b
TGF-b
P.M.
P
P
Smad 2/3 P
Smad 4
Smad 2/3 P
Smad 4
Smad 7
Target
genes
Negative
feedback loop
52
II. Transcriptional regulation by Wnt/wingless
Wnt
LRP
Frizzled
Dishevelled
GSK-3b
P Axin
APC
b-catenin
ZZZ
Dishevelled
GSK-3b
b-catenin
Groucho
myc G1
TCF Target
genes
b-catenin
TCF
MMP7
Target genes
53
Recommended reading
Chapter 15
879-941
946-954
Alberts et al. 5th edition
"All science is either physics
or stamp
collecting"
My own
Ernest Rutherford
favorite
(1871-1937, protein!
Nobelprize1908)
Signal transducing
proteins are often
targets of therapeutic
drugs or infections agents
54