Upload
marlin
View
53
Download
1
Tags:
Embed Size (px)
DESCRIPTION
Cell biology 2014 (revised 29/1 -14). Lecture 4 & 5:. Mechanisms of cell communication part I & II . Cell Biology interactive media ”video” or ” interactive ”. Differentiate. Proliferate. Secrete. Die. Move. x. x. Cell communication. Signal molecules/proteins. - PowerPoint PPT Presentation
Citation preview
Cell Biology interactive media ”video” or ”interactive” 1
Lecture 4 & 5:
Cell biology 2014 (revised 29/1 -14)
Cell communication
Differentiate Proliferate
xx
DieSecreteMove
Signal molecules/proteins
2
All diseases involve changes of normal cells. In some cases, these changes may affect other cells of the individual
Events during cell communication
Regulated synthesis…..….... or regulated release of a signaling molecule
(Transport of signaling molecule to target cells)
Binding of the signaling molecule to a specific receptor on/in a target cell
Activation of a transduction chain
1.
2.
3.
4.
Target cell response5.
Termination of signal6.
1.
2.
Target cell
3.
4.
5.
Producer cell
3
6.“hormone" = to urge on/impulse
Signaling receptor diversity
The mammalian genome encodes for thousandsof signaling receptors - Many of these are targets for drugs
Tissue specific expression: Each individual animal cellexpress only some of these receptors
4
Membrane permeability
O• Hydrophobic molecules
Na+
• Charged
molecules
IonsAmino acids
Cl-
• “Large” uncharged polar molecules
Glucose
5
Cortisol TestosteroneCholesterol
O N O
O
ON C C
Localization of signaling receptors
Receptor on plasma membrane
Receptor in cytosol Receptor in nucleus
Hydrophilic molecule
(and proteins)
Hydrophobicmolecules
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
6
hydrophobic lipohilic non-polar (often used as synonyms)
Receptor agonists and antagonistsOther 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
OHOH
CHCH2NHCH3
OH
OH
CHCH2NHCH3
OH
Adrenalin Phenylephrine(natural) (selective
agonist)
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
A
B
C
D
E
Five modes of cell communication
Neuron
Bloodstream
B Paracrine C AutocrineD Endocrine E Neuronal/synaptic
Signaling by secreted ligands:
Contact dependent signaling: Ligands on the cell surfaceA
8
Surface receptorIntra-cellular receptors
"crinis" = secrete
Signaling cell Target cell
Receptor/ligand
Contact-dependent signaling uses ligands and receptors that are plasma membrane-bound:- Persistent signals (uni- or bidirectional) - Directed toward neighboring cells
A Contact-dependent signaling
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 controlSurvival
Gap Junctions permit free passage of smallmolecules between adjacent cells
Important for e.g., synchronous heart contraction
Cadherin
Gap junction
Integrin10
Paracrine signaling involves secretion of a ligand that act locally on cells with the appropriate receptors:
Local effect "para" = near
Signaling cell
Adjacent target cells
B Paracrine signaling
11
Signaling and target cell
Autocrine signaling impliesthat a cell secretes a ligand that it responds to itself
C Autocrine signaling
?
12
"autos" = self
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 through the blood systems before reaching a target cell
Endocrine cellsecreting
Distant target cells
D Endocrine signaling
13
Signaling cell
"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
Target cell
Cell body of a neuronAxon
Synapse
Release of neurotransmittor
E Neuronal/synaptic signaling
14
kidney
*Gland
Neuroendocrine integration
Hormone secreting glands in the brain link neuronal signals and peripheral endocrine glands.
Fight-or-flight reflex: the Hypothalamic- Pituitary-Adrenal (HPA) system
The adrenal gland responds to both the hormone (ACTH) and a nerve signal
ACTH Adrenal Cortex cortisolIncreased 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 15
*Gland
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
“Ryss 5a”: A mix of synthetic anabolic steroids ( muscle growth)
Membrane permeable
Membraneimpermeable
16
Fast versus slow signal transduction events
Altered protein function
Cell response
An altered cytoplasmic signaling protein
DNA
mRNA
Altered gene expression
Alteredproteinlevel
mRNA
Protein
Slow (minutes to hours)
Fast (<seconds)
17
Signal
Signaling with nitric oxide gas
CH2 CH2 CH2
O
NO2
O
NO2
O
NO2
• 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)
18
Nitroglycerin is converted in blood to NO (used totreat coronary artery disease since 1878)
• Skeletal muscle
• Cardiac muscle
• Smooth muscle cells: i) surrounds hollow organs –
intestines and blood vessels ii) arrector pili muscles
attached to hair follicles
Three types of cells dedicated to contraction
19
All three muscle cell types contains filaments consisting of actin and myosin, which may
contract and slide apart
Endothelial cell
Smooth muscle cell
Blood vessel
Neuron
Arginine
NO (Nitric oxide)
Vasodilatation through nitric oxide signaling
Acetylcholine
Relaxation of smooth muscle cell
Diffusion to adjacentsmooth muscle cell
Increased blood flow20
”2nd messengers”
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+
Ca2+
Ca2+Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Video 15.1-calcium_signaling 21
Adenylylcyclase
Guanylyl cyclase
Ca2+
=1 mM
=10 nM
Effect of nitric oxide on smooth muscle cells
P P P
P PP +
Cyclic GMP
GTP
Guanylyl cyclase
P
GMP
Cyclic-GMP phosphodiesterase
(constitutively active)
Viagraä
Activation of an “in-ward” Ca2+-pump in membranes of
intra-cellular Ca2+-stores
Nitric oxide
Low [Ca2+] makes contractile filaments (actin and myosin)
slide apart
Relaxation of smoothmuscle cells and
increased blood flow
22
Signaling by intracellular receptors – part I
Target genes
NLS
NLS
NLSCombined receptor/transcription factor
Hydrophobic ligand (e.g. Cortisol)
Plasma membrane
Binding displaces a protein that masks an NLS on the cortisol receptor
1.
2.
Receptor translocationinto the nucleus specific transcription
3.
1.
2.
3.
Cortisol diffuse through the plasma membrane
23
= DNA
Signaling by intracellular receptors – part II
Plasma membrane
Target genes
Target genes
Inhibitor
Inhibitor
1.
3.
2.
3.
2.
1. The DNA-binding receptor/transcription factor is inactive
The ligand (e.g. sex hormones) diffuses into the nucleus
The ligand displaces the inhibitor
24
Hydrophobic ligand
General principle of cell surface receptor signaling
1.
3.
Reception
2. Signal transductioncascade comprising:i. molecular switchesii. 2nd messengers
Response Metabolicenzyme
Gene regulatoryprotein
Etc.
Receptor
Signal molecule(Ligand)
25
P. M.
Cytosol
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 phosphorylationThe most common ‘on-off’ switch is provided by protein phosphorylation
OH
Serine, threonineor tyrosine
Serine, threonineor tyrosine
O O
O -P
O
Kinase
-
Phosphatase
26
+ ATP
Kinase : ~1000 protein kinase genes in vertebrates. Some have only a single substrate. Others are “multi-functional” and may have >10 substrates
GTP
GDP
Guanine-nucleotide Exchange Factor (GEF)
GTPase Activating Protein (GAP)
Inactive Active
P
GDP
II. Molecular switches in signal transduction2. GTP binding proteins (G-proteins)Another ‘on-off’ switch is provided byregulatable GTP-binding and hydrolysis
GDPGTP >>GTP
27
Molecular_models 15.5-Ras (one PO4 makes the diff.)
Signal transduction cascades
MetabolismGene regulation Etc.
P
GTP
A single cell surface receptor may activate several signal transduction pathways
This involves various G-proteins, 2nd messengers and protein kinases
Protein kinases at the end of a cascade mayhave many substrates
Response:
28
P P
Kinase
Ca2+cGMPcAMP
P. M.
Three main classes of cell-surface receptors
G-protein coupled receptors
Receptors with intrinsic enzymatic activity
ZZZ ZZZ
ZZZ
Ion channel coupled receptors
Ion
Ion
Ion
Ligand
29
3. G-proteins may regulate enzymes or ion channels
G-protein coupled receptors (GPCR)
A hallmark of GPCR´s is 7 trans-membrane spanning regions
ZZZ
1. Ligand binding conformational change
2. A specific G-protein is recruited and activated G
30
Down-stream effectors of various G-proteins
Cyclic AMPAdenylylcyclase
ATPGuanylyl cyclase
Cyclic GMP
GTP
Phospholipase C
Ca2+
Increase incytosolic and activation of protein kinase C
Ion
Ion
Ion
1. 2.
3. 4. Ion channels
31
I. Regulation of hetero-trimeric G-proteins
GTP
GDP
Inactive Active
P
GDP
GTP
a abg
b
g+
a-subunit and/or b,g-subunit can activate or suppress
different downstream targets
Complex dissociate upon GTP binding
=GAP
GDPGTP >>
32
RGS
=GEF
RGS: Regulator of G-protein Signaling
P.M.
b g
P.M. GDPab g
GTPab
g +
GDP GTP
Ligand binding causes a conformational change
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
GDPa
No ligand (default state)
33
II. Regulation of hetero-trimeric G-proteins
GTPa
P.M.b
gGDP
a
P
b g
GDPa+
The intrinsic GTP hydrolysis is slow but RGS, an a-subunit specific GAP, catalyzes hydrolysis. This terminates the signal
RGS
GTPas
GTPai
Phospholipase C-b(PLC-b)
GTPaq
Adenylyl cyclase
A family of a-subunits with distinct functions
Anim. 15.3-G-protein_signaling
34III. Regulation of hetero-trimeric G-proteins
Alberts et al: Table 15-3 (tissue specificity)
Adenylyl cyclase activation by the as-subunit of G-proteins
GTPas
P.M.
P P P
P PP +
Cyclic AMP
ATP
P
AMP
Adenylyl cyclase
Cyclic-AMP phosphodiesterase
(constitutively active)
Caffeine
35
Inactive PKA
Cyclic AMP
Active PKA
Target genes
CREB
CREB
P
Cyclic AMP second messenger signaling
Glycogen phosporylase
PGlycogen
phosporylase
Cyclic AMP activates Protein kinase A (PKA), which can regulate: Metabolism Gene transcription 1.2.
1.
2.
Glucose-1- phosphate
Glycogen36
Summary of the cyclic AMP signaling cascade
Cyclic AMP
CREB
P
Adenylylcyclase
PKA
Regulates metabolismATP
PRegulates transcription
Glycogen breakdown
P
GTPas
Anim. 15.4-cAMP_signaling 37
-Regulated DNA binding
P
Glycogen:- Stored in muscles and liver- Rapidly available energy sourceWork/stress adrenalin cAMP PKA Glycogen breakdown
Alberts et al: Table 15-1 (tissue specific response)
GEF (GPCR)
Signal induced cleavage of phospholipids
Variable
Phosphate
GlycerolFatty acid
Fatty acidPhospholipase A1
Phospholipase A2
Phospholipase C
Phospholipase D
External signals may activate distinct phospholipases that cleave phospholipids at specific sites and thereby catalyze the formation of various molecules with signaling properties
Soluble compounds release into the
cytosol38
Precursors for various signaling
substances
Phospholipase C activation generates two 2nd messengers
aq
Fatty acid
Fatty acid
Glycerol
Fatty acid
Fatty acidGlycerol
OH
Diacylglycerol (DAG)
Phosphatidylinositol 4,5- bisphosphate, PI (4,5)P2
Inner leaflet ofplasma membrane
Phospho-lipase C-b(PLC-b) P
PP
PP
P
Inositol 1,4,5-triphosphate, IP3
aq-subunit activates PLC1.
2. PLC cleaves PIP2, generating thetwo 2nd messengers DAG and IP3
1.
2.
GTP
39
PP
P
IP3
OH
Inner leaflet ofplasma membrane
Ca2+ Ca2+
IP3 regulated Ca2+ channel
PKCCa2+
Ca2+
PKC Ca2+
CalmodulinCa2+
Ca2+
Ca2+
Ca2+
Calmodulin
Calmodulin regulated Ca2+ pump in ER
Ca2+
DAG
1.
2.
DAG recruits PKC to plasmamembraneIP3 mediate release of Ca2+ from ER
3. DAG and Ca2+ activates PKC
1.
2.
3.
4. Ca2+ activates calmodulin to terminate signal by pumping Ca2+
back into ER
4.
Role of the 2nd messengers IP3 and DAG
40
Ca2+/calmodulin dependent protein kinase (CaMK)
P
CalmodulinCa2+
Ca2+
Ca2+
Ca2+
Calmodulin
Ca 2+
Ca 2+
Ca 2+
Ca 2+
Calmodulin
Ca 2+
Ca 2+ Ca 2+
Ca 2+
Autophosphorylation
DephosphorylationCatalytic
Inhibitory
Calmodulin
ActivatedFully active
P
Partially active
Resting state
Inactive
Ca2+
Increasedcytosolic
41
Molecular_models 15.6-calmodulin
IP3
OH
Ca2+
PKC
CalmodulinCa2+
Ca2+
Ca2+
Ca2+
DAGPLC-b
PP
P
PP
P
CaMK
Summary of G-protein signaling through PLC-β
aq
GTP
Other regulated enzymes
Ca2+
STOP
Termination of signalCa2+
Etc!
42
Both PKC and CaMK have many potential (tissue specific) substrates
GEF (GPCR)
Enzyme linked receptors
Tyr P Ser/Thr
Many variants on this theme – here we focus on:Receptor tyrosine kinasesReceptor serine/threonine kinases
PHomo-dimers Hetero-dimers
Single pass transmembrane receptors. Ligand binding cause dimer formation and consequent “auto”-phosphorylation
43Jenkinson : RTK - dimerizationAlberts et al: Table 15-4 (tissue specific RTK’s)
Signaling through Receptor Tyrosine Kinases
Tyr
Kinase domain
Tyr
Kinase domain
Tyr
Kinase domain
Tyr
Kinase domain
Inactive receptor monomers
Active receptor dimer
Ligand binding causes receptor dimerization
P P
P. M.
Trans-phosphorylation of tyrosine residues
Single pass transmembrane protein
TyrTyr
TyrTyr TyrTyrP P
TyrTyrP P
44
Cis- prefix means "on this side"Trans- prefix means "across"
TyrTyrP P
TyrTyrP P
Tyr
Kinase domain
Tyr
Kinase domain
P P SH2 SH3
GTPRas
Ras GEF (Sos)
Regions containing phospho-Tyr may serve as specific docking sites for SH2 domain-containing signaling proteins (SH = Src Homology domain)
SH2-proteins binds at specific phospho-tyrosines
P3
These can be enzymes….
Phosphatidyl-inositol (PI) Monomeric G-protein
GDPRas
PI-3 Kinase
45
………….or they act as adaptors forsignaling proteins
Fig. 15-55
Phosphorylation cascade downstream of RasGTP
Ras
MekMek
Raf
P
Erk (MAPK)
P. M.
Erk P
Target genes
P
2.
Erk P
PCytosolic
target proteins
1.
1. Altered protein function
Altered gene expression2.
46
Termination of RTK/Ras/MAPK pathwayReceptor and ligand internalization
GDP GTPRas Ras
Erk (MAPK) Erk
P
1. 2. Ras GTP hydrolysis
Ras GAP
3. Dephosphorylation
Phosphatase
Note: Signalingreceptors arerarely recycled
47Anim. 13.3-receptor_endocytosis (Note: vesicle fusion with endosome)
Fusion withendosome
Fusion withprimary lysosome degradation
3
4
5P
PI kinase PIP kinasePI(4,5)P2
P
PI – phosphorylation cycles on inositol ring position 4 & 5
P PP
P
PI(4)PInositol
Extracellular space
CytosolPhosphatidylinositol (PI)
I. PI-kinases act at specific positions of the inositol ring
PhosphateGlycerol
Fatty
aci
d
Fatty
aci
d
Inositol
48
II. PI-3 kinase completes a PH-domain binding site
PI(3,4,5)P3
P3 3
PI(4,5)P2
PTEN
PI-3 Kinase
Activated receptor recruits and activates PI-3 kinase 1.
2.
PI-3 kinase
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)
1.2.
3. PTEN removes phosphorylation on position 3 on PI(3,4,5)P3 to terminate signal
3.PP
PP
P
Phosphatidyl-inositol (PI)
49
P P
III. PKB/Akt activation downstream of PI-3 kinase
P PPDK1 PKB/Akt
P
3 3
PH-domainsPKB/AktP
P P3 3
PDK1
PKB/Akt
PP P PPP PP
PDK1 phosphorylates PKB/Akt thereby mediating its activation
2.
1. PI(3,4,5)P3 brings PDK1 and PKB/Aktinto proximity through their PH-domains
1.2.
50
PP P P
IV. Different signaling pathways – same target
PPI-3 K
PP
3
GDPa
b g
GTPa b
g+
PI-3 K
Both G-protein- and RTK signaling may result in generation of PI(3,4,5)P3
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 via a bg-subunit binding domain or a SH2 domain 51
P
I. Transcriptional regulation by TGF-b / BMP
P.M.
Target genes
P PSmad 2/3 P
Smad 4
Smad 2/3 PSmad 4
Type I receptor
Smad 7 Negative feedback loop
Type II receptor: Ser/Thr kinase
52
TGF-b TGF-bTGF-b
II. Transcriptional regulation by Wnt/winglessWnt
DishevelledLRP Frizzled
Target genesTCFb-catenin
GSK-3b
b-cateninP
Ub
UbUb
Target genes
TCF
Groucho
AxinAPC
GSK-3b
b-cateninAxinAPC
G1myc
MMP7
Dishevelled
ZZZ
53
54
Signal transducing proteins are oftentargets of therapeutic drugs or infections agents
My own favorite protein!
"All science is either physics or stamp collecting"
Ernest Rutherford(1871-1937, Nobelprize1908)
Recommended readingChapter 15879-941946-954
Alberts et al. 5th edition